An integrated genetic linkage map of watermelon and genetic diversity based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers

USDA-ARS?s Scientific Manuscript database

Watermelon (Citrullus lanatus var. lanatus) is an important vegetable fruit throughout the world. A high number of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers should provide large coverage of the watermelon genome and high phylogenetic resolution of germplasm acces...

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Two Low Coverage Bird Genomes and a Comparison of Reference-Guided versus De Novo Genome Assemblies

PubMed Central

Card, Daren C.; Schield, Drew R.; Reyes-Velasco, Jacobo; Fujita, Matthew K.; Andrew, Audra L.; Oyler-McCance, Sara J.; Fike, Jennifer A.; Tomback, Diana F.; Ruggiero, Robert P.; Castoe, Todd A.

2014-01-01

As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5–5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies. PMID:25192061

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

A HIGH COVERAGE GENOME SEQUENCE FROM AN ARCHAIC DENISOVAN INDIVIDUAL

PubMed Central

Meyer, Matthias; Kircher, Martin; Gansauge, Marie-Theres; Li, Heng; Racimo, Fernando; Mallick, Swapan; Schraiber, Joshua G.; Jay, Flora; Prüfer, Kay; de Filippo, Cesare; Sudmant, Peter H.; Alkan, Can; Fu, Qiaomei; Do, Ron; Rohland, Nadin; Tandon, Arti; Siebauer, Michael; Green, Richard E.; Bryc, Katarzyna; Briggs, Adrian W.; Stenzel, Udo; Dabney, Jesse; Shendure, Jay; Kitzman, Jacob; Hammer, Michael F.; Shunkov, Michael V.; Derevianko, Anatoli P.; Patterson, Nick; Andrés, Aida M.; Eichler, Evan E.; Slatkin, Montgomery; Reich, David; Kelso, Janet; Pääbo, Svante

2013-01-01

We present a DNA library preparation method that has allowed us to reconstruct a high coverage (30X) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans. PMID:22936568

MSeq-CNV: accurate detection of Copy Number Variation from Sequencing of Multiple samples.

PubMed

Malekpour, Seyed Amir; Pezeshk, Hamid; Sadeghi, Mehdi

2018-03-05

Currently a few tools are capable of detecting genome-wide Copy Number Variations (CNVs) based on sequencing of multiple samples. Although aberrations in mate pair insertion sizes provide additional hints for the CNV detection based on multiple samples, the majority of the current tools rely only on the depth of coverage. Here, we propose a new algorithm (MSeq-CNV) which allows detecting common CNVs across multiple samples. MSeq-CNV applies a mixture density for modeling aberrations in depth of coverage and abnormalities in the mate pair insertion sizes. Each component in this mixture density applies a Binomial distribution for modeling the number of mate pairs with aberration in the insertion size and also a Poisson distribution for emitting the read counts, in each genomic position. MSeq-CNV is applied on simulated data and also on real data of six HapMap individuals with high-coverage sequencing, in 1000 Genomes Project. These individuals include a CEU trio of European ancestry and a YRI trio of Nigerian ethnicity. Ancestry of these individuals is studied by clustering the identified CNVs. MSeq-CNV is also applied for detecting CNVs in two samples with low-coverage sequencing in 1000 Genomes Project and six samples form the Simons Genome Diversity Project.

Recovering complete and draft population genomes from metagenome datasets

DOE PAGES

Sangwan, Naseer; Xia, Fangfang; Gilbert, Jack A.

2016-03-08

Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem ofmore » chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution.« less

Recovering complete and draft population genomes from metagenome datasets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sangwan, Naseer; Xia, Fangfang; Gilbert, Jack A.

Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem ofmore » chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution.« less

Novel chytrid lineages dominate fungal sequences in diverse marine and freshwater habitats

NASA Astrophysics Data System (ADS)

Comeau, André M.; Vincent, Warwick F.; Bernier, Louis; Lovejoy, Connie

2016-07-01

In aquatic environments, fungal communities remain little studied despite their taxonomic and functional diversity. To extend the ecological coverage of this group, we conducted an in-depth analysis of fungal sequences within our collection of 3.6 million V4 18S rRNA pyrosequences originating from 319 individual marine (including sea-ice) and freshwater samples from libraries generated within diverse projects studying Arctic and temperate biomes in the past decade. Among the ~1.7 million post-filtered reads of highest taxonomic and phylogenetic quality, 23,263 fungal sequences were identified. The overall mean proportion was 1.35%, but with large variability; for example, from 0.01 to 59% of total sequences for Arctic seawater samples. Almost all sample types were dominated by Chytridiomycota-like sequences, followed by moderate-to-minor contributions of Ascomycota, Cryptomycota and Basidiomycota. Species and/or strain richness was high, with many novel sequences and high niche separation. The affinity of the most common reads to phytoplankton parasites suggests that aquatic fungi deserve renewed attention for their role in algal succession and carbon cycling.

Genomic Sequencing: Assessing The Health Care System, Policy, And Big-Data Implications

PubMed Central

Phillips, Kathryn A.; Trosman, Julia; Kelley, Robin K.; Pletcher, Mark J.; Douglas, Michael P.; Weldon, Christine B.

2014-01-01

New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a “big data” technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. PMID:25006153

Genomic sequencing: assessing the health care system, policy, and big-data implications.

PubMed

Phillips, Kathryn A; Trosman, Julia R; Kelley, Robin K; Pletcher, Mark J; Douglas, Michael P; Weldon, Christine B

2014-07-01

New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a "big data" technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. Project HOPE—The People-to-People Health Foundation, Inc.

A user's guide to quantitative and comparative analysis of metagenomic datasets.

PubMed

Luo, Chengwei; Rodriguez-R, Luis M; Konstantinidis, Konstantinos T

2013-01-01

Metagenomics has revolutionized microbiological studies during the past decade and provided new insights into the diversity, dynamics, and metabolic potential of natural microbial communities. However, metagenomics still represents a field in development, and standardized tools and approaches to handle and compare metagenomes have not been established yet. An important reason accounting for the latter is the continuous changes in the type of sequencing data available, for example, long versus short sequencing reads. Here, we provide a guide to bioinformatic pipelines developed to accomplish the following tasks, focusing primarily on those developed by our team: (i) assemble a metagenomic dataset; (ii) determine the level of sequence coverage obtained and the amount of sequencing required to obtain complete coverage; (iii) identify the taxonomic affiliation of a metagenomic read or assembled contig; and (iv) determine differentially abundant genes, pathways, and species between different datasets. Most of these pipelines do not depend on the type of sequences available or can be easily adjusted to fit different types of sequences, and are freely available (for instance, through our lab Web site: http://www.enve-omics.gatech.edu/). The limitations of current approaches, as well as the computational aspects that can be further improved, will also be briefly discussed. The work presented here provides practical guidelines on how to perform metagenomic analysis of microbial communities characterized by varied levels of diversity and establishes approaches to handle the resulting data, independent of the sequencing platform employed. © 2013 Elsevier Inc. All rights reserved.

Sequential strand displacement beacon for detection of DNA coverage on functionalized gold nanoparticles.

PubMed

Paliwoda, Rebecca E; Li, Feng; Reid, Michael S; Lin, Yanwen; Le, X Chris

2014-06-17

Functionalizing nanomaterials for diverse analytical, biomedical, and therapeutic applications requires determination of surface coverage (or density) of DNA on nanomaterials. We describe a sequential strand displacement beacon assay that is able to quantify specific DNA sequences conjugated or coconjugated onto gold nanoparticles (AuNPs). Unlike the conventional fluorescence assay that requires the target DNA to be fluorescently labeled, the sequential strand displacement beacon method is able to quantify multiple unlabeled DNA oligonucleotides using a single (universal) strand displacement beacon. This unique feature is achieved by introducing two short unlabeled DNA probes for each specific DNA sequence and by performing sequential DNA strand displacement reactions. Varying the relative amounts of the specific DNA sequences and spacing DNA sequences during their coconjugation onto AuNPs results in different densities of the specific DNA on AuNP, ranging from 90 to 230 DNA molecules per AuNP. Results obtained from our sequential strand displacement beacon assay are consistent with those obtained from the conventional fluorescence assays. However, labeling of DNA with some fluorescent dyes, e.g., tetramethylrhodamine, alters DNA density on AuNP. The strand displacement strategy overcomes this problem by obviating direct labeling of the target DNA. This method has broad potential to facilitate more efficient design and characterization of novel multifunctional materials for diverse applications.

Metagenomic analysis of viral diversity in respiratory samples from patients with respiratory tract infections in Kuwait.

PubMed

Madi, Nada; Al-Nakib, Widad; Mustafa, Abu Salim; Habibi, Nazima

2018-03-01

A metagenomic approach based on target independent next-generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next-generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non-respiratory viruses were detected in 14% of the clinical samples, while sequences of non-human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real-Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections. © 2017 Wiley Periodicals, Inc.

Clonal evolution in breast cancer revealed by single nucleus genome sequencing.

PubMed

Wang, Yong; Waters, Jill; Leung, Marco L; Unruh, Anna; Roh, Whijae; Shi, Xiuqing; Chen, Ken; Scheet, Paul; Vattathil, Selina; Liang, Han; Multani, Asha; Zhang, Hong; Zhao, Rui; Michor, Franziska; Meric-Bernstam, Funda; Navin, Nicholas E

2014-08-14

Sequencing studies of breast tumour cohorts have identified many prevalent mutations, but provide limited insight into the genomic diversity within tumours. Here we developed a whole-genome and exome single cell sequencing approach called nuc-seq that uses G2/M nuclei to achieve 91% mean coverage breadth. We applied this method to sequence single normal and tumour nuclei from an oestrogen-receptor-positive (ER(+)) breast cancer and a triple-negative ductal carcinoma. In parallel, we performed single nuclei copy number profiling. Our data show that aneuploid rearrangements occurred early in tumour evolution and remained highly stable as the tumour masses clonally expanded. In contrast, point mutations evolved gradually, generating extensive clonal diversity. Using targeted single-molecule sequencing, many of the diverse mutations were shown to occur at low frequencies (<10%) in the tumour mass. Using mathematical modelling we found that the triple-negative tumour cells had an increased mutation rate (13.3×), whereas the ER(+) tumour cells did not. These findings have important implications for the diagnosis, therapeutic treatment and evolution of chemoresistance in breast cancer.

Diversity arrays technology (DArT) markers in apple for genetic linkage maps.

PubMed

Schouten, Henk J; van de Weg, W Eric; Carling, Jason; Khan, Sabaz Ali; McKay, Steven J; van Kaauwen, Martijn P W; Wittenberg, Alexander H J; Koehorst-van Putten, Herma J J; Noordijk, Yolanda; Gao, Zhongshan; Rees, D Jasper G; Van Dyk, Maria M; Jaccoud, Damian; Considine, Michael J; Kilian, Andrzej

2012-03-01

Diversity Arrays Technology (DArT) provides a high-throughput whole-genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. The work presented here details the development and performance of a DArT genotyping array for apple. This is the first paper on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high-throughput method for obtaining accurate and reproducible marker data, despite the low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. The standard complexity reduction method, based on the methylation-sensitive PstI restriction enzyme, resulted in a high frequency of markers, although there was 52-54% redundancy due to the repeated sampling of highly similar sequences. Sequencing of the marker clones showed that they are significantly enriched for low-copy, genic regions. The genome coverage using the standard method was 55-76%. For improved genome coverage, an alternative complexity reduction method was examined, which resulted in less redundancy and additional segregating markers. The DArT markers proved to be of high quality and were very suitable for genetic mapping at low cost for the apple, providing moderate genome coverage. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9579-5) contains supplementary material, which is available to authorized users.

Identification of copy number variants in whole-genome data using Reference Coverage Profiles

PubMed Central

Glusman, Gustavo; Severson, Alissa; Dhankani, Varsha; Robinson, Max; Farrah, Terry; Mauldin, Denise E.; Stittrich, Anna B.; Ament, Seth A.; Roach, Jared C.; Brunkow, Mary E.; Bodian, Dale L.; Vockley, Joseph G.; Shmulevich, Ilya; Niederhuber, John E.; Hood, Leroy

2015-01-01

The identification of DNA copy numbers from short-read sequencing data remains a challenge for both technical and algorithmic reasons. The raw data for these analyses are measured in tens to hundreds of gigabytes per genome; transmitting, storing, and analyzing such large files is cumbersome, particularly for methods that analyze several samples simultaneously. We developed a very efficient representation of depth of coverage (150–1000× compression) that enables such analyses. Current methods for analyzing variants in whole-genome sequencing (WGS) data frequently miss copy number variants (CNVs), particularly hemizygous deletions in the 1–100 kb range. To fill this gap, we developed a method to identify CNVs in individual genomes, based on comparison to joint profiles pre-computed from a large set of genomes. We analyzed depth of coverage in over 6000 high quality (>40×) genomes. The depth of coverage has strong sequence-specific fluctuations only partially explained by global parameters like %GC. To account for these fluctuations, we constructed multi-genome profiles representing the observed or inferred diploid depth of coverage at each position along the genome. These Reference Coverage Profiles (RCPs) take into account the diverse technologies and pipeline versions used. Normalization of the scaled coverage to the RCP followed by hidden Markov model (HMM) segmentation enables efficient detection of CNVs and large deletions in individual genomes. Use of pre-computed multi-genome coverage profiles improves our ability to analyze each individual genome. We make available RCPs and tools for performing these analyses on personal genomes. We expect the increased sensitivity and specificity for individual genome analysis to be critical for achieving clinical-grade genome interpretation. PMID:25741365

Graph-based optimization of epitope coverage for vaccine antigen design

DOE PAGES

Theiler, James Patrick; Korber, Bette Tina Marie

2017-01-29

Epigraph is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population. Potential epitopes are defined as short contiguous stretches of proteins, comparable in length to T-cell epitopes. This optimal coverage problem can be formulated in terms of a directed graph, with candidate antigens represented as paths that traverse this graph. Epigraph protein sequences can also be used as the basis for designing peptides for experimental evaluation of immune responses in natural infections to highly variable proteins. The epigraph tool suite also enables rapidmore » characterization of populations of diverse sequences from an immunological perspective. Fundamental distance measures are based on immunologically relevant shared potential epitope frequencies, rather than simple Hamming or phylogenetic distances. Here, we provide a mathematical description of the epigraph algorithm, include a comparison of different heuristics that can be used when graphs are not acyclic, and we describe an additional tool we have added to the web-based epigraph tool suite that provides frequency summaries of all distinct potential epitopes in a population. Lastly, we also show examples of the graphical output and summary tables that can be generated using the epigraph tool suite and explain their content and applications.« less

Graph-based optimization of epitope coverage for vaccine antigen design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Theiler, James Patrick; Korber, Bette Tina Marie

Epigraph is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population. Potential epitopes are defined as short contiguous stretches of proteins, comparable in length to T-cell epitopes. This optimal coverage problem can be formulated in terms of a directed graph, with candidate antigens represented as paths that traverse this graph. Epigraph protein sequences can also be used as the basis for designing peptides for experimental evaluation of immune responses in natural infections to highly variable proteins. The epigraph tool suite also enables rapidmore » characterization of populations of diverse sequences from an immunological perspective. Fundamental distance measures are based on immunologically relevant shared potential epitope frequencies, rather than simple Hamming or phylogenetic distances. Here, we provide a mathematical description of the epigraph algorithm, include a comparison of different heuristics that can be used when graphs are not acyclic, and we describe an additional tool we have added to the web-based epigraph tool suite that provides frequency summaries of all distinct potential epitopes in a population. Lastly, we also show examples of the graphical output and summary tables that can be generated using the epigraph tool suite and explain their content and applications.« less

Pangenome and taxonomic analysis of Salmonella enterica subspecies enterica

USDA-ARS?s Scientific Manuscript database

Salmonella enterica subspecies enterica (S. enterica ssp. I) contains almost all the major pathogens in this genus. We sequenced 354 new S. enterica ssp. I genomes using paired end 100 base reads to ~80-fold coverage. These genomes were chosen to maximize genetic diversity, representing at least 100...

Determining the specific microbial populations and their spatial distribution within the stromatolite ecosystem of Shark Bay.

PubMed

Goh, Falicia; Allen, Michelle A; Leuko, Stefan; Kawaguchi, Tomohiro; Decho, Alan W; Burns, Brendan P; Neilan, Brett A

2009-04-01

The stromatolites at Shark Bay, Western Australia, are analogues of some of the oldest evidence of life on Earth. The aim of this study was to identify and spatially characterize the specific microbial communities associated with Shark Bay intertidal columnar stromatolites. Conventional culturing methods and construction of 16S rDNA clone libraries from community genomic DNA with both universal and specific PCR primers were employed. The estimated coverage, richness and diversity of stromatolite microbial populations were compared with earlier studies on these ecosystems. The estimated coverage for all clone libraries indicated that population coverage was comprehensive. Phylogenetic analyses of stromatolite and surrounding seawater sequences were performed in ARB with the Greengenes database of full-length non-chimaeric 16S rRNA genes. The communities identified exhibited extensive diversity. The most abundant sequences from the stromatolites were alpha- and gamma-proteobacteria (58%), whereas the cyanobacterial community was characterized by sequences related to the genera Euhalothece, Gloeocapsa, Gloeothece, Chroococcidiopsis, Dermocarpella, Acaryochloris, Geitlerinema and Schizothrix. All clones from the archaeal-specific clone libraries were related to the halophilic archaea; however, no archaeal sequence was identified from the surrounding seawater. Fluorescence in situ hybridization also revealed stromatolite surfaces to be dominated by unicellular cyanobacteria, in contrast to the sub-surface archaea and sulphate-reducing bacteria. This study is the first to compare the microbial composition of morphologically similar stromatolites over time and examine the spatial distribution of specific microorganismic groups in these intertidal structures and the surrounding seawater at Shark Bay. The results provide a platform for identifying the key microbial physiology groups and their potential roles in modern stromatolite morphogenesis and ecology.

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

PubMed

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

Detection of active transposable elements in Arabidopsis thaliana using Oxford Nanopore Sequencing technology.

PubMed

Debladis, Emilie; Llauro, Christel; Carpentier, Marie-Christine; Mirouze, Marie; Panaud, Olivier

2017-07-17

Transposables elements (TEs) contribute to both structural and functional dynamics of most eukaryotic genomes. Because of their propensity to densely populate plant and animal genomes, the precise estimation of the impact of transposition on genomic diversity has been considered as one of the main challenges of today's genomics. The recent development of NGS (next generation sequencing) technologies has open new perspectives in population genomics by providing new methods for high throughput detection of Transposable Elements-associated Structural Variants (TEASV). However, these have relied on Illumina platform that generates short reads (up to 350 nucleotides). This limitation in size of sequence reads can cause high false discovery rate (FDR) and therefore limit the power of detection of TEASVs, especially in the case of large, complex genomes. The newest sequencing technologies, such as Oxford Nanopore Technologies (ONT) can generate kilobases-long reads thus representing a promising tool for TEASV detection in plant and animals. We present the results of a pilot experiment for TEASV detection on the model plant species Arabidopsis thaliana using ONT sequencing and show that it can be used efficiently to detect TE movements. We generated a ~0.8X genome coverage of a met1-derived epigenetic recombinant inbred line (epiRIL) using a MinIon device with R7 chemistry. We were able to detect nine new copies of the LTR-retrotransposon Evadé (EVD). We also evidenced the activity of the DNA transposon CACTA, CAC1. Even at a low sequence coverage (0.8X), ONT sequencing allowed us to reliably detect several TE insertions in Arabidopsis thaliana genome. The long read length allowed a precise and un-ambiguous mapping of the structural variations caused by the activity of TEs. This suggests that the trade-off between read length and genome coverage for TEASV detection may be in favor of the former. Should the technology be further improved both in terms of lower error rate and operation costs, it could be efficiently used in diversity studies at population level.

Streptococcus pyogenes strains in Sao Paulo, Brazil: molecular characterization as a basis for StreptInCor coverage capacity analysis.

PubMed

Freschi de Barros, Samar; De Amicis, Karine Marafigo; Alencar, Raquel; Smeesters, Pierre Robert; Trunkel, Ariel; Postól, Edilberto; Almeida Junior, João Nóbrega; Rossi, Flavia; Pignatari, Antonio Carlos Campos; Kalil, Jorge; Guilherme, Luiza

2015-08-05

Several human diseases are caused by Streptococcus pyogenes, ranging from common infections to autoimmunity. Characterization of the most prevalent strains worldwide is a useful tool for evaluating the coverage capacity of vaccines under development. In this study, a collection of S. pyogenes strains from Sao Paulo, Brazil, was analyzed to describe the diversity of strains and assess the vaccine coverage capacity of StreptInCor. Molecular epidemiology of S. pyogenes strains was performed by emm-genotyping the 229 isolates from different clinical sites, and PCR was used for superantigen profile analysis. The emm-pattern and tissue tropism for these M types were also predicted and compared based on the emm-cluster classification. The strains were fit into 12 different emm-clusters, revealing a diverse phylogenetic origin and, consequently, different mechanisms of infection and escape of the host immune system. Forty-eight emm-types were distinguished in 229 samples, and the 10 most frequently observed types accounted for 69 % of all isolates, indicating a diverse profile of circulating strains comparable to other countries under development. A similar proportion of E and A-C emm-patterns were observed, whereas pattern D was less frequent, indicating that the strains of this collection primarily had a tissue tropism for the throat. In silico analysis of the coverage capacity of StreptInCor, an M protein-conserved regionally based vaccine candidate developed by our group, had a range of 94.5 % to 59.7 %, with a mean of 71.0 % identity between the vaccine antigen and the predicted amino acid sequence of the emm-types included here. This is the first report of S. pyogenes strain characterization in Sao Paulo, one of the largest cities in the world; thus, the strain panel described here is a representative sample for vaccine coverage capacity analysis. Our results enabled evaluation of StreptInCor candidate vaccine coverage capacity against diverse M-types, indicating that the vaccine candidate likely would induce protection against the diverse strains worldwide.

High-throughput sequencing of the chloroplast and mitochondrion of Chlamydomonas reinhardtii to generate improved de novo assemblies, analyze expression patterns and transcript speciation, and evaluate diversity among laboratory strains and wild isolates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallaher, Sean D.; Fitz-Gibbon, Sorel T.; Strenkert, Daniela

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Gen-ome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genomemore » and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar tran-scripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3’-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellargenomes, which is 1/10th as much genetic diversity as is found in the nucleus.« less

Genomic diversity of the human intestinal parasite Entamoeba histolytica

PubMed Central

2012-01-01

Background Entamoeba histolytica is a significant cause of disease worldwide. However, little is known about the genetic diversity of the parasite. We re-sequenced the genomes of ten laboratory cultured lines of the eukaryotic pathogen Entamoeba histolytica in order to develop a picture of genetic diversity across the genome. Results The extreme nucleotide composition bias and repetitiveness of the E. histolytica genome provide a challenge for short-read mapping, yet we were able to define putative single nucleotide polymorphisms in a large portion of the genome. The results suggest a rather low level of single nucleotide diversity, although genes and gene families with putative roles in virulence are among the more polymorphic genes. We did observe large differences in coverage depth among genes, indicating differences in gene copy number between genomes. We found evidence indicating that recombination has occurred in the history of the sequenced genomes, suggesting that E. histolytica may reproduce sexually. Conclusions E. histolytica displays a relatively low level of nucleotide diversity across its genome. However, large differences in gene family content and gene copy number are seen among the sequenced genomes. The pattern of polymorphism indicates that E. histolytica reproduces sexually, or has done so in the past, which has previously been suggested but not proven. PMID:22630046

Candidate mosaic proteins for a pan-filoviral cytotoxic T-Cell lymphocyte vaccine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fenimore, Paul W; Fischer, William M; Kuiken, Carla

The extremely high fatality rates of many filovirus (FILV) strains the recurrent but rarely identified origin of human epidemics, the only partly identified viral reservoirs and the continuing non-human primate epizootics in Africa make a broadly-protective filovirus vaccine highly desirable. Cytotoxic T-cells (CTL) have been shown to be protective in mice, guinea pigs and non-human primates. In murine models the cytotoxic T-cell epitopes that are protective against Ebola virus have been mapped and in non-human primates CTL-mediated protection between viral strains (John Dye: specify) has been demonstrated using two filoviral proteins, nucleoprotein (NP) and glycoprotein (GP). These immunological results suggestmore » that the CTL avenue of immunity deserves consideration for a vaccine. The poorly-understood viral reservoirs means that it is difficult to predict what strains are likely to cause epidemics. Thus, there is a premium on developing a pan-filoviral vaccine. The genetic diversity of FILV is large, roughly the same scale as human immunodeficiency virus (HIV). This presents a serious challenge for the vaccine designer because a traditional vaccine aspiring to pan-filoviral coverage is likely to require the inclusion of many antigenic reagents. A recent method for optimizing cytotoxic T-cell lymphocyte epitope coverage with mosaic antigens was successful in improving potential CTL epitope coverage against HIV and may be useful in the context of very different viruses, such as the filoviruses discussed here. Mosaic proteins are recombinants composed of fragments of wild-type proteins joined at locations resulting in exclusively natural k-mers, 9 {le} k {le} 15, and having approximately the same length as the wild-type proteins. The use of mosaic antigens is motivated by three conjectures: (1) optimizing a mosaic protein to maximize coverage of k-mers found in a set of reference proteins will give better odds of including broadly-protective CTL epitopes in a vaccine than is possible with a wild-type protein, (2) reducing the number of low-prevalence k-mers minimizes the likelihood of undesirable immunodominance, and (3) excluding exogenous k-mers will result in mosaic proteins whose processing for presentation is close to what occurs with wild-type proteins. The first and second applications of the mosaic method were to HIV and Hepatitis C Virus (HCV). HIV is the virus with the largest number of known sequences, and consequently a plethora of information for the CTL vaccine designer to incorporate into their mosaics. Experience with HIV and HCV mosaics supports the validity of the three conjectures above. The available FILV sequences are probably closer to the minimum amount of information needed to make a meaningful mosaic vaccine candidate. There were 532 protein sequences in the National Institutes of Health GenPept database in November 2007 when our reference set was downloaded. These sequences come from both Ebola and Marburg viruses (EBOV and MARV), representing transcripts of all 7 genes. The coverage of viral diversity by the 7 genes is variable, with genes 1 (nucleoprotein, NP), 4 (glycoprotein, GP; soluble glycoprotein, sGP) and 7 (polymerase, L) giving the best coverage. Broadly-protective vaccine candidates for diverse viruses, such as HIV or Hepatitis C virus (HCV) have required pools of antigens. FILV is similar in this regard. While we have designed CTL mosaic proteins using all 7 types of filoviral proteins, only NP, GP and L proteins are reported here. If it were important to include other proteins in a mosaic CTL vaccine, additional sequences would be required to cover the space of known viral diversity.« less

The study of human Y chromosome variation through ancient DNA.

PubMed

Kivisild, Toomas

2017-05-01

High throughput sequencing methods have completely transformed the study of human Y chromosome variation by offering a genome-scale view on genetic variation retrieved from ancient human remains in context of a growing number of high coverage whole Y chromosome sequence data from living populations from across the world. The ancient Y chromosome sequences are providing us the first exciting glimpses into the past variation of male-specific compartment of the genome and the opportunity to evaluate models based on previously made inferences from patterns of genetic variation in living populations. Analyses of the ancient Y chromosome sequences are challenging not only because of issues generally related to ancient DNA work, such as DNA damage-induced mutations and low content of endogenous DNA in most human remains, but also because of specific properties of the Y chromosome, such as its highly repetitive nature and high homology with the X chromosome. Shotgun sequencing of uniquely mapping regions of the Y chromosomes to sufficiently high coverage is still challenging and costly in poorly preserved samples. To increase the coverage of specific target SNPs capture-based methods have been developed and used in recent years to generate Y chromosome sequence data from hundreds of prehistoric skeletal remains. Besides the prospects of testing directly as how much genetic change in a given time period has accompanied changes in material culture the sequencing of ancient Y chromosomes allows us also to better understand the rate at which mutations accumulate and get fixed over time. This review considers genome-scale evidence on ancient Y chromosome diversity that has recently started to accumulate in geographic areas favourable to DNA preservation. More specifically the review focuses on examples of regional continuity and change of the Y chromosome haplogroups in North Eurasia and in the New World.

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

DOE PAGES

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; ...

2014-01-01

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

Improved imputation accuracy of rare and low-frequency variants using population-specific high-coverage WGS-based imputation reference panel.

PubMed

Mitt, Mario; Kals, Mart; Pärn, Kalle; Gabriel, Stacey B; Lander, Eric S; Palotie, Aarno; Ripatti, Samuli; Morris, Andrew P; Metspalu, Andres; Esko, Tõnu; Mägi, Reedik; Palta, Priit

2017-06-01

Genetic imputation is a cost-efficient way to improve the power and resolution of genome-wide association (GWA) studies. Current publicly accessible imputation reference panels accurately predict genotypes for common variants with minor allele frequency (MAF)≥5% and low-frequency variants (0.5≤MAF<5%) across diverse populations, but the imputation of rare variation (MAF<0.5%) is still rather limited. In the current study, we evaluate imputation accuracy achieved with reference panels from diverse populations with a population-specific high-coverage (30 ×) whole-genome sequencing (WGS) based reference panel, comprising of 2244 Estonian individuals (0.25% of adult Estonians). Although the Estonian-specific panel contains fewer haplotypes and variants, the imputation confidence and accuracy of imputed low-frequency and rare variants was significantly higher. The results indicate the utility of population-specific reference panels for human genetic studies.

Improved imputation accuracy of rare and low-frequency variants using population-specific high-coverage WGS-based imputation reference panel

PubMed Central

Mitt, Mario; Kals, Mart; Pärn, Kalle; Gabriel, Stacey B; Lander, Eric S; Palotie, Aarno; Ripatti, Samuli; Morris, Andrew P; Metspalu, Andres; Esko, Tõnu; Mägi, Reedik; Palta, Priit

2017-01-01

Genetic imputation is a cost-efficient way to improve the power and resolution of genome-wide association (GWA) studies. Current publicly accessible imputation reference panels accurately predict genotypes for common variants with minor allele frequency (MAF)≥5% and low-frequency variants (0.5≤MAF<5%) across diverse populations, but the imputation of rare variation (MAF<0.5%) is still rather limited. In the current study, we evaluate imputation accuracy achieved with reference panels from diverse populations with a population-specific high-coverage (30 ×) whole-genome sequencing (WGS) based reference panel, comprising of 2244 Estonian individuals (0.25% of adult Estonians). Although the Estonian-specific panel contains fewer haplotypes and variants, the imputation confidence and accuracy of imputed low-frequency and rare variants was significantly higher. The results indicate the utility of population-specific reference panels for human genetic studies. PMID:28401899

A-to-I RNA editing occurs at over a hundred million genomic sites, located in a majority of human genes.

PubMed

Bazak, Lily; Haviv, Ami; Barak, Michal; Jacob-Hirsch, Jasmine; Deng, Patricia; Zhang, Rui; Isaacs, Farren J; Rechavi, Gideon; Li, Jin Billy; Eisenberg, Eli; Levanon, Erez Y

2014-03-01

RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity are not yet clear. Here, we analyzed large-scale RNA-seq data and detected ∼1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultradeep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual antisense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.

Deep sampling of the Palomero maize transcriptome by a high throughput strategy of pyrosequencing.

PubMed

Vega-Arreguín, Julio C; Ibarra-Laclette, Enrique; Jiménez-Moraila, Beatriz; Martínez, Octavio; Vielle-Calzada, Jean Philippe; Herrera-Estrella, Luis; Herrera-Estrella, Alfredo

2009-07-06

In-depth sequencing analysis has not been able to determine the overall complexity of transcriptional activity of a plant organ or tissue sample. In some cases, deep parallel sequencing of Expressed Sequence Tags (ESTs), although not yet optimized for the sequencing of cDNAs, has represented an efficient procedure for validating gene prediction and estimating overall gene coverage. This approach could be very valuable for complex plant genomes. In addition, little emphasis has been given to efforts aiming at an estimation of the overall transcriptional universe found in a multicellular organism at a specific developmental stage. To explore, in depth, the transcriptional diversity in an ancient maize landrace, we developed a protocol to optimize the sequencing of cDNAs and performed 4 consecutive GS20-454 pyrosequencing runs of a cDNA library obtained from 2 week-old Palomero Toluqueño maize plants. The protocol reported here allowed obtaining over 90% of informative sequences. These GS20-454 runs generated over 1.5 Million reads, representing the largest amount of sequences reported from a single plant cDNA library. A collection of 367,391 quality-filtered reads (30.09 Mb) from a single run was sufficient to identify transcripts corresponding to 34% of public maize ESTs databases; total sequences generated after 4 filtered runs increased this coverage to 50%. Comparisons of all 1.5 Million reads to the Maize Assembled Genomic Islands (MAGIs) provided evidence for the transcriptional activity of 11% of MAGIs. We estimate that 5.67% (86,069 sequences) do not align with public ESTs or annotated genes, potentially representing new maize transcripts. Following the assembly of 74.4% of the reads in 65,493 contigs, real-time PCR of selected genes confirmed a predicted correlation between the abundance of GS20-454 sequences and corresponding levels of gene expression. A protocol was developed that significantly increases the number, length and quality of cDNA reads using massive 454 parallel sequencing. We show that recurrent 454 pyrosequencing of a single cDNA sample is necessary to attain a thorough representation of the transcriptional universe present in maize, that can also be used to estimate transcript abundance of specific genes. This data suggests that the molecular and functional diversity contained in the vast native landraces remains to be explored, and that large-scale transcriptional sequencing of a presumed ancestor of the modern maize varieties represents a valuable approach to characterize the functional diversity of maize for future agricultural and evolutionary studies.

[Diversity of beta-proteobacterial ammonia-oxidizing bacteria and ammonia-oxidizing archaea in shrimp farm sediment].

PubMed

Gao, Lihai; Lin, Weitie

2011-01-01

In order to study the diversity of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in shrimp farm sediment. Total microbial DNA was directly extracted from the shrimp farm sediment. The clone library of amoA genes were constructed with beta-Proteobacterial-AOB and AOA specific primers. The library was screened by PCR-restriction fragment length polymorphism (RFLP) analysis and clones with unique RFLP patterns were sequenced. Phylogenetic analyses of the amoA gene fragments showed that all AOB sequences from shrimp farm sediment were affiliated with Nitrosomonas (61.54%) or Nitrosomonas-like (38. 46%) species and grouped into Nitrosomonas communis cluster, Nitrosomonas sp. Nm148 cluster, Nitrosomonas oligotropha cluster. All AOA sequences belonged to the kingdom Crenarchaeote except that one Operational Taxa Unit (OTU) sequence was Unclassified-Archaea and fell within cluster S (soil origin). AOB and AOA species composition included 13 OTUs and 9 OTUs. The clone coverage of bacterial and archaeal amoA genes was 73.47% and 90.43%. The Shannon-Wiener index, Evenness index, Simpson index and Richness index of AOB were higher than those of AOA. These findings represent the first detailed examination of archaeal amoA diversity in shrimp farm sediment and demonstrate that diverse communities of Crenarchaeote capable of ammonia oxidation are present within shrimp farm sediment, where they may be actively involved in nitrification.

A quantitative and qualitative comparison of illumina MiSeq and 454 amplicon sequencing for genotyping the highly polymorphic major histocompatibility complex (MHC) in a non-model species.

PubMed

Razali, Haslina; O'Connor, Emily; Drews, Anna; Burke, Terry; Westerdahl, Helena

2017-07-28

High-throughput sequencing enables high-resolution genotyping of extremely duplicated genes. 454 amplicon sequencing (454) has become the standard technique for genotyping the major histocompatibility complex (MHC) genes in non-model organisms. However, illumina MiSeq amplicon sequencing (MiSeq), which offers a much higher read depth, is now superseding 454. The aim of this study was to quantitatively and qualitatively evaluate the performance of MiSeq in relation to 454 for genotyping MHC class I alleles using a house sparrow (Passer domesticus) dataset with pedigree information. House sparrows provide a good study system for this comparison as their MHC class I genes have been studied previously and, consequently, we had prior expectations concerning the number of alleles per individual. We found that 454 and MiSeq performed equally well in genotyping amplicons with low diversity, i.e. amplicons from individuals that had fewer than 6 alleles. Although there was a higher rate of failure in the 454 dataset in resolving amplicons with higher diversity (6-9 alleles), the same genotypes were identified by both 454 and MiSeq in 98% of cases. We conclude that low diversity amplicons are equally well genotyped using either 454 or MiSeq, but the higher coverage afforded by MiSeq can lead to this approach outperforming 454 in amplicons with higher diversity.

Conservation in gene encoding Mycobacterium tuberculosis antigen Rv2660 and a high predicted population coverage of H56 multistage vaccine in South Africa.

PubMed

Perez-Martinez, Angy P; Ong, Edison; Zhang, Lixin; Marrs, Carl F; He, Yongqun; Yang, Zhenhua

2017-11-01

H56/AERAS-456+IC31 (H56), composed of two early secretion proteins, Ag85B and ESAT-6, and a latency associated protein, Rv2660, and the IC31 Intercell adjuvant, is a new fusion subunit vaccine candidate designed to induce immunity against both new infection and reactivation of latent tuberculosis infection. Efficacy of subunit vaccines may be affected by the diversity of vaccine antigens among clinical strains and the extent of recognition by the diverse HLA molecules in the recipient population. Although a previous study showed the conservative nature of Ag85B- and ESAT-6-encoding genes, genetic diversity of Rv2660c that encodes RV2660 is largely unknown. The population coverage of H56 as a whole yet remains to be assessed. The present study was conducted to address these important knowledge gaps. DNA sequence analysis of Rv2660c found no variation among 83 of the 84 investigated clinical strains belonging to four genetic lineages. H56 was predicted to have as high as 99.6% population coverage in the South Africa population using the Immune Epitope Database (IEDB) Population Coverage Tool. Further comparison of H56 population coverage between South African Blacks and Caucasians based on the phenotypic frequencies of binding MHC Class I and Class II supertype alleles found that all of the nine MHC-I and six of eight MHC-II human leukocyte antigen (HLA) supertype alleles analyzed were significantly differentially expressed between the two subpopulations. This finding suggests the presence of race-specific functional binding motifs of MHC-I and MHC-II HLA alleles, which, in turn, highlights the importance of including diverse populations in vaccine clinical evaluation. In conclusion, H56 vaccine is predicted to have a promising population coverage in South Africa; this study demonstrates the utility of integrating comparative genomics and bioinformatics in bridging animal and clinical studies of novel TB vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.

Metatranscriptomics of N2-fixing cyanobacteria in the Amazon River plume

PubMed Central

Hilton, Jason A; Satinsky, Brandon M; Doherty, Mary; Zielinski, Brian; Zehr, Jonathan P

2015-01-01

Biological N2 fixation is an important nitrogen source for surface ocean microbial communities. However, nearly all information on the diversity and gene expression of organisms responsible for oceanic N2 fixation in the environment has come from targeted approaches that assay only a small number of genes and organisms. Using genomes of diazotrophic cyanobacteria to extract reads from extensive meta-genomic and -transcriptomic libraries, we examined diazotroph diversity and gene expression from the Amazon River plume, an area characterized by salinity and nutrient gradients. Diazotroph genome and transcript sequences were most abundant in the transitional waters compared with lower salinity or oceanic water masses. We were able to distinguish two genetically divergent phylotypes within the Hemiaulus-associated Richelia sequences, which were the most abundant diazotroph sequences in the data set. Photosystem (PS)-II transcripts in Richelia populations were much less abundant than those in Trichodesmium, and transcripts from several Richelia PS-II genes were absent, indicating a prominent role for cyclic electron transport in Richelia. In addition, there were several abundant regulatory transcripts, including one that targets a gene involved in PS-I cyclic electron transport in Richelia. High sequence coverage of the Richelia transcripts, as well as those from Trichodesmium populations, allowed us to identify expressed regions of the genomes that had been overlooked by genome annotations. High-coverage genomic and transcription analysis enabled the characterization of distinct phylotypes within diazotrophic populations, revealed a distinction in a core process between dominant populations and provided evidence for a prominent role for noncoding RNAs in microbial communities. PMID:25514535

Effect of condensed tannins on bovine rumen protist diversity based on 18S rRNA gene sequences.

PubMed

Tan, Hui Yin; Sieo, Chin Chin; Abdullah, Norhani; Liang, Juan Boo; Huang, Xiao Dan; Ho, Yin Wan

2013-01-01

Molecular diversity of protists from bovine rumen fluid incubated with condensed tannins of Leucaena leucocephala hybrid-Rendang at 20 mg/500 mg dry matter (treatment) or without condensed tannins (control) was investigated using 18S rRNA gene library. Clones from the control library were distributed within nine genera, but clones from the condensed tannin treatment clone library were related to only six genera. Diversity estimators such as abundance-based coverage estimation and Chao1 showed significant differences between the two libraries, although no differences were found based on Shannon-Weaver index and Libshuff. © 2012 The Author(s) Journal of Eukaryotic Microbiology © 2012 International Society of Protistologists.

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing

PubMed Central

2011-01-01

Background Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. Results A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. Conclusions The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models. PMID:21542930

Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing.

PubMed

Straub, Shannon C K; Fishbein, Mark; Livshultz, Tatyana; Foster, Zachary; Parks, Matthew; Weitemier, Kevin; Cronn, Richard C; Liston, Aaron

2011-05-04

Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.

Rapid isolation of microsatellite DNAs and identification of polymorphic mitochondrial DNA regions in the fish rotan (Perccottus glenii) invading European Russia

USGS Publications Warehouse

King, Timothy L.; Eackles, Michael S.; Reshetnikov, Andrey N.

2015-01-01

Human-mediated translocations and subsequent large-scale colonization by the invasive fish rotan (Perccottus glenii Dybowski, 1877; Perciformes, Odontobutidae), also known as Amur or Chinese sleeper, has resulted in dramatic transformations of small lentic ecosystems. However, no detailed genetic information exists on population structure, levels of effective movement, or relatedness among geographic populations of P. glenii within the European part of the range. We used massively parallel genomic DNA shotgun sequencing on the semiconductor-based Ion Torrent Personal Genome Machine (PGM) sequencing platform to identify nuclear microsatellite and mitochondrial DNA sequences in P. glenii from European Russia. Here we describe the characterization of nine nuclear microsatellite loci, ascertain levels of allelic diversity, heterozygosity, and demographic status of P. glenii collected from Ilev, Russia, one of several initial introduction points in European Russia. In addition, we mapped sequence reads to the complete P. glenii mitochondrial DNA sequence to identify polymorphic regions. Nuclear microsatellite markers developed for P. glenii yielded sufficient genetic diversity to: (1) produce unique multilocus genotypes; (2) elucidate structure among geographic populations; and (3) provide unique perspectives for analysis of population sizes and historical demographics. Among 4.9 million filtered P. glenii Ion Torrent PGM sequence reads, 11,304 mapped to the mitochondrial genome (NC_020350). This resulted in 100 % coverage of this genome to a mean coverage depth of 102X. A total of 130 variable sites were observed between the publicly available genome from China and the studied composite mitochondrial genome. Among these, 82 were diagnostic and monomorphic between the mitochondrial genomes and distributed among 15 genome regions. The polymorphic sites (N = 48) were distributed among 11 mitochondrial genome regions. Our results also indicate that sequence reads generated from two three-hour runs on the Ion Torrent PGM can generate a sufficient number of nuclear and mitochondrial markers to improve understanding of the evolutionary and ecological dynamics of non-model and in particular, invasive species.

Virome Assembly and Annotation: A Surprise in the Namib Desert

PubMed Central

Hesse, Uljana; van Heusden, Peter; Kirby, Bronwyn M.; Olonade, Israel; van Zyl, Leonardo J.; Trindade, Marla

2017-01-01

Sequencing, assembly, and annotation of environmental virome samples is challenging. Methodological biases and differences in species abundance result in fragmentary read coverage; sequence reconstruction is further complicated by the mosaic nature of viral genomes. In this paper, we focus on biocomputational aspects of virome analysis, emphasizing latent pitfalls in sequence annotation. Using simulated viromes that mimic environmental data challenges we assessed the performance of five assemblers (CLC-Workbench, IDBA-UD, SPAdes, RayMeta, ABySS). Individual analyses of relevant scaffold length fractions revealed shortcomings of some programs in reconstruction of viral genomes with excessive read coverage (IDBA-UD, RayMeta), and in accurate assembly of scaffolds ≥50 kb (SPAdes, RayMeta, ABySS). The CLC-Workbench assembler performed best in terms of genome recovery (including highly covered genomes) and correct reconstruction of large scaffolds; and was used to assemble a virome from a copper rich site in the Namib Desert. We found that scaffold network analysis and cluster-specific read reassembly improved reconstruction of sequences with excessive read coverage, and that strict data filtering for non-viral sequences prior to downstream analyses was essential. In this study we describe novel viral genomes identified in the Namib Desert copper site virome. Taxonomic affiliations of diverse proteins in the dataset and phylogenetic analyses of circovirus-like proteins indicated links to the marine habitat. Considering additional evidence from this dataset we hypothesize that viruses may have been carried from the Atlantic Ocean into the Namib Desert by fog and wind, highlighting the impact of the extended environment on an investigated niche in metagenome studies. PMID:28167933

Genomic encyclopedia of bacteria and archaea: sequencing a myriad of type strains.

PubMed

Kyrpides, Nikos C; Hugenholtz, Philip; Eisen, Jonathan A; Woyke, Tanja; Göker, Markus; Parker, Charles T; Amann, Rudolf; Beck, Brian J; Chain, Patrick S G; Chun, Jongsik; Colwell, Rita R; Danchin, Antoine; Dawyndt, Peter; Dedeurwaerdere, Tom; DeLong, Edward F; Detter, John C; De Vos, Paul; Donohue, Timothy J; Dong, Xiu-Zhu; Ehrlich, Dusko S; Fraser, Claire; Gibbs, Richard; Gilbert, Jack; Gilna, Paul; Glöckner, Frank Oliver; Jansson, Janet K; Keasling, Jay D; Knight, Rob; Labeda, David; Lapidus, Alla; Lee, Jung-Sook; Li, Wen-Jun; Ma, Juncai; Markowitz, Victor; Moore, Edward R B; Morrison, Mark; Meyer, Folker; Nelson, Karen E; Ohkuma, Moriya; Ouzounis, Christos A; Pace, Norman; Parkhill, Julian; Qin, Nan; Rossello-Mora, Ramon; Sikorski, Johannes; Smith, David; Sogin, Mitch; Stevens, Rick; Stingl, Uli; Suzuki, Ken-Ichiro; Taylor, Dorothea; Tiedje, Jim M; Tindall, Brian; Wagner, Michael; Weinstock, George; Weissenbach, Jean; White, Owen; Wang, Jun; Zhang, Lixin; Zhou, Yu-Guang; Field, Dawn; Whitman, William B; Garrity, George M; Klenk, Hans-Peter

2014-08-01

Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.

Genomic Encyclopedia of Bacteria and Archaea: Sequencing a Myriad of Type Strains

PubMed Central

Kyrpides, Nikos C.; Hugenholtz, Philip; Eisen, Jonathan A.; Woyke, Tanja; Göker, Markus; Parker, Charles T.; Amann, Rudolf; Beck, Brian J.; Chain, Patrick S. G.; Chun, Jongsik; Colwell, Rita R.; Danchin, Antoine; Dawyndt, Peter; Dedeurwaerdere, Tom; DeLong, Edward F.; Detter, John C.; De Vos, Paul; Donohue, Timothy J.; Dong, Xiu-Zhu; Ehrlich, Dusko S.; Fraser, Claire; Gibbs, Richard; Gilbert, Jack; Gilna, Paul; Glöckner, Frank Oliver; Jansson, Janet K.; Keasling, Jay D.; Knight, Rob; Labeda, David; Lapidus, Alla; Lee, Jung-Sook; Li, Wen-Jun; MA, Juncai; Markowitz, Victor; Moore, Edward R. B.; Morrison, Mark; Meyer, Folker; Nelson, Karen E.; Ohkuma, Moriya; Ouzounis, Christos A.; Pace, Norman; Parkhill, Julian; Qin, Nan; Rossello-Mora, Ramon; Sikorski, Johannes; Smith, David; Sogin, Mitch; Stevens, Rick; Stingl, Uli; Suzuki, Ken-ichiro; Taylor, Dorothea; Tiedje, Jim M.; Tindall, Brian; Wagner, Michael; Weinstock, George; Weissenbach, Jean; White, Owen; Wang, Jun; Zhang, Lixin; Zhou, Yu-Guang; Field, Dawn; Whitman, William B.; Garrity, George M.; Klenk, Hans-Peter

2014-01-01

Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment. PMID:25093819

Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat.

PubMed

Harris, J Kirk; Caporaso, J Gregory; Walker, Jeffrey J; Spear, John R; Gold, Nicholas J; Robertson, Charles E; Hugenholtz, Philip; Goodrich, Julia; McDonald, Daniel; Knights, Dan; Marshall, Paul; Tufo, Henry; Knight, Rob; Pace, Norman R

2013-01-01

The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ∼1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ∼119,000 nearly full-length sequences and 28,000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.

Whole genome sequencing data and de novo draft assemblies for 66 teleost species

PubMed Central

Malmstrøm, Martin; Matschiner, Michael; Tørresen, Ole K.; Jakobsen, Kjetill S.; Jentoft, Sissel

2017-01-01

Teleost fishes comprise more than half of all vertebrate species, yet genomic data are only available for 0.2% of their diversity. Here, we present whole genome sequencing data for 66 new species of teleosts, vastly expanding the availability of genomic data for this important vertebrate group. We report on de novo assemblies based on low-coverage (9–39×) sequencing and present detailed methodology for all analyses. To facilitate further utilization of this data set, we present statistical analyses of the gene space completeness and verify the expected phylogenetic position of the sequenced genomes in a large mitogenomic context. We further present a nuclear marker set used for phylogenetic inference and evaluate each gene tree in relation to the species tree to test for homogeneity in the phylogenetic signal. Collectively, these analyses illustrate the robustness of this highly diverse data set and enable extensive reuse of the selected phylogenetic markers and the genomic data in general. This data set covers all major teleost lineages and provides unprecedented opportunities for comparative studies of teleosts. PMID:28094797

Demographic history and rare allele sharing among human populations.

PubMed

Gravel, Simon; Henn, Brenna M; Gutenkunst, Ryan N; Indap, Amit R; Marth, Gabor T; Clark, Andrew G; Yu, Fuli; Gibbs, Richard A; Bustamante, Carlos D

2011-07-19

High-throughput sequencing technology enables population-level surveys of human genomic variation. Here, we examine the joint allele frequency distributions across continental human populations and present an approach for combining complementary aspects of whole-genome, low-coverage data and targeted high-coverage data. We apply this approach to data generated by the pilot phase of the Thousand Genomes Project, including whole-genome 2-4× coverage data for 179 samples from HapMap European, Asian, and African panels as well as high-coverage target sequencing of the exons of 800 genes from 697 individuals in seven populations. We use the site frequency spectra obtained from these data to infer demographic parameters for an Out-of-Africa model for populations of African, European, and Asian descent and to predict, by a jackknife-based approach, the amount of genetic diversity that will be discovered as sample sizes are increased. We predict that the number of discovered nonsynonymous coding variants will reach 100,000 in each population after ∼1,000 sequenced chromosomes per population, whereas ∼2,500 chromosomes will be needed for the same number of synonymous variants. Beyond this point, the number of segregating sites in the European and Asian panel populations is expected to overcome that of the African panel because of faster recent population growth. Overall, we find that the majority of human genomic variable sites are rare and exhibit little sharing among diverged populations. Our results emphasize that replication of disease association for specific rare genetic variants across diverged populations must overcome both reduced statistical power because of rarity and higher population divergence.

Demographic history and rare allele sharing among human populations

PubMed Central

Gravel, Simon; Henn, Brenna M.; Gutenkunst, Ryan N.; Indap, Amit R.; Marth, Gabor T.; Clark, Andrew G.; Yu, Fuli; Gibbs, Richard A.; Bustamante, Carlos D.; Altshuler, David L.; Durbin, Richard M.; Abecasis, Gonçalo R.; Bentley, David R.; Chakravarti, Aravinda; Clark, Andrew G.; Collins, Francis S.; De La Vega, Francisco M.; Donnelly, Peter; Egholm, Michael; Flicek, Paul; Gabriel, Stacey B.; Gibbs, Richard A.; Knoppers, Bartha M.; Lander, Eric S.; Lehrach, Hans; Mardis, Elaine R.; McVean, Gil A.; Nickerson, Debbie A.; Peltonen, Leena; Schafer, Alan J.; Sherry, Stephen T.; Wang, Jun; Wilson, Richard K.; Gibbs, Richard A.; Deiros, David; Metzker, Mike; Muzny, Donna; Reid, Jeff; Wheeler, David; Wang, Jun; Li, Jingxiang; Jian, Min; Li, Guoqing; Li, Ruiqiang; Liang, Huiqing; Tian, Geng; Wang, Bo; Wang, Jian; Wang, Wei; Yang, Huanming; Zhang, Xiuqing; Zheng, Huisong; Lander, Eric S.; Altshuler, David L.; Ambrogio, Lauren; Bloom, Toby; Cibulskis, Kristian; Fennell, Tim J.; Gabriel, Stacey B.; Jaffe, David B.; Shefler, Erica; Sougnez, Carrie L.; Bentley, David R.; Gormley, Niall; Humphray, Sean; Kingsbury, Zoya; Koko-Gonzales, Paula; Stone, Jennifer; McKernan, Kevin J.; Costa, Gina L.; Ichikawa, Jeffry K.; Lee, Clarence C.; Sudbrak, Ralf; Lehrach, Hans; Borodina, Tatiana A.; Dahl, Andreas; Davydov, Alexey N.; Marquardt, Peter; Mertes, Florian; Nietfeld, Wilfiried; Rosenstiel, Philip; Schreiber, Stefan; Soldatov, Aleksey V.; Timmermann, Bernd; Tolzmann, Marius; Egholm, Michael; Affourtit, Jason; Ashworth, Dana; Attiya, Said; Bachorski, Melissa; Buglione, Eli; Burke, Adam; Caprio, Amanda; Celone, Christopher; Clark, Shauna; Conners, David; Desany, Brian; Gu, Lisa; Guccione, Lorri; Kao, Kalvin; Kebbel, Andrew; Knowlton, Jennifer; Labrecque, Matthew; McDade, Louise; Mealmaker, Craig; Minderman, Melissa; Nawrocki, Anne; Niazi, Faheem; Pareja, Kristen; Ramenani, Ravi; Riches, David; Song, Wanmin; Turcotte, Cynthia; Wang, Shally; Mardis, Elaine R.; Wilson, Richard K.; Dooling, David; Fulton, Lucinda; Fulton, Robert; Weinstock, George; Durbin, Richard M.; Burton, John; Carter, David M.; Churcher, Carol; Coffey, Alison; Cox, Anthony; Palotie, Aarno; Quail, Michael; Skelly, Tom; Stalker, James; Swerdlow, Harold P.; Turner, Daniel; De Witte, Anniek; Giles, Shane; Gibbs, Richard A.; Wheeler, David; Bainbridge, Matthew; Challis, Danny; Sabo, Aniko; Yu, Fuli; Yu, Jin; Wang, Jun; Fang, Xiaodong; Guo, Xiaosen; Li, Ruiqiang; Li, Yingrui; Luo, Ruibang; Tai, Shuaishuai; Wu, Honglong; Zheng, Hancheng; Zheng, Xiaole; Zhou, Yan; Li, Guoqing; Wang, Jian; Yang, Huanming; Marth, Gabor T.; Garrison, Erik P.; Huang, Weichun; Indap, Amit; Kural, Deniz; Lee, Wan-Ping; Leong, Wen Fung; Quinlan, Aaron R.; Stewart, Chip; Stromberg, Michael P.; Ward, Alistair N.; Wu, Jiantao; Lee, Charles; Mills, Ryan E.; Shi, Xinghua; Daly, Mark J.; DePristo, Mark A.; Altshuler, David L.; Ball, Aaron D.; Banks, Eric; Bloom, Toby; Browning, Brian L.; Cibulskis, Kristian; Fennell, Tim J.; Garimella, Kiran V.; Grossman, Sharon R.; Handsaker, Robert E.; Hanna, Matt; Hartl, Chris; Jaffe, David B.; Kernytsky, Andrew M.; Korn, Joshua M.; Li, Heng; Maguire, Jared R.; McCarroll, Steven A.; McKenna, Aaron; Nemesh, James C.; Philippakis, Anthony A.; Poplin, Ryan E.; Price, Alkes; Rivas, Manuel A.; Sabeti, Pardis C.; Schaffner, Stephen F.; Shefler, Erica; Shlyakhter, Ilya A.; Cooper, David N.; Ball, Edward V.; Mort, Matthew; Phillips, Andrew D.; Stenson, Peter D.; Sebat, Jonathan; Makarov, Vladimir; Ye, Kenny; Yoon, Seungtai C.; Bustamante, Carlos D.; Clark, Andrew G.; Boyko, Adam; Degenhardt, Jeremiah; Gravel, Simon; Gutenkunst, Ryan N.; Kaganovich, Mark; Keinan, Alon; Lacroute, Phil; Ma, Xin; Reynolds, Andy; Clarke, Laura; Flicek, Paul; Cunningham, Fiona; Herrero, Javier; Keenen, Stephen; Kulesha, Eugene; Leinonen, Rasko; McLaren, William M.; Radhakrishnan, Rajesh; Smith, Richard E.; Zalunin, Vadim; Zheng-Bradley, Xiangqun; Korbel, Jan O.; Stütz, Adrian M.; Humphray, Sean; Bauer, Markus; Cheetham, R. Keira; Cox, Tony; Eberle, Michael; James, Terena; Kahn, Scott; Murray, Lisa; Chakravarti, Aravinda; Ye, Kai; De La Vega, Francisco M.; Fu, Yutao; Hyland, Fiona C. L.; Manning, Jonathan M.; McLaughlin, Stephen F.; Peckham, Heather E.; Sakarya, Onur; Sun, Yongming A.; Tsung, Eric F.; Batzer, Mark A.; Konkel, Miriam K.; Walker, Jerilyn A.; Sudbrak, Ralf; Albrecht, Marcus W.; Amstislavskiy, Vyacheslav S.; Herwig, Ralf; Parkhomchuk, Dimitri V.; Sherry, Stephen T.; Agarwala, Richa; Khouri, Hoda M.; Morgulis, Aleksandr O.; Paschall, Justin E.; Phan, Lon D.; Rotmistrovsky, Kirill E.; Sanders, Robert D.; Shumway, Martin F.; Xiao, Chunlin; McVean, Gil A.; Auton, Adam; Iqbal, Zamin; Lunter, Gerton; Marchini, Jonathan L.; Moutsianas, Loukas; Myers, Simon; Tumian, Afidalina; Desany, Brian; Knight, James; Winer, Roger; Craig, David W.; Beckstrom-Sternberg, Steve M.; Christoforides, Alexis; Kurdoglu, Ahmet A.; Pearson, John V.; Sinari, Shripad A.; Tembe, Waibhav D.; Haussler, David; Hinrichs, Angie S.; Katzman, Sol J.; Kern, Andrew; Kuhn, Robert M.; Przeworski, Molly; Hernandez, Ryan D.; Howie, Bryan; Kelley, Joanna L.; Melton, S. Cord; Abecasis, Gonçalo R.; Li, Yun; Anderson, Paul; Blackwell, Tom; Chen, Wei; Cookson, William O.; Ding, Jun; Kang, Hyun Min; Lathrop, Mark; Liang, Liming; Moffatt, Miriam F.; Scheet, Paul; Sidore, Carlo; Snyder, Matthew; Zhan, Xiaowei; Zöllner, Sebastian; Awadalla, Philip; Casals, Ferran; Idaghdour, Youssef; Keebler, John; Stone, Eric A.; Zilversmit, Martine; Jorde, Lynn; Xing, Jinchuan; Eichler, Evan E.; Aksay, Gozde; Alkan, Can; Hajirasouliha, Iman; Hormozdiari, Fereydoun; Kidd, Jeffrey M.; Sahinalp, S. Cenk; Sudmant, Peter H.; Mardis, Elaine R.; Chen, Ken; Chinwalla, Asif; Ding, Li; Koboldt, Daniel C.; McLellan, Mike D.; Dooling, David; Weinstock, George; Wallis, John W.; Wendl, Michael C.; Zhang, Qunyuan; Durbin, Richard M.; Albers, Cornelis A.; Ayub, Qasim; Balasubramaniam, Senduran; Barrett, Jeffrey C.; Carter, David M.; Chen, Yuan; Conrad, Donald F.; Danecek, Petr; Dermitzakis, Emmanouil T.; Hu, Min; Huang, Ni; Hurles, Matt E.; Jin, Hanjun; Jostins, Luke; Keane, Thomas M.; Le, Si Quang; Lindsay, Sarah; Long, Quan; MacArthur, Daniel G.; Montgomery, Stephen B.; Parts, Leopold; Stalker, James; Tyler-Smith, Chris; Walter, Klaudia; Zhang, Yujun; Gerstein, Mark B.; Snyder, Michael; Abyzov, Alexej; Balasubramanian, Suganthi; Bjornson, Robert; Du, Jiang; Grubert, Fabian; Habegger, Lukas; Haraksingh, Rajini; Jee, Justin; Khurana, Ekta; Lam, Hugo Y. K.; Leng, Jing; Mu, Xinmeng Jasmine; Urban, Alexander E.; Zhang, Zhengdong; Li, Yingrui; Luo, Ruibang; Marth, Gabor T.; Garrison, Erik P.; Kural, Deniz; Quinlan, Aaron R.; Stewart, Chip; Stromberg, Michael P.; Ward, Alistair N.; Wu, Jiantao; Lee, Charles; Mills, Ryan E.; Shi, Xinghua; McCarroll, Steven A.; Banks, Eric; DePristo, Mark A.; Handsaker, Robert E.; Hartl, Chris; Korn, Joshua M.; Li, Heng; Nemesh, James C.; Sebat, Jonathan; Makarov, Vladimir; Ye, Kenny; Yoon, Seungtai C.; Degenhardt, Jeremiah; Kaganovich, Mark; Clarke, Laura; Smith, Richard E.; Zheng-Bradley, Xiangqun; Korbel, Jan O.; Humphray, Sean; Cheetham, R. Keira; Eberle, Michael; Kahn, Scott; Murray, Lisa; Ye, Kai; De La Vega, Francisco M.; Fu, Yutao; Peckham, Heather E.; Sun, Yongming A.; Batzer, Mark A.; Konkel, Miriam K.; Walker, Jerilyn A.; Xiao, Chunlin; Iqbal, Zamin; Desany, Brian; Blackwell, Tom; Snyder, Matthew; Xing, Jinchuan; Eichler, Evan E.; Aksay, Gozde; Alkan, Can; Hajirasouliha, Iman; Hormozdiari, Fereydoun; Kidd, Jeffrey M.; Chen, Ken; Chinwalla, Asif; Ding, Li; McLellan, Mike D.; Wallis, John W.; Hurles, Matt E.; Conrad, Donald F.; Walter, Klaudia; Zhang, Yujun; Gerstein, Mark B.; Snyder, Michael; Abyzov, Alexej; Du, Jiang; Grubert, Fabian; Haraksingh, Rajini; Jee, Justin; Khurana, Ekta; Lam, Hugo Y. K.; Leng, Jing; Mu, Xinmeng Jasmine; Urban, Alexander E.; Zhang, Zhengdong; Gibbs, Richard A.; Bainbridge, Matthew; Challis, Danny; Coafra, Cristian; Dinh, Huyen; Kovar, Christie; Lee, Sandy; Muzny, Donna; Nazareth, Lynne; Reid, Jeff; Sabo, Aniko; Yu, Fuli; Yu, Jin; Marth, Gabor T.; Garrison, Erik P.; Indap, Amit; Leong, Wen Fung; Quinlan, Aaron R.; Stewart, Chip; Ward, Alistair N.; Wu, Jiantao; Cibulskis, Kristian; Fennell, Tim J.; Gabriel, Stacey B.; Garimella, Kiran V.; Hartl, Chris; Shefler, Erica; Sougnez, Carrie L.; Wilkinson, Jane; Clark, Andrew G.; Gravel, Simon; Grubert, Fabian; Clarke, Laura; Flicek, Paul; Smith, Richard E.; Zheng-Bradley, Xiangqun; Sherry, Stephen T.; Khouri, Hoda M.; Paschall, Justin E.; Shumway, Martin F.; Xiao, Chunlin; McVean, Gil A.; Katzman, Sol J.; Abecasis, Gonçalo R.; Blackwell, Tom; Mardis, Elaine R.; Dooling, David; Fulton, Lucinda; Fulton, Robert; Koboldt, Daniel C.; Durbin, Richard M.; Balasubramaniam, Senduran; Coffey, Allison; Keane, Thomas M.; MacArthur, Daniel G.; Palotie, Aarno; Scott, Carol; Stalker, James; Tyler-Smith, Chris; Gerstein, Mark B.; Balasubramanian, Suganthi; Chakravarti, Aravinda; Knoppers, Bartha M.; Abecasis, Gonçalo R.; Bustamante, Carlos D.; Gharani, Neda; Gibbs, Richard A.; Jorde, Lynn; Kaye, Jane S.; Kent, Alastair; Li, Taosha; McGuire, Amy L.; McVean, Gil A.; Ossorio, Pilar N.; Rotimi, Charles N.; Su, Yeyang; Toji, Lorraine H.; TylerSmith, Chris; Brooks, Lisa D.; Felsenfeld, Adam L.; McEwen, Jean E.; Abdallah, Assya; Juenger, Christopher R.; Clemm, Nicholas C.; Collins, Francis S.; Duncanson, Audrey; Green, Eric D.; Guyer, Mark S.; Peterson, Jane L.; Schafer, Alan J.; Abecasis, Gonçalo R.; Altshuler, David L.; Auton, Adam; Brooks, Lisa D.; Durbin, Richard M.; Gibbs, Richard A.; Hurles, Matt E.; McVean, Gil A.

2011-01-01

High-throughput sequencing technology enables population-level surveys of human genomic variation. Here, we examine the joint allele frequency distributions across continental human populations and present an approach for combining complementary aspects of whole-genome, low-coverage data and targeted high-coverage data. We apply this approach to data generated by the pilot phase of the Thousand Genomes Project, including whole-genome 2–4× coverage data for 179 samples from HapMap European, Asian, and African panels as well as high-coverage target sequencing of the exons of 800 genes from 697 individuals in seven populations. We use the site frequency spectra obtained from these data to infer demographic parameters for an Out-of-Africa model for populations of African, European, and Asian descent and to predict, by a jackknife-based approach, the amount of genetic diversity that will be discovered as sample sizes are increased. We predict that the number of discovered nonsynonymous coding variants will reach 100,000 in each population after ∼1,000 sequenced chromosomes per population, whereas ∼2,500 chromosomes will be needed for the same number of synonymous variants. Beyond this point, the number of segregating sites in the European and Asian panel populations is expected to overcome that of the African panel because of faster recent population growth. Overall, we find that the majority of human genomic variable sites are rare and exhibit little sharing among diverged populations. Our results emphasize that replication of disease association for specific rare genetic variants across diverged populations must overcome both reduced statistical power because of rarity and higher population divergence. PMID:21730125

Insights into the genetic structure and diversity of 38 South Asian Indians from deep whole-genome sequencing.

PubMed

Wong, Lai-Ping; Lai, Jason Kuan-Han; Saw, Woei-Yuh; Ong, Rick Twee-Hee; Cheng, Anthony Youzhi; Pillai, Nisha Esakimuthu; Liu, Xuanyao; Xu, Wenting; Chen, Peng; Foo, Jia-Nee; Tan, Linda Wei-Lin; Koo, Seok-Hwee; Soong, Richie; Wenk, Markus Rene; Lim, Wei-Yen; Khor, Chiea-Chuen; Little, Peter; Chia, Kee-Seng; Teo, Yik-Ying

2014-05-01

South Asia possesses a significant amount of genetic diversity due to considerable intergroup differences in culture and language. There have been numerous reports on the genetic structure of Asian Indians, although these have mostly relied on genotyping microarrays or targeted sequencing of the mitochondria and Y chromosomes. Asian Indians in Singapore are primarily descendants of immigrants from Dravidian-language-speaking states in south India, and 38 individuals from the general population underwent deep whole-genome sequencing with a target coverage of 30X as part of the Singapore Sequencing Indian Project (SSIP). The genetic structure and diversity of these samples were compared against samples from the Singapore Sequencing Malay Project and populations in Phase 1 of the 1,000 Genomes Project (1 KGP). SSIP samples exhibited greater intra-population genetic diversity and possessed higher heterozygous-to-homozygous genotype ratio than other Asian populations. When compared against a panel of well-defined Asian Indians, the genetic makeup of the SSIP samples was closely related to South Indians. However, even though the SSIP samples clustered distinctly from the Europeans in the global population structure analysis with autosomal SNPs, eight samples were assigned to mitochondrial haplogroups that were predominantly present in Europeans and possessed higher European admixture than the remaining samples. An analysis of the relative relatedness between SSIP with two archaic hominins (Denisovan, Neanderthal) identified higher ancient admixture in East Asian populations than in SSIP. The data resource for these samples is publicly available and is expected to serve as a valuable complement to the South Asian samples in Phase 3 of 1 KGP.

Comprehensive Survey of Genetic Diversity in Chloroplast Genomes and 45S nrDNAs within Panax ginseng Species

PubMed Central

Kim, Kyunghee; Lee, Sang-Choon; Lee, Junki; Lee, Hyun Oh; Joh, Ho Jun; Kim, Nam-Hoon; Park, Hyun-Seung; Yang, Tae-Jin

2015-01-01

We report complete sequences of chloroplast (cp) genome and 45S nuclear ribosomal DNA (45S nrDNA) for 11 Panax ginseng cultivars. We have obtained complete sequences of cp and 45S nrDNA, the representative barcoding target sequences for cytoplasm and nuclear genome, respectively, based on low coverage NGS sequence of each cultivar. The cp genomes sizes ranged from 156,241 to 156,425 bp and the major size variation was derived from differences in copy number of tandem repeats in the ycf1 gene and in the intergenic regions of rps16-trnUUG and rpl32-trnUAG. The complete 45S nrDNA unit sequences were 11,091 bp, representing a consensus single transcriptional unit with an intergenic spacer region. Comparative analysis of these sequences as well as those previously reported for three Chinese accessions identified very rare but unique polymorphism in the cp genome within P. ginseng cultivars. There were 12 intra-species polymorphisms (six SNPs and six InDels) among 14 cultivars. We also identified five SNPs from 45S nrDNA of 11 Korean ginseng cultivars. From the 17 unique informative polymorphic sites, we developed six reliable markers for analysis of ginseng diversity and cultivar authentication. PMID:26061692

Microbial Community and Biochemical Dynamics of Biological Soil Crusts across a Gradient of Surface Coverage in the Central Mojave Desert.

PubMed

Mogul, Rakesh; Vaishampayan, Parag; Bashir, Mina; McKay, Chris P; Schubert, Keith; Bornaccorsi, Rosalba; Gomez, Ernesto; Tharayil, Sneha; Payton, Geoffrey; Capra, Juliana; Andaya, Jessica; Bacon, Leonard; Bargoma, Emily; Black, David; Boos, Katie; Brant, Michaela; Chabot, Michael; Chau, Danny; Cisneros, Jessica; Chu, Geoff; Curnutt, Jane; DiMizio, Jessica; Engelbrecht, Christian; Gott, Caroline; Harnoto, Raechel; Hovanesian, Ruben; Johnson, Shane; Lavergne, Britne; Martinez, Gabriel; Mans, Paul; Morales, Ernesto; Oei, Alex; Peplow, Gary; Piaget, Ryan; Ponce, Nicole; Renteria, Eduardo; Rodriguez, Veronica; Rodriguez, Joseph; Santander, Monica; Sarmiento, Khamille; Scheppelmann, Allison; Schroter, Gavin; Sexton, Devan; Stephenson, Jenin; Symer, Kristin; Russo-Tait, Tatiane; Weigel, Bill; Wilhelm, Mary B

2017-01-01

In this study, we expand upon the biogeography of biological soil crusts (BSCs) and provide molecular insights into the microbial community and biochemical dynamics along the vertical BSC column structure, and across a transect of increasing BSC surface coverage in the central Mojave Desert, CA, United States. Next generation sequencing reveals a bacterial community profile that is distinct among BSCs in the southwestern United States. Distribution of major phyla in the BSC topsoils included Cyanobacteria (33 ± 8%), Proteobacteria (26 ± 6%), and Chloroflexi (12 ± 4%), with Phormidium being the numerically dominant genus. Furthermore, BSC subsurfaces contained Proteobacteria (23 ± 5%), Actinobacteria (20 ± 5%), and Chloroflexi (18 ± 3%), with an unidentified genus from Chloroflexi (AKIW781, order) being numerically dominant. Across the transect, changes in distribution at the phylum ( p < 0.0439) and genus ( p < 0.006) levels, including multiple biochemical and geochemical trends ( p < 0.05), positively correlated with increasing BSC surface coverage. This included increases in (a) Chloroflexi abundance, (b) abundance and diversity of Cyanobacteria, (b) OTU-level diversity in the topsoil, (c) OTU-level differentiation between the topsoil and subsurface, (d) intracellular ATP abundances and catalase activities, and (e) enrichments in clay, silt, and varying elements, including S, Mn, Co, As, and Pb, in the BSC topsoils. In sum, these studies suggest that BSCs from regions of differing surface coverage represent early successional stages, which exhibit increasing bacterial diversity, metabolic activities, and capacity to restructure the soil. Further, these trends suggest that BSC successional maturation and colonization across the transect are inhibited by metals/metalloids such as B, Ca, Ti, Mn, Co, Ni, Mo, and Pb.

Microbial Community and Biochemical Dynamics of Biological Soil Crusts across a Gradient of Surface Coverage in the Central Mojave Desert

PubMed Central

Mogul, Rakesh; Vaishampayan, Parag; Bashir, Mina; McKay, Chris P.; Schubert, Keith; Bornaccorsi, Rosalba; Gomez, Ernesto; Tharayil, Sneha; Payton, Geoffrey; Capra, Juliana; Andaya, Jessica; Bacon, Leonard; Bargoma, Emily; Black, David; Boos, Katie; Brant, Michaela; Chabot, Michael; Chau, Danny; Cisneros, Jessica; Chu, Geoff; Curnutt, Jane; DiMizio, Jessica; Engelbrecht, Christian; Gott, Caroline; Harnoto, Raechel; Hovanesian, Ruben; Johnson, Shane; Lavergne, Britne; Martinez, Gabriel; Mans, Paul; Morales, Ernesto; Oei, Alex; Peplow, Gary; Piaget, Ryan; Ponce, Nicole; Renteria, Eduardo; Rodriguez, Veronica; Rodriguez, Joseph; Santander, Monica; Sarmiento, Khamille; Scheppelmann, Allison; Schroter, Gavin; Sexton, Devan; Stephenson, Jenin; Symer, Kristin; Russo-Tait, Tatiane; Weigel, Bill; Wilhelm, Mary B.

2017-01-01

In this study, we expand upon the biogeography of biological soil crusts (BSCs) and provide molecular insights into the microbial community and biochemical dynamics along the vertical BSC column structure, and across a transect of increasing BSC surface coverage in the central Mojave Desert, CA, United States. Next generation sequencing reveals a bacterial community profile that is distinct among BSCs in the southwestern United States. Distribution of major phyla in the BSC topsoils included Cyanobacteria (33 ± 8%), Proteobacteria (26 ± 6%), and Chloroflexi (12 ± 4%), with Phormidium being the numerically dominant genus. Furthermore, BSC subsurfaces contained Proteobacteria (23 ± 5%), Actinobacteria (20 ± 5%), and Chloroflexi (18 ± 3%), with an unidentified genus from Chloroflexi (AKIW781, order) being numerically dominant. Across the transect, changes in distribution at the phylum (p < 0.0439) and genus (p < 0.006) levels, including multiple biochemical and geochemical trends (p < 0.05), positively correlated with increasing BSC surface coverage. This included increases in (a) Chloroflexi abundance, (b) abundance and diversity of Cyanobacteria, (b) OTU-level diversity in the topsoil, (c) OTU-level differentiation between the topsoil and subsurface, (d) intracellular ATP abundances and catalase activities, and (e) enrichments in clay, silt, and varying elements, including S, Mn, Co, As, and Pb, in the BSC topsoils. In sum, these studies suggest that BSCs from regions of differing surface coverage represent early successional stages, which exhibit increasing bacterial diversity, metabolic activities, and capacity to restructure the soil. Further, these trends suggest that BSC successional maturation and colonization across the transect are inhibited by metals/metalloids such as B, Ca, Ti, Mn, Co, Ni, Mo, and Pb. PMID:29109701

Urbanization Reduces Transfer of Diverse Environmental Microbiota Indoors

PubMed Central

Parajuli, Anirudra; Grönroos, Mira; Siter, Nathan; Puhakka, Riikka; Vari, Heli K.; Roslund, Marja I.; Jumpponen, Ari; Nurminen, Noora; Laitinen, Olli H.; Hyöty, Heikki; Rajaniemi, Juho; Sinkkonen, Aki

2018-01-01

Expanding urbanization is a major factor behind rapidly declining biodiversity. It has been proposed that in urbanized societies, the rarity of contact with diverse environmental microbiota negatively impacts immune function and ultimately increases the risk for allergies and other immune-mediated disorders. Surprisingly, the basic assumption that urbanization reduces exposure to environmental microbiota and its transfer indoors has rarely been examined. We investigated if the land use type around Finnish homes affects the diversity, richness, and abundance of bacterial communities indoors. Debris deposited on standardized doormats was collected in 30 rural and 26 urban households in and near the city of Lahti, Finland, in August 2015. Debris was weighed, bacterial community composition determined by high throughput sequencing of bacterial 16S ribosomal RNA (rRNA) gene on the Illumina MiSeq platform, and the percentage of four different land use types (i.e., built area, forest, transitional, and open area) within 200 m and 2000 m radiuses from each household was characterized. The quantity of doormat debris was inversely correlated with coverage of built area. The diversity of total bacterial, Proteobacterial, Actinobacterial, Bacteroidetes, and Firmicutes communities decreased as the percentage of built area increased. Their richness followed the same pattern except for Firmicutes for which no association was observed. The relative abundance of Proteobacteria and particularly Gammaproteobacteria increased, whereas that of Actinobacteria decreased with increasing built area. Neither Phylum Firmicutes nor Bacteroidetes varied with coverage of built area. Additionally, the relative abundance of potentially pathogenic bacterial families and genera increased as the percentage of built area increased. Interestingly, having domestic animals (including pets) only altered the association between the richness of Gammaproteobacteria and diversity of Firmicutes with the built area coverage suggesting that animal ownership minimally affects transfer of environmental microbiota indoors from the living environment. These results support the hypothesis that people living in densely built areas are less exposed to diverse environmental microbiota than people living in more sparsely built areas. PMID:29467728

Microbial diversity of an anoxic zone of a hydroelectric power station reservoir in Brazilian Amazonia.

PubMed

Graças, Diego A; Miranda, Paulo R; Baraúna, Rafael A; McCulloch, John A; Ghilardi, Rubens; Schneider, Maria Paula C; Silva, Artur

2011-11-01

Microbial diversity was evaluated in an anoxic zone of Tucuruí Hydroelectric Power Station reservoir in Brazilian Amazonia using a culture-independent approach by amplifying and sequencing fragments of the 16S rRNA gene using metagenomic DNA as a template. Samples obtained from the photic, aphotic (40 m) and sediment (60 m) layers were used to construct six 16S rDNA libraries containing a total of 1,152 clones. The sediment, aphotic and photic layers presented 64, 33 and 35 unique archaeal operational taxonomic units (OTUs). The estimated richness of these layers was evaluated to be 153, 106 and 79 archaeal OTUs, respectively, using the abundance-based coverage estimator (ACE) and 114, 83 and 77 OTUs using the Chao1 estimator. For bacterial sequences, 114, 69 and 57 OTUs were found in the sediment, aphotic and photic layers, which presented estimated richnesses of 1,414, 522 and 197 OTUs (ACE) and 1,059, 1,014 and 148 OTUs (Chao1), respectively. Phylogenetic analyses of the sequences obtained revealed a high richness of microorganisms which participate in the carbon cycle, namely, methanogenic archaea and methanotrophic proteobacteria. Most sequences obtained belong to non-culturable prokaryotes. The present study offers the first glimpse of the huge microbial diversity of an anoxic area of a man-made lacustrine environment in the tropics.

Genetic and antigenic diversity of Theileria parva in cattle in Eastern and Southern zones of Tanzania. A study to support control of East Coast fever.

PubMed

Elisa, Mwega; Hasan, Salih Dia; Moses, Njahira; Elpidius, Rukambile; Skilton, Robert; Gwakisa, Paul

2015-04-01

This study investigated the genetic and antigenic diversity of Theileria parva in cattle from the Eastern and Southern zones of Tanzania. Thirty-nine (62%) positive samples were genotyped using 14 mini- and microsatellite markers with coverage of all four T. parva chromosomes. Wright's F index (F(ST) = 0 × 094) indicated a high level of panmixis. Linkage equilibrium was observed in the two zones studied, suggesting existence of a panmyctic population. In addition, sequence analysis of CD8+ T-cell target antigen genes Tp1 revealed a single protein sequence in all samples analysed, which is also present in the T. parva Muguga strain, which is a component of the FAO1 vaccine. All Tp2 epitope sequences were identical to those in the T. parva Muguga strain, except for one variant of a Tp2 epitope, which is found in T. parva Kiambu 5 strain, also a component the FAO1 vaccine. Neighbour joining tree of the nucleotide sequences of Tp2 showed clustering according to geographical origin. Our results show low genetic and antigenic diversity of T. parva within the populations analysed. This has very important implications for the development of sustainable control measures for T. parva in Eastern and Southern zones of Tanzania, where East Coast fever is endemic.

Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform

PubMed Central

Van Nostrand, Joy D.; Ning, Daliang; Sun, Bo; Xue, Kai; Liu, Feifei; Deng, Ye; Liang, Yuting; Zhou, Jizhong

2017-01-01

Illumina’s MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered, the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1–3%, p<0.001). Increasing the sequencing depth to 160,000 reads by deep sequencing increased OTU overlap both when sequence abundance was considered (95%) and when not (44%). However, if singletons were not removed the overlap between two technical replicates (not considering sequence abundance) plateaus at 39% with 30,000 sequences. Diversity measures were not affected by the low overlap as α-diversities were similar among technical replicates while β-diversities (Bray-Curtis) were much smaller among technical replicates than among treatment replicates (e.g., 0.269 vs. 0.374). Higher diversity coverage, but lower OTU overlap, was observed when replicates were sequenced in separate runs. Detrended correspondence analysis indicated that while there was considerable variation among technical replicates, the reproducibility was sufficient for detecting treatment effects for the samples examined. These results suggest that although there is variation among technical replicates, amplicon sequencing on MiSeq is useful for analyzing microbial community structure if used appropriately and with caution. For example, including technical replicates, removing spurious sequences and unrepresentative OTUs, using a clustering method with a high stringency for OTU generation, estimating treatment effects at higher taxonomic levels, and adapting the unique molecular identifier (UMI) and other newly developed methods to lower PCR and sequencing error and to identify true low abundance rare species all can increase reproducibility. PMID:28453559

Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wen, Chongqing; Wu, Liyou; Qin, Yujia

Illumina's MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered,more » the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1-3%, p<0.001). Increasing the sequencing depth to 160,000 reads by deep sequencing increased OTU overlap both when sequence abundance was considered (95%) and when not (44%). However, if singletons were not removed the overlap between two technical replicates (not considering sequence abundance) plateaus at 39% with 30,000 sequences. Diversity measures were not affected by the low overlap as α-diversities were similar among technical replicates while β-diversities (Bray-Curtis) were much smaller among technical replicates than among treatment replicates (e.g., 0.269 vs. 0.374). Higher diversity coverage, but lower OTU overlap, was observed when replicates were sequenced in separate runs. Detrended correspondence analysis indicated that while there was considerable variation among technical replicates, the reproducibility was sufficient for detecting treatment effects for the samples examined. These results suggest that although there is variation among technical replicates, amplicon sequencing on MiSeq is useful for analyzing microbial community structure if used appropriately and with caution. For example, including technical replicates, removing spurious sequences and unrepresentative OTUs, using a clustering method with a high stringency for OTU generation, estimating treatment effects at higher taxonomic levels, and adapting the unique molecular identifier (UMI) and other newly developed methods to lower PCR and sequencing error and to identify true low abundance rare species all can increase reproducibility.« less

Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform

DOE PAGES

Wen, Chongqing; Wu, Liyou; Qin, Yujia; ...

2017-04-28

Illumina's MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered,more » the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1-3%, p<0.001). Increasing the sequencing depth to 160,000 reads by deep sequencing increased OTU overlap both when sequence abundance was considered (95%) and when not (44%). However, if singletons were not removed the overlap between two technical replicates (not considering sequence abundance) plateaus at 39% with 30,000 sequences. Diversity measures were not affected by the low overlap as α-diversities were similar among technical replicates while β-diversities (Bray-Curtis) were much smaller among technical replicates than among treatment replicates (e.g., 0.269 vs. 0.374). Higher diversity coverage, but lower OTU overlap, was observed when replicates were sequenced in separate runs. Detrended correspondence analysis indicated that while there was considerable variation among technical replicates, the reproducibility was sufficient for detecting treatment effects for the samples examined. These results suggest that although there is variation among technical replicates, amplicon sequencing on MiSeq is useful for analyzing microbial community structure if used appropriately and with caution. For example, including technical replicates, removing spurious sequences and unrepresentative OTUs, using a clustering method with a high stringency for OTU generation, estimating treatment effects at higher taxonomic levels, and adapting the unique molecular identifier (UMI) and other newly developed methods to lower PCR and sequencing error and to identify true low abundance rare species all can increase reproducibility.« less

Evaluation of the reproducibility of amplicon sequencing with Illumina MiSeq platform.

PubMed

Wen, Chongqing; Wu, Liyou; Qin, Yujia; Van Nostrand, Joy D; Ning, Daliang; Sun, Bo; Xue, Kai; Liu, Feifei; Deng, Ye; Liang, Yuting; Zhou, Jizhong

2017-01-01

Illumina's MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered, the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1-3%, p<0.001). Increasing the sequencing depth to 160,000 reads by deep sequencing increased OTU overlap both when sequence abundance was considered (95%) and when not (44%). However, if singletons were not removed the overlap between two technical replicates (not considering sequence abundance) plateaus at 39% with 30,000 sequences. Diversity measures were not affected by the low overlap as α-diversities were similar among technical replicates while β-diversities (Bray-Curtis) were much smaller among technical replicates than among treatment replicates (e.g., 0.269 vs. 0.374). Higher diversity coverage, but lower OTU overlap, was observed when replicates were sequenced in separate runs. Detrended correspondence analysis indicated that while there was considerable variation among technical replicates, the reproducibility was sufficient for detecting treatment effects for the samples examined. These results suggest that although there is variation among technical replicates, amplicon sequencing on MiSeq is useful for analyzing microbial community structure if used appropriately and with caution. For example, including technical replicates, removing spurious sequences and unrepresentative OTUs, using a clustering method with a high stringency for OTU generation, estimating treatment effects at higher taxonomic levels, and adapting the unique molecular identifier (UMI) and other newly developed methods to lower PCR and sequencing error and to identify true low abundance rare species all can increase reproducibility.

Genomic Encyclopedia of Bacteria and Archaea: Sequencing a Myriad of Type Strains

DOE PAGES

Kyrpides, Nikos C.; Hugenholtz, Philip; Eisen, Jonathan A.; ...

2014-08-05

Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overallmore » known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently~11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.« less

In-solution hybridization for mammalian mitogenome enrichment: pros, cons and challenges associated with multiplexing degraded DNA.

PubMed

Hawkins, Melissa T R; Hofman, Courtney A; Callicrate, Taylor; McDonough, Molly M; Tsuchiya, Mirian T N; Gutiérrez, Eliécer E; Helgen, Kristofer M; Maldonado, Jesus E

2016-09-01

Here, we present a set of RNA-based probes for whole mitochondrial genome in-solution enrichment, targeting a diversity of mammalian mitogenomes. This probes set was designed from seven mammalian orders and tested to determine the utility for enriching degraded DNA. We generated 63 mitogenomes representing five orders and 22 genera of mammals that yielded varying coverage ranging from 0 to >5400X. Based on a threshold of 70% mitogenome recovery and at least 10× average coverage, 32 individuals or 51% of samples were considered successful. The estimated sequence divergence of samples from the probe sequences used to construct the array ranged up to nearly 20%. Sample type was more predictive of mitogenome recovery than sample age. The proportion of reads from each individual in multiplexed enrichments was highly skewed, with each pool having one sample that yielded a majority of the reads. Recovery across each mitochondrial gene varied with most samples exhibiting regions with gaps or ambiguous sites. We estimated the ability of the probes to capture mitogenomes from a diversity of mammalian taxa not included here by performing a clustering analysis of published sequences for 100 taxa representing most mammalian orders. Our study demonstrates that a general array can be cost and time effective when there is a need to screen a modest number of individuals from a variety of taxa. We also address the practical concerns for using such a tool, with regard to pooling samples, generating high quality mitogenomes and detail a pipeline to remove chimeric molecules. © 2015 John Wiley & Sons Ltd.

Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample.

PubMed

Luo, Chengwei; Tsementzi, Despina; Kyrpides, Nikos; Read, Timothy; Konstantinidis, Konstantinos T

2012-01-01

Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R(2)>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.

Evaluation of six primer pairs targeting the nuclear rRNA operon for characterization of arbuscular mycorrhizal fungal (AMF) communities using 454 pyrosequencing.

PubMed

Van Geel, Maarten; Busschaert, Pieter; Honnay, Olivier; Lievens, Bart

2014-11-01

In the last few years, 454 pyrosequencing-based analysis of arbuscular mycorrhizal fungal (AMF; Glomeromycota) communities has tremendously increased our knowledge of the distribution and diversity of AMF. Nonetheless, comparing results between different studies is difficult, as different target genes (or regions thereof) and primer combinations, with potentially dissimilar specificities and efficacies, are being utilized. In this study we evaluated six primer pairs that have previously been used in AMF studies (NS31-AM1, AMV4.5NF-AMDGR, AML1-AML2, NS31-AML2, FLR3-LSUmBr and Glo454-NDL22) for their use in 454 pyrosequencing based on both an in silico approach and 454 pyrosequencing of AMF communities from apple tree roots. Primers were evaluated in terms of (i) in silico coverage of Glomeromycota fungi, (ii) the number of high-quality sequences obtained, (iii) selectivity for AMF species, (iv) reproducibility and (v) ability to accurately describe AMF communities. We show that primer pairs AMV4.5NF-AMDGR, AML1-AML2 and NS31-AML2 outperformed the other tested primer pairs in terms of number of Glomeromycota reads (AMF specificity and coverage). Additionally, these primer pairs were found to have no or only few mismatches to AMF sequences and were able to consistently describe AMF communities from apple roots. However, whereas most high-quality AMF sequences were obtained for AMV4.5NF-AMDGR, our results also suggest that this primer pair favored amplification of Glomeraceae sequences at the expense of Ambisporaceae, Claroideoglomeraceae and Paraglomeraceae sequences. Furthermore, we demonstrate the complementary specificity of AMV4.5NF-AMDGR with AML1-AML2, and of AMV4.5NF-AMDGR with NS31-AML2, making these primer combinations highly suitable for tandem use in covering the diversity of AMF communities. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of malaria transmission reduction by insecticide-treated bed nets (ITNs) on the genetic diversity of Plasmodium falciparum merozoite surface protein (MSP-1) and circumsporozoite (CSP) in western Kenya.

PubMed

Kariuki, Simon K; Njunge, James; Muia, Ann; Muluvi, Geofrey; Gatei, Wangeci; Ter Kuile, Feiko; Terlouw, Dianne J; Hawley, William A; Phillips-Howard, Penelope A; Nahlen, Bernard L; Lindblade, Kim A; Hamel, Mary J; Slutsker, Laurence; Shi, Ya Ping

2013-08-27

Although several studies have investigated the impact of reduced malaria transmission due to insecticide-treated bed nets (ITNs) on the patterns of morbidity and mortality, there is limited information on their effect on parasite diversity. Sequencing was used to investigate the effect of ITNs on polymorphisms in two genes encoding leading Plasmodium falciparum vaccine candidate antigens, the 19 kilodalton blood stage merozoite surface protein-1 (MSP-1(19kDa)) and the Th2R and Th3R T-cell epitopes of the pre-erythrocytic stage circumsporozoite protein (CSP) in a large community-based ITN trial site in western Kenya. The number and frequency of haplotypes as well as nucleotide and haplotype diversity were compared among parasites obtained from children <5 years old prior to the introduction of ITNs (1996) and after 5 years of high coverage ITN use (2001). A total of 12 MSP-1(19kDa) haplotypes were detected in 1996 and 2001. The Q-KSNG-L and E-KSNG-L haplotypes corresponding to the FVO and FUP strains of P. falciparum were the most prevalent (range 32-37%), with an overall haplotype diversity of > 0.7. No MSP-1(19kDa) 3D7 sequence-types were detected in 1996 and the frequency was less than 4% in 2001. The CSP Th2R and Th3R domains were highly polymorphic with a total of 26 and 14 haplotypes, respectively detected in 1996 and 34 and 13 haplotypes in 2001, with an overall haplotype diversity of > 0.9 and 0.75 respectively. The frequency of the most predominant Th2R and Th3R haplotypes was 14 and 36%, respectively. The frequency of Th2R and Th3R haplotypes corresponding to the 3D7 parasite strain was less than 4% at both time points. There was no significant difference in nucleotide and haplotype diversity in parasite isolates collected at both time points. High diversity in these two genes has been maintained overtime despite marked reductions in malaria transmission due to ITNs use. The frequency of 3D7 sequence-types was very low in this area. These findings provide information that could be useful in the design of future malaria vaccines for deployment in endemic areas with high ITN coverage and in interpretation of efficacy data for malaria vaccines based on 3D7 parasite strains.

Termite hindguts and the ecology of microbial communities in the sequencing age.

PubMed

Tai, Vera; Keeling, Patrick J

2013-01-01

Advances in high-throughput nucleic acid sequencing have improved our understanding of microbial communities in a number of ways. Deeper sequence coverage provides the means to assess diversity at the resolution necessary to recover ecological and biogeographic patterns, and at the same time single-cell genomics provides detailed information about the interactions between members of a microbial community. Given the vastness and complexity of microbial ecosystems, such analyses remain challenging for most environments, so greater insight can also be drawn from analysing less dynamic ecosystems. Here, we outline the advantages of one such environment, the wood-digesting hindgut communities of termites and cockroaches, and how it is a model to examine and compare both protist and bacterial communities. Beyond the analysis of diversity, our understanding of protist community ecology will depend on using statistically sound sampling regimes at biologically relevant scales, transitioning from discovery-based to experimental ecology, incorporating single-cell microbiology and other data sources, and continued development of analytical tools. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

The History of Bordetella pertussis Genome Evolution Includes Structural Rearrangement

PubMed Central

Peng, Yanhui; Loparev, Vladimir; Batra, Dhwani; Bowden, Katherine E.; Burroughs, Mark; Cassiday, Pamela K.; Davis, Jamie K.; Johnson, Taccara; Juieng, Phalasy; Knipe, Kristen; Mathis, Marsenia H.; Pruitt, Andrea M.; Rowe, Lori; Sheth, Mili; Tondella, M. Lucia; Williams, Margaret M.

2017-01-01

ABSTRACT Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genomic structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine the potential evolution of the chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. The observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigating disease resurgence and molecular epidemiology. IMPORTANCE Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though the coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen's evolution. The chromosome of B. pertussis includes a large number of repetitive mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-encoding genes, which otherwise exhibit little nucleotide sequence diversity. By comparing the complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology. PMID:28167525

Modified RNA-seq method for microbial community and diversity analysis using rRNA in different types of environmental samples

PubMed Central

Yan, Yong-Wei; Zou, Bin; Zhu, Ting; Hozzein, Wael N.

2017-01-01

RNA-seq-based SSU (small subunit) rRNA (ribosomal RNA) analysis has provided a better understanding of potentially active microbial community within environments. However, for RNA-seq library construction, high quantities of purified RNA are typically required. We propose a modified RNA-seq method for SSU rRNA-based microbial community analysis that depends on the direct ligation of a 5’ adaptor to RNA before reverse-transcription. The method requires only a low-input quantity of RNA (10–100 ng) and does not require a DNA removal step. The method was initially tested on three mock communities synthesized with enriched SSU rRNA of archaeal, bacterial and fungal isolates at different ratios, and was subsequently used for environmental samples of high or low biomass. For high-biomass salt-marsh sediments, enriched SSU rRNA and total nucleic acid-derived RNA-seq datasets revealed highly consistent community compositions for all of the SSU rRNA sequences, and as much as 46.4%-59.5% of 16S rRNA sequences were suitable for OTU (operational taxonomic unit)-based community and diversity analyses with complete coverage of V1-V2 regions. OTU-based community structures for the two datasets were also highly consistent with those determined by all of the 16S rRNA reads. For low-biomass samples, total nucleic acid-derived RNA-seq datasets were analyzed, and highly active bacterial taxa were also identified by the OTU-based method, notably including members of the previously underestimated genus Nitrospira and phylum Acidobacteria in tap water, members of the phylum Actinobacteria on a shower curtain, and members of the phylum Cyanobacteria on leaf surfaces. More than half of the bacterial 16S rRNA sequences covered the complete region of primer 8F, and non-coverage rates as high as 38.7% were obtained for phylum-unclassified sequences, providing many opportunities to identify novel bacterial taxa. This modified RNA-seq method will provide a better snapshot of diverse microbial communities, most notably by OTU-based analysis, even communities with low-biomass samples. PMID:29016661

Blood from a turnip: tissue origin of low-coverage shotgun sequencing libraries affects recovery of mitogenome sequences

USGS Publications Warehouse

Barker, F. Keith; Oyler-McCance, Sara; Tomback, Diana F.

2015-01-01

Next generation sequencing methods allow rapid, economical accumulation of data that have many applications, even at relatively low levels of genome coverage. However, the utility of shotgun sequencing data sets for specific goals may vary depending on the biological nature of the samples sequenced. We show that the ability to assemble mitogenomes from three avian samples of two different tissue types varies widely. In particular, data with coverage typical of microsatellite development efforts (∼1×) from DNA extracted from avian blood failed to cover even 50% of the mitogenome, relative to at least 500-fold coverage from muscle-derived data. Researchers should consider possible applications of their data and select the tissue source for their work accordingly. Practitioners analyzing low-coverage shotgun sequencing data (including for microsatellite locus development) should consider the potential benefits of mitogenome assembly, including internal barcode verification of species identity, mitochondrial primer development, and phylogenetics.

Using high throughput sequencing to explore the biodiversity in oral bacterial communities.

PubMed

Diaz, P I; Dupuy, A K; Abusleme, L; Reese, B; Obergfell, C; Choquette, L; Dongari-Bagtzoglou, A; Peterson, D E; Terzi, E; Strausbaugh, L D

2012-06-01

High throughput sequencing of 16S ribosomal RNA gene amplicons is a cost-effective method for characterization of oral bacterial communities. However, before undertaking large-scale studies, it is necessary to understand the technique-associated limitations and intrinsic variability of the oral ecosystem. In this work we evaluated bias in species representation using an in vitro-assembled mock community of oral bacteria. We then characterized the bacterial communities in saliva and buccal mucosa of five healthy subjects to investigate the power of high throughput sequencing in revealing their diversity and biogeography patterns. Mock community analysis showed primer and DNA isolation biases and an overestimation of diversity that was reduced after eliminating singleton operational taxonomic units (OTUs). Sequencing of salivary and mucosal communities found a total of 455 OTUs (0.3% dissimilarity) with only 78 of these present in all subjects. We demonstrate that this variability was partly the result of incomplete richness coverage even at great sequencing depths, and so comparing communities by their structure was more effective than comparisons based solely on membership. With respect to oral biogeography, we found inter-subject variability in community structure was lower than site differences between salivary and mucosal communities within subjects. These differences were evident at very low sequencing depths and were mostly caused by the abundance of Streptococcus mitis and Gemella haemolysans in mucosa. In summary, we present an experimental and data analysis framework that will facilitate design and interpretation of pyrosequencing-based studies. Despite challenges associated with this technique, we demonstrate its power for evaluation of oral diversity and biogeography patterns. © 2012 John Wiley & Sons A/S.

Bacterial communities in Great Barrier Reef calcareous sediments: Contrasting 16S rDNA libraries from nearshore and outer shelf reefs

NASA Astrophysics Data System (ADS)

Uthicke, S.; McGuire, K.

2007-03-01

Bacterial communities in eight 16S rDNA clone libraries from calcareous sediments were investigated to provide an assessment of the bacterial diversity on sediments of the Great Barrier Reef (GBR) and to investigate differences due to decreased water quality. Sample effort was spread across two locations on each of four coral reefs, with two reefs located nearshore and two reefs on the outer shelf to allow robust statistical comparison of nearshore reefs (subjected to enhanced runoff) and outer shelf reefs (pristine conditions). Out of 221 non-chimeric sequences, 189 (85.5%) were unique and only one sequence occurred in more than one library. Rarefaction analyses and coverage calculations indicated that only a small fraction of the diversity was sampled. Cluster analyses and comparison to published sequences indicated that sequences retrieved belonged to the α, γ and δ subdivision of the Proteobacteria (6.8, 29.4 and 13.6% of the total, respectively), Cytophaga-Flavobacteria-Bacteroidetes (CFB) group (20.4%), Cyanobacteria (5.4%), Planctomycetaceae (7.7%), Verrucomicrobiaceae (6.8%), Acidobacteriaceae (2.7%). Analysis of Similarity (ANOSIM, based on grouping all retrieved sequences into 9 phylogenetic groups) indicated that subtle differences do exist in the community composition between nearshore and outer shelf reefs. Similarity percentage analysis (SIMPER) indicated that Acidobacteriaceae and Cyanobacteriaceae were the main contributors to the dissimilarity. A significant difference between bacteria on nearshore and outer shelf reefs also existed on the molecular level ( FST = 0.008, p = 0.007 for all samples, 0.006, p = 0.022 when repeated sequences within libraries were removed). Thus, bacterial communities on carbonate sediments investigated were highly diverse and differences in community composition may provide important leads for the search for indicator species or communities for water quality differences.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

Protein family clustering for structural genomics.

PubMed

Yan, Yongpan; Moult, John

2005-10-28

A major goal of structural genomics is the provision of a structural template for a large fraction of protein domains. The magnitude of this task depends on the number and nature of protein sequence families. With a large number of bacterial genomes now fully sequenced, it is possible to obtain improved estimates of the number and diversity of families in that kingdom. We have used an automated clustering procedure to group all sequences in a set of genomes into protein families. Bench-marking shows the clustering method is sensitive at detecting remote family members, and has a low level of false positives. This comprehensive protein family set has been used to address the following questions. (1) What is the structure coverage for currently known families? (2) How will the number of known apparent families grow as more genomes are sequenced? (3) What is a practical strategy for maximizing structure coverage in future? Our study indicates that approximately 20% of known families with three or more members currently have a representative structure. The study indicates also that the number of apparent protein families will be considerably larger than previously thought: We estimate that, by the criteria of this work, there will be about 250,000 protein families when 1000 microbial genomes have been sequenced. However, the vast majority of these families will be small, and it will be possible to obtain structural templates for 70-80% of protein domains with an achievable number of representative structures, by systematically sampling the larger families.

Assessment of species diversity and distribution of an ancient diatom lineage using a DNA metabarcoding approach.

PubMed

Nanjappa, Deepak; Audic, Stephane; Romac, Sarah; Kooistra, Wiebe H C F; Zingone, Adriana

2014-01-01

Continuous efforts to estimate actual diversity and to trace the species distribution and ranges in the natural environments have gone in equal pace with advancements of the technologies in the study of microbial species diversity from microscopic observations to DNA-based barcoding. DNA metabarcoding based on Next Generation Sequencing (NGS) constitutes the latest advancement in these efforts. Here we use NGS data from different sites to investigate the geographic range of six species of the diatom family Leptocylindraceae and to identify possible new taxa within the family. We analysed the V4 and V9 regions of the nuclear-encoded SSU rDNA gene region in the NGS database of the European ERA-Biodiversa project BioMarKs, collected in plankton and sediments at six coastal sites in European coastal waters, as well as environmental sequences from the NCBI database. All species known in the family Leptocylindraceae were detected in both datasets, but the much larger Illumina V9 dataset showed a higher species coverage at the various sites than the 454 V4 dataset. Sequences identical or similar to the references of Leptocylindrus aporus, L. convexus, L. danicus/hargravesii and Tenuicylindrus belgicus were found in the Mediterranean Sea, North Atlantic Ocean and Black Sea as well as at locations outside Europe. Instead, sequences identical or close to that of L. minimus were found in the North Atlantic Ocean and the Black Sea but not in the Mediterranean Sea, while sequences belonging to a yet undescribed taxon were encountered only in Oslo Fjord and Baffin Bay. Identification of Leptocylindraceae species in NGS datasets has expanded our knowledge of the species biogeographic distribution and of the overall diversity of this diatom family. Individual species appear to be widespread, but not all of them are found everywhere. Despite the sequencing depth allowed by NGS and the wide geographic area covered by this study, the diversity of this ancient diatom family appears to be low, at least at the level of the marker used in this study.

Assessment of Species Diversity and Distribution of an Ancient Diatom Lineage Using a DNA Metabarcoding Approach

PubMed Central

Nanjappa, Deepak; Audic, Stephane; Romac, Sarah; Kooistra, Wiebe H. C. F.; Zingone, Adriana

2014-01-01

Background Continuous efforts to estimate actual diversity and to trace the species distribution and ranges in the natural environments have gone in equal pace with advancements of the technologies in the study of microbial species diversity from microscopic observations to DNA-based barcoding. DNA metabarcoding based on Next Generation Sequencing (NGS) constitutes the latest advancement in these efforts. Here we use NGS data from different sites to investigate the geographic range of six species of the diatom family Leptocylindraceae and to identify possible new taxa within the family. Methodology/Principal Findings We analysed the V4 and V9 regions of the nuclear-encoded SSU rDNA gene region in the NGS database of the European ERA-Biodiversa project BioMarKs, collected in plankton and sediments at six coastal sites in European coastal waters, as well as environmental sequences from the NCBI database. All species known in the family Leptocylindraceae were detected in both datasets, but the much larger Illumina V9 dataset showed a higher species coverage at the various sites than the 454 V4 dataset. Sequences identical or similar to the references of Leptocylindrus aporus, L. convexus, L. danicus/hargravesii and Tenuicylindrus belgicus were found in the Mediterranean Sea, North Atlantic Ocean and Black Sea as well as at locations outside Europe. Instead, sequences identical or close to that of L. minimus were found in the North Atlantic Ocean and the Black Sea but not in the Mediterranean Sea, while sequences belonging to a yet undescribed taxon were encountered only in Oslo Fjord and Baffin Bay. Conclusions/Significance Identification of Leptocylindraceae species in NGS datasets has expanded our knowledge of the species biogeographic distribution and of the overall diversity of this diatom family. Individual species appear to be widespread, but not all of them are found everywhere. Despite the sequencing depth allowed by NGS and the wide geographic area covered by this study, the diversity of this ancient diatom family appears to be low, at least at the level of the marker used in this study. PMID:25133638

The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific

PubMed Central

Rusch, Douglas B; Halpern, Aaron L; Sutton, Granger; Heidelberg, Karla B; Williamson, Shannon; Yooseph, Shibu; Wu, Dongying; Eisen, Jonathan A; Hoffman, Jeff M; Remington, Karin; Beeson, Karen; Tran, Bao; Smith, Hamilton; Baden-Tillson, Holly; Stewart, Clare; Thorpe, Joyce; Freeman, Jason; Andrews-Pfannkoch, Cynthia; Venter, Joseph E; Li, Kelvin; Kravitz, Saul; Heidelberg, John F; Utterback, Terry; Rogers, Yu-Hui; Falcón, Luisa I; Souza, Valeria; Bonilla-Rosso, Germán; Eguiarte, Luis E; Karl, David M; Sathyendranath, Shubha; Platt, Trevor; Bermingham, Eldredge; Gallardo, Victor; Tamayo-Castillo, Giselle; Ferrari, Michael R; Strausberg, Robert L; Nealson, Kenneth; Friedman, Robert; Frazier, Marvin; Venter, J. Craig

2007-01-01

The world's oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed “fragment recruitment,” addressed questions of genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes and gene families. A second method, termed “extreme assembly,” made possible the assembly and reconstruction of large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3) hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into genetically isolated populations that have overlapping but independent distributions, implying distinct environmental preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show how they may be grouped into several community types. Specific functional adaptations can be identified both within individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or absence of the phosphate-binding gene PstS. PMID:17355176

Determination of a Screening Metric for High Diversity DNA Libraries.

PubMed

Guido, Nicholas J; Handerson, Steven; Joseph, Elaine M; Leake, Devin; Kung, Li A

2016-01-01

The fields of antibody engineering, enzyme optimization and pathway construction rely increasingly on screening complex variant DNA libraries. These highly diverse libraries allow researchers to sample a maximized sequence space; and therefore, more rapidly identify proteins with significantly improved activity. The current state of the art in synthetic biology allows for libraries with billions of variants, pushing the limits of researchers' ability to qualify libraries for screening by measuring the traditional quality metrics of fidelity and diversity of variants. Instead, when screening variant libraries, researchers typically use a generic, and often insufficient, oversampling rate based on a common rule-of-thumb. We have developed methods to calculate a library-specific oversampling metric, based on fidelity, diversity, and representation of variants, which informs researchers, prior to screening the library, of the amount of oversampling required to ensure that the desired fraction of variant molecules will be sampled. To derive this oversampling metric, we developed a novel alignment tool to efficiently measure frequency counts of individual nucleotide variant positions using next-generation sequencing data. Next, we apply a method based on the "coupon collector" probability theory to construct a curve of upper bound estimates of the sampling size required for any desired variant coverage. The calculated oversampling metric will guide researchers to maximize their efficiency in using highly variant libraries.

Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy

DOE PAGES

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa; ...

2014-09-01

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

Improved hybrid de novo genome assembly of domesticated apple (Malus x domestica).

PubMed

Li, Xuewei; Kui, Ling; Zhang, Jing; Xie, Yinpeng; Wang, Liping; Yan, Yan; Wang, Na; Xu, Jidi; Li, Cuiying; Wang, Wen; van Nocker, Steve; Dong, Yang; Ma, Fengwang; Guan, Qingmei

2016-08-08

Domesticated apple (Malus × domestica Borkh) is a popular temperate fruit with high nutrient levels and diverse flavors. In 2012, global apple production accounted for at least one tenth of all harvested fruits. A high-quality apple genome assembly is crucial for the selection and breeding of new cultivars. Currently, a single reference genome is available for apple, assembled from 16.9 × genome coverage short reads via Sanger and 454 sequencing technologies. Although a useful resource, this assembly covers only ~89 % of the non-repetitive portion of the genome, and has a relatively short (16.7 kb) contig N50 length. These downsides make it difficult to apply this reference in transcriptive or whole-genome re-sequencing analyses. Here we present an improved hybrid de novo genomic assembly of apple (Golden Delicious), which was obtained from 76 Gb (~102 × genome coverage) Illumina HiSeq data and 21.7 Gb (~29 × genome coverage) PacBio data. The final draft genome is approximately 632.4 Mb, representing ~ 90 % of the estimated genome. The contig N50 size is 111,619 bp, representing a 7 fold improvement. Further annotation analyses predicted 53,922 protein-coding genes and 2,765 non-coding RNA genes. The new apple genome assembly will serve as a valuable resource for investigating complex apple traits at the genomic level. It is not only suitable for genome editing and gene cloning, but also for RNA-seq and whole-genome re-sequencing studies.

A global reference for human genetic variation

PubMed Central

2016-01-01

The 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies. PMID:26432245

Ultraviolet, Infrared, and High-Low Energy Photodissociation of Post-Translationally Modified Peptides

NASA Astrophysics Data System (ADS)

Halim, Mohammad A.; MacAleese, Luke; Lemoine, Jérôme; Antoine, Rodolphe; Dugourd, Philippe; Girod, Marion

2018-02-01

Mass spectrometry-based methods have made significant progress in characterizing post-translational modifications in peptides and proteins; however, certain aspects regarding fragmentation methods must still be improved. A good technique is expected to provide excellent sequence information, locate PTM sites, and retain the labile PTM groups. To address these issues, we investigate 10.6 μm IRMPD, 213 nm UVPD, and combined UV and IR photodissociation, known as HiLoPD (high-low photodissociation), for phospho-, sulfo-, and glyco-peptide cations. IRMPD shows excellent backbone fragmentation and produces equal numbers of N- and C-terminal ions. The results reveal that 213 nm UVPD and HiLoPD methods can provide diverse backbone fragmentation producing a/x, b/y, and c/z ions with excellent sequence coverage, locate PTM sites, and offer reasonable retention efficiency for phospho- and glyco-peptides. Excellent sequence coverage is achieved for sulfo-peptides and the position of the SO3 group can be pinpointed; however, widespread SO3 losses are detected irrespective of the methods used herein. Based on the overall performance achieved, we believe that 213 nm UVPD and HiLoPD can serve as alternative options to collision activation and electron transfer dissociations for phospho- and glyco-proteomics.

Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections.

PubMed

Hammoumi, Saliha; Vallaeys, Tatiana; Santika, Ayi; Leleux, Philippe; Borzym, Ewa; Klopp, Christophe; Avarre, Jean-Christophe

2016-01-01

Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp, Cyprinus carpio L. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×10 7 . The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3.

Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections

PubMed Central

Hammoumi, Saliha; Vallaeys, Tatiana; Santika, Ayi; Leleux, Philippe; Borzym, Ewa; Klopp, Christophe

2016-01-01

Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp, Cyprinus carpio L. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×107. The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3. PMID:27703859

Bone protein “extractomics”: comparing the efficiency of bone protein extractions of Gallus gallus in tandem mass spectrometry, with an eye towards paleoproteomics

PubMed Central

DeHart, Caroline J.; Schweitzer, Mary H.; Thomas, Paul M.; Kelleher, Neil L.

2016-01-01

Proteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recovered by mass spectrometry (MS) analyses. Characterizing these different “extractomes” will provide critical data for development of novel and more efficient protein extraction methodologies for fossils. Here, we analyze 22 unique sub-extractions of chicken bone and directly compare individual extraction components for their total protein yield and diversity and coverage of bone proteins identified by MS. We extracted proteins using different combinations and ratios of demineralizing reagents, protein-solubilizing reagents, and post-extraction buffer removal methods, then evaluated tryptic digests from 20 µg aliquots of each fraction by tandem MS/MS on a 12T FT-ICR mass spectrometer. We compared total numbers of peptide spectral matches, peptides, and proteins identified from each fraction, the redundancy of protein identifications between discrete steps of extraction methods, and the sequence coverage obtained for select, abundant proteins. Although both alpha chains of collagen I (the most abundant protein in bone) were found in all fractions, other collagenous and non-collagenous proteins (e.g., apolipoprotein, osteonectin, hemoglobin) were differentially identified. We found that when a standardized amount of extracted proteins was analyzed, extraction steps that yielded the most protein (by weight) from bone were often not the ones that produced the greatest diversity of bone proteins, or the highest degree of protein coverage. Generally, the highest degrees of diversity and coverage were obtained from demineralization fractions, and the proteins found in the subsequent solubilization fractions were highly redundant with those in the previous fraction. Based on these data, we identify future directions and parameters to consider (e.g., proteins targeted, amount of sample required) when applying discrete parts of these protocols to fossils. PMID:27812413

Variant calling in low-coverage whole genome sequencing of a Native American population sample.

PubMed

Bizon, Chris; Spiegel, Michael; Chasse, Scott A; Gizer, Ian R; Li, Yun; Malc, Ewa P; Mieczkowski, Piotr A; Sailsbery, Josh K; Wang, Xiaoshu; Ehlers, Cindy L; Wilhelmsen, Kirk C

2014-01-30

The reduction in the cost of sequencing a human genome has led to the use of genotype sampling strategies in order to impute and infer the presence of sequence variants that can then be tested for associations with traits of interest. Low-coverage Whole Genome Sequencing (WGS) is a sampling strategy that overcomes some of the deficiencies seen in fixed content SNP array studies. Linkage-disequilibrium (LD) aware variant callers, such as the program Thunder, may provide a calling rate and accuracy that makes a low-coverage sequencing strategy viable. We examined the performance of an LD-aware variant calling strategy in a population of 708 low-coverage whole genome sequences from a community sample of Native Americans. We assessed variant calling through a comparison of the sequencing results to genotypes measured in 641 of the same subjects using a fixed content first generation exome array. The comparison was made using the variant calling routines GATK Unified Genotyper program and the LD-aware variant caller Thunder. Thunder was found to improve concordance in a coverage dependent fashion, while correctly calling nearly all of the common variants as well as a high percentage of the rare variants present in the sample. Low-coverage WGS is a strategy that appears to collect genetic information intermediate in scope between fixed content genotyping arrays and deep-coverage WGS. Our data suggests that low-coverage WGS is a viable strategy with a greater chance of discovering novel variants and associations than fixed content arrays for large sample association analyses.

A Simple Method to Decode the Complete 18-5.8-28S rRNA Repeated Units of Green Algae by Genome Skimming.

PubMed

Lin, Geng-Ming; Lai, Yu-Heng; Audira, Gilbert; Hsiao, Chung-Der

2017-11-06

Green algae, Chlorella ellipsoidea , Haematococcus pluvialis and Aegagropila linnaei (Phylum Chlorophyta) were simultaneously decoded by a genomic skimming approach within 18-5.8-28S rRNA region. Whole genomic DNAs were isolated from green algae and directly subjected to low coverage genome skimming sequencing. After de novo assembly and mapping, the size of complete 18-5.8-28S rRNA repeated units for three green algae were ranged from 5785 to 6028 bp, which showed high nucleotide diversity (π is around 0.5-0.6) within ITS1 and ITS2 (Internal Transcribed Spacer) regions. Previously, the evolutional diversity of algae has been difficult to decode due to the inability design universal primers that amplify specific marker genes across diverse algal species. In this study, our method provided a rapid and universal approach to decode the 18-5.8-28S rRNA repeat unit in three green algal species. In addition, the completely sequenced 18-5.8-28S rRNA repeated units provided a solid nuclear marker for phylogenetic and evolutionary analysis for green algae for the first time.

Single haplotype assembly of the human genome from a hydatidiform mole.

PubMed

Steinberg, Karyn Meltz; Schneider, Valerie A; Graves-Lindsay, Tina A; Fulton, Robert S; Agarwala, Richa; Huddleston, John; Shiryev, Sergey A; Morgulis, Aleksandr; Surti, Urvashi; Warren, Wesley C; Church, Deanna M; Eichler, Evan E; Wilson, Richard K

2014-12-01

A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can improve assembly, even the longest available reads do not resolve all regions. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly. © 2014 Steinberg et al.; Published by Cold Spring Harbor Laboratory Press.

Single haplotype assembly of the human genome from a hydatidiform mole

PubMed Central

Steinberg, Karyn Meltz; Schneider, Valerie A.; Graves-Lindsay, Tina A.; Fulton, Robert S.; Agarwala, Richa; Huddleston, John; Shiryev, Sergey A.; Morgulis, Aleksandr; Surti, Urvashi; Warren, Wesley C.; Church, Deanna M.; Eichler, Evan E.; Wilson, Richard K.

2014-01-01

A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can improve assembly, even the longest available reads do not resolve all regions. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly. PMID:25373144

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

PubMed

Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

2017-07-12

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Genome sequence of Phytophthora ramorum: implications for management

Treesearch

Brett Tyler; Sucheta Tripathy; Nik Grunwald; Kurt Lamour; Kelly Ivors; Matteo Garbelotto; Daniel Rokhsar; Nik Putnam; Igor Grigoriev; Jeffrey Boore

2006-01-01

A draft genome sequence has been determined for Phytophthora ramorum, together with a draft sequence of the soybean pathogen Phytophthora sojae. The P. ramorum genome was sequenced to a depth of 7-fold coverage, while the P. sojae genome was sequenced to a depth of 9-fold coverage. The genome...

Environmental metabarcodes for insects: in silico PCR reveals potential for taxonomic bias.

PubMed

Clarke, Laurence J; Soubrier, Julien; Weyrich, Laura S; Cooper, Alan

2014-11-01

Studies of insect assemblages are suited to the simultaneous DNA-based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally possess appropriate taxonomic coverage to avoid PCR-amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers [16S and cytochrome oxidase c subunit I (COI)] and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR-amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR-amplified the blend using five primer sets, targeting either COI or 16S, with high-throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers. © 2014 John Wiley & Sons Ltd.

Genome-wide ancestry and divergence patterns from low-coverage sequencing data reveal a complex history of admixture in wild baboons

PubMed Central

Wall, Jeffrey D; Schlebusch, Stephen A; Alberts, Susan C; Cox, Laura A; Snyder-Mackler, Noah; Nevonen, Kimberly; Carbone, Lucia; Tung, Jenny

2017-01-01

Naturally occurring admixture has now been documented in every major primate lineage, suggesting its key role in primate evolutionary history. Active primate hybrid zones can provide valuable insight into this process. Here, we investigate the history of admixture in one of the best-studied natural primate hybrid zones, between yellow baboons (Papio cynocephalus) and anubis baboons (Papio anubis) in the Amboseli ecosystem of Kenya. We generated a new genome assembly for yellow baboon and low coverage genome-wide resequencing data from yellow baboons, anubis baboons, and known hybrids (n=44). Using a novel composite likelihood method for estimating local ancestry from low coverage data, we found high levels of genetic diversity and genetic differentiation between the parent taxa, and excellent agreement between genome-scale ancestry estimates and a priori pedigree, life history, and morphology-based estimates (r2=0.899). However, even putatively unadmixed Amboseli yellow individuals carried a substantial proportion of anubis ancestry, presumably due to historical admixture. Further, the distribution of shared versus fixed differences between a putatively unadmixed Amboseli yellow baboon and an unadmixed anubis baboon, both sequenced at high coverage, are inconsistent with simple isolation-migration or equilibrium migration models. Our findings suggest a complex process of intermittent contact that has occurred multiple times in baboon evolutionary history, despite no obvious fitness costs to hybrids or major geographic or behavioral barriers. In combination with the extensive phenotypic data available for baboon hybrids, our results provide valuable context for understanding the history of admixture in primates, including in our own lineage. PMID:27145036

Calibrating snakehead diversity with DNA barcodes: expanding taxonomic coverage to enable identification of potential and established invasive species.

PubMed

Serrao, Natasha R; Steinke, Dirk; Hanner, Robert H

2014-01-01

Detecting and documenting the occurrence of invasive species outside their native range requires tools to support their identification. This can be challenging for taxa with diverse life stages and/or problematic or unresolved morphological taxonomies. DNA barcoding provides a potent method for identifying invasive species, as it allows for species identification at all life stages, including fragmentary remains. It also provides an efficient interim taxonomic framework for quantifying cryptic genetic diversity by parsing barcode sequences into discontinuous haplogroup clusters (typical of reproductively isolated species) and labelling them with unique alphanumeric identifiers. Snakehead fishes are a diverse group of opportunistic predators endemic to Asia and Africa that may potentially pose significant threats as aquatic invasive species. At least three snakehead species (Channa argus, C. maculata, and C. marulius) are thought to have entered North America through the aquarium and live-food fish markets, and have established populations, yet their origins remain unclear. The objectives of this study were to assemble a library of DNA barcode sequences derived from expert identified reference specimens in order to determine the identity and aid invasion pathway analysis of the non-indigenous species found in North America using DNA barcodes. Sequences were obtained from 121 tissue samples representing 25 species and combined with public records from GenBank for a total of 36 putative species, which then partitioned into 49 discrete haplogroups. Multiple divergent clusters were observed within C. gachua, C. marulius, C. punctata and C. striata suggesting the potential presence of cryptic species diversity within these lineages. Our findings demonstrate that DNA barcoding is a valuable tool for species identification in challenging and under-studied taxonomic groups such as snakeheads, and provides a useful framework for inferring invasion pathway analysis.

Hyperbaric biofilms on engineering surfaces formed in the deep sea.

PubMed

Meier, Alexandra; Tsaloglou, Nefeli-Maria; Mowlem, Matthew C; Keevil, C William; Connelly, Douglas P

2013-01-01

Biofouling is a major problem for long-term deployment of sensors in the marine environment. This study showed that significant biofilm formation occurred on a variety of artificial materials (glass, copper, Delrin(™) and poly-methyl methacrylate [PMMA]) deployed for 10 days at a depth of 4700 m in the Cayman Trough. Biofilm surface coverage was used as an indicator of biomass. The lowest biofilm coverage was on copper and PMMA. Molecular analyses indicated that bacteria dominated the biofilms found on copper, Delrin(™) and PMMA with 75, 55 and 73% coverage, respectively. Archea (66%) were dominant on the glass surface simulating interior sensor conditions, whereas Eukarya comprised the highest percentage of microflora (75%) on the glass simulating the exterior of sensors. Analysis of Denaturing Gradient Gel Electrophoresis profiles indicated that copper and Delrin(™) shared the same community diversity, which was not the case for glass and PMMA, or between PMMA and copper/Delrin(™). Sequence alignment matches belonged exclusively to uncultivable microorganisms, most of which were not further classified. One extracted sequence found on glass was associated with Cowellia sp., while another extracted from the PMMA surface was associated with a bacterium in the Alterominidaceae, both γ-proteobacteria. The results demonstrate the necessity of understanding biofilm formation in the deep sea and the potential need for mitigation strategies for any kind of long-term deployment of remote sensors in the marine environment.

High-resolution mitochondrial DNA analysis sheds light on human diversity, cultural interactions, and population mobility in Northwestern Amazonia.

PubMed

Arias, Leonardo; Barbieri, Chiara; Barreto, Guillermo; Stoneking, Mark; Pakendorf, Brigitte

2018-02-01

Northwestern Amazonia (NWA) is a center of high linguistic and cultural diversity. Several language families and linguistic isolates occur in this region, as well as different subsistence patterns, with some groups being foragers and others agriculturalists. In addition, speakers of Eastern Tukanoan languages are known for practicing linguistic exogamy, a marriage system in which partners are taken from different language groups. In this study, we use high-resolution mitochondrial DNA sequencing to investigate the impact of this linguistic and cultural diversity on the genetic relationships and population structure of NWA groups. We collected saliva samples from individuals representing 40 different NWA ethnolinguistic groups and sequenced 439 complete mitochondrial genomes to an average coverage of 1,030×. The mtDNA data revealed that NWA populations have high genetic diversity with extensive sharing of haplotypes among groups. Moreover, groups who practice linguistic exogamy have higher genetic diversity, while the foraging Nukak have lower genetic diversity. We also find that rivers play a more important role than either geography or language affiliation in structuring the genetic relationships of populations. Contrary to the view of NWA as a pristine area inhabited by small human populations living in isolation, our data support a view of high diversity and contact among different ethnolinguistic groups, with movement along rivers probably facilitating this contact. Additionally, we provide evidence for the impact of cultural practices, such as linguistic exogamy, on patterns of genetic variation. Overall, this study provides new data and insights into a remote and little-studied region of the world. © 2017 Wiley Periodicals, Inc.

A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in Neoromicia bats within South Africa.

PubMed

Geldenhuys, Marike; Mortlock, Marinda; Weyer, Jacqueline; Bezuidt, Oliver; Seamark, Ernest C J; Kearney, Teresa; Gleasner, Cheryl; Erkkila, Tracy H; Cui, Helen; Markotter, Wanda

2018-01-01

Species within the Neoromicia bat genus are abundant and widely distributed in Africa. It is common for these insectivorous bats to roost in anthropogenic structures in urban regions. Additionally, Neoromicia capensis have previously been identified as potential hosts for Middle East respiratory syndrome (MERS)-related coronaviruses. This study aimed to ascertain the gastrointestinal virome of these bats, as viruses excreted in fecal material or which may be replicating in rectal or intestinal tissues have the greatest opportunities of coming into contact with other hosts. Samples were collected in five regions of South Africa over eight years. Initial virome composition was determined by viral metagenomic sequencing by pooling samples and enriching for viral particles. Libraries were sequenced on the Illumina MiSeq and NextSeq500 platforms, producing a combined 37 million reads. Bioinformatics analysis of the high throughput sequencing data detected the full genome of a novel species of the Circoviridae family, and also identified sequence data from the Adenoviridae, Coronaviridae, Herpesviridae, Parvoviridae, Papillomaviridae, Phenuiviridae, and Picornaviridae families. Metagenomic sequencing data was insufficient to determine the viral diversity of certain families due to the fragmented coverage of genomes and lack of suitable sequencing depth, as some viruses were detected from the analysis of reads-data only. Follow up conventional PCR assays targeting conserved gene regions for the Adenoviridae, Coronaviridae, and Herpesviridae families were used to confirm metagenomic data and generate additional sequences to determine genetic diversity. The complete coding genome of a MERS-related coronavirus was recovered with additional amplicon sequencing on the MiSeq platform. The new genome shared 97.2% overall nucleotide identity to a previous Neoromicia-associated MERS-related virus, also from South Africa. Conventional PCR analysis detected diverse adenovirus and herpesvirus sequences that were widespread throughout Neoromicia populations in South Africa. Furthermore, similar adenovirus sequences were detected within these populations throughout several years. With the exception of the coronaviruses, the study represents the first report of sequence data from several viral families within a Southern African insectivorous bat genus; highlighting the need for continued investigations in this regard.

A metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in Neoromicia bats within South Africa

PubMed Central

Geldenhuys, Marike; Mortlock, Marinda; Weyer, Jacqueline; Bezuidt, Oliver; Seamark, Ernest C. J.; Kearney, Teresa; Gleasner, Cheryl; Erkkila, Tracy H.; Cui, Helen; Markotter, Wanda

2018-01-01

Species within the Neoromicia bat genus are abundant and widely distributed in Africa. It is common for these insectivorous bats to roost in anthropogenic structures in urban regions. Additionally, Neoromicia capensis have previously been identified as potential hosts for Middle East respiratory syndrome (MERS)-related coronaviruses. This study aimed to ascertain the gastrointestinal virome of these bats, as viruses excreted in fecal material or which may be replicating in rectal or intestinal tissues have the greatest opportunities of coming into contact with other hosts. Samples were collected in five regions of South Africa over eight years. Initial virome composition was determined by viral metagenomic sequencing by pooling samples and enriching for viral particles. Libraries were sequenced on the Illumina MiSeq and NextSeq500 platforms, producing a combined 37 million reads. Bioinformatics analysis of the high throughput sequencing data detected the full genome of a novel species of the Circoviridae family, and also identified sequence data from the Adenoviridae, Coronaviridae, Herpesviridae, Parvoviridae, Papillomaviridae, Phenuiviridae, and Picornaviridae families. Metagenomic sequencing data was insufficient to determine the viral diversity of certain families due to the fragmented coverage of genomes and lack of suitable sequencing depth, as some viruses were detected from the analysis of reads-data only. Follow up conventional PCR assays targeting conserved gene regions for the Adenoviridae, Coronaviridae, and Herpesviridae families were used to confirm metagenomic data and generate additional sequences to determine genetic diversity. The complete coding genome of a MERS-related coronavirus was recovered with additional amplicon sequencing on the MiSeq platform. The new genome shared 97.2% overall nucleotide identity to a previous Neoromicia-associated MERS-related virus, also from South Africa. Conventional PCR analysis detected diverse adenovirus and herpesvirus sequences that were widespread throughout Neoromicia populations in South Africa. Furthermore, similar adenovirus sequences were detected within these populations throughout several years. With the exception of the coronaviruses, the study represents the first report of sequence data from several viral families within a Southern African insectivorous bat genus; highlighting the need for continued investigations in this regard. PMID:29579103

A High-Coverage Yersinia pestis Genome from a Sixth-Century Justinianic Plague Victim

PubMed Central

Feldman, Michal; Harbeck, Michaela; Keller, Marcel; Spyrou, Maria A.; Rott, Andreas; Trautmann, Bernd; Scholz, Holger C.; Päffgen, Bernd; Peters, Joris; McCormick, Michael; Bos, Kirsten; Herbig, Alexander; Krause, Johannes

2016-01-01

The Justinianic Plague, which started in the sixth century and lasted to the mid eighth century, is thought to be the first of three historically documented plague pandemics causing massive casualties. Historical accounts and molecular data suggest the bacterium Yersinia pestis as its etiological agent. Here we present a new high-coverage (17.9-fold) Y. pestis genome obtained from a sixth-century skeleton recovered from a southern German burial site close to Munich. The reconstructed genome enabled the detection of 30 unique substitutions as well as structural differences that have not been previously described. We report indels affecting a lacl family transcription regulator gene as well as nonsynonymous substitutions in the nrdE, fadJ, and pcp genes, that have been suggested as plague virulence determinants or have been shown to be upregulated in different models of plague infection. In addition, we identify 19 false positive substitutions in a previously published lower-coverage Y. pestis genome from another archaeological site of the same time period and geographical region that is otherwise genetically identical to the high-coverage genome sequence reported here, suggesting low-genetic diversity of the plague during the sixth century in rural southern Germany. PMID:27578768

Mosaic vaccines elicit CD8+ T cell responses in monkeys that confer immune coverage of diverse HIV strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fischer, Will; Korber, Bette

2009-01-01

Creation of a successful HIV vaccine will require the development of a strategy to generate cellular immunity with sufficient cross-clade breadth to deal with the extreme genetic diversity of the virus. Polyvalent mosaic immunogens derived from in silica recombination of natural strains of HIV are designed to induce cellular immune responses that maximally cover the sequence diversity of circulating virus isolates. Immunization of rhesus monkeys with plasmid DNA and recombinant vaccinia virus vaccine constructs expressing either consensus immunogens or polyvalent mosaic immunogens elicited a CD4+ T lymphocyte-biased response with comparably broad epitope-specific total T lymphocyte specificities. However, immunization with themore » mosaic immunogens induced HIV-specific CD8+ T lymphocyte responses with markedly greater depth and breadth. Therefore, the use of polyvalent mosaic immunogens is a promising strategy for a global vaccine for HIV.« less

A recent bottleneck of Y chromosome diversity coincides with a global change in culture

PubMed Central

Saag, Lauri; Vicente, Mário; Sayres, Melissa A. Wilson; Järve, Mari; Talas, Ulvi Gerst; Rootsi, Siiri; Ilumäe, Anne-Mai; Mägi, Reedik; Mitt, Mario; Pagani, Luca; Puurand, Tarmo; Faltyskova, Zuzana; Clemente, Florian; Cardona, Alexia; Metspalu, Ene; Sahakyan, Hovhannes; Yunusbayev, Bayazit; Hudjashov, Georgi; DeGiorgio, Michael; Loogväli, Eva-Liis; Eichstaedt, Christina; Eelmets, Mikk; Chaubey, Gyaneshwer; Tambets, Kristiina; Litvinov, Sergei; Mormina, Maru; Xue, Yali; Ayub, Qasim; Zoraqi, Grigor; Korneliussen, Thorfinn Sand; Akhatova, Farida; Lachance, Joseph; Tishkoff, Sarah; Momynaliev, Kuvat; Ricaut, François-Xavier; Kusuma, Pradiptajati; Razafindrazaka, Harilanto; Pierron, Denis; Cox, Murray P.; Sultana, Gazi Nurun Nahar; Willerslev, Rane; Muller, Craig; Westaway, Michael; Lambert, David; Skaro, Vedrana; Kovačevic´, Lejla; Turdikulova, Shahlo; Dalimova, Dilbar; Khusainova, Rita; Trofimova, Natalya; Akhmetova, Vita; Khidiyatova, Irina; Lichman, Daria V.; Isakova, Jainagul; Pocheshkhova, Elvira; Sabitov, Zhaxylyk; Barashkov, Nikolay A.; Nymadawa, Pagbajabyn; Mihailov, Evelin; Seng, Joseph Wee Tien; Evseeva, Irina; Migliano, Andrea Bamberg; Abdullah, Syafiq; Andriadze, George; Primorac, Dragan; Atramentova, Lubov; Utevska, Olga; Yepiskoposyan, Levon; Marjanovic´, Damir; Kushniarevich, Alena; Behar, Doron M.; Gilissen, Christian; Vissers, Lisenka; Veltman, Joris A.; Balanovska, Elena; Derenko, Miroslava; Malyarchuk, Boris; Metspalu, Andres; Fedorova, Sardana; Eriksson, Anders; Manica, Andrea; Mendez, Fernando L.; Karafet, Tatiana M.; Veeramah, Krishna R.; Bradman, Neil; Hammer, Michael F.; Osipova, Ludmila P.; Balanovsky, Oleg; Khusnutdinova, Elza K.; Johnsen, Knut; Remm, Maido; Thomas, Mark G.; Tyler-Smith, Chris; Underhill, Peter A.; Willerslev, Eske; Nielsen, Rasmus; Metspalu, Mait; Villems, Richard

2015-01-01

It is commonly thought that human genetic diversity in non-African populations was shaped primarily by an out-of-Africa dispersal 50–100 thousand yr ago (kya). Here, we present a study of 456 geographically diverse high-coverage Y chromosome sequences, including 299 newly reported samples. Applying ancient DNA calibration, we date the Y-chromosomal most recent common ancestor (MRCA) in Africa at 254 (95% CI 192–307) kya and detect a cluster of major non-African founder haplogroups in a narrow time interval at 47–52 kya, consistent with a rapid initial colonization model of Eurasia and Oceania after the out-of-Africa bottleneck. In contrast to demographic reconstructions based on mtDNA, we infer a second strong bottleneck in Y-chromosome lineages dating to the last 10 ky. We hypothesize that this bottleneck is caused by cultural changes affecting variance of reproductive success among males. PMID:25770088

BRILIA: Integrated Tool for High-Throughput Annotation and Lineage Tree Assembly of B-Cell Repertoires.

PubMed

Lee, Donald W; Khavrutskii, Ilja V; Wallqvist, Anders; Bavari, Sina; Cooper, Christopher L; Chaudhury, Sidhartha

2016-01-01

The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from Variable (V), Diversity (D), and Joining (J) (VDJ) recombination and somatic hypermutation (SHM) during B-cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable complementarity determining region 3 (CDR3), which plays a critical role in antigen specificity and binding affinity. Tracking the selection and mutation of the CDR3 can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present B-cell repertoire inductive lineage and immunosequence annotator (BRILIA), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94 and 70% accuracy, respectively, and it has a 90% SHM-positive prediction rate in the CDR3 of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B-cell clusters and yielded inconsistent SHM frequencies. We demonstrate BRILIA's utility in B-cell repertoire studies related to VDJ gene usage, mechanisms for adenosine mutations, and SHM hot spot motifs. Furthermore, we show that the complete gene usage annotation and SHM identification across the entire CDR3 are essential for studying the B-cell affinity maturation process through immunosequencing methods.

Genomic treasure troves: complete genome sequencing of herbarium and insect museum specimens.

PubMed

Staats, Martijn; Erkens, Roy H J; van de Vossenberg, Bart; Wieringa, Jan J; Kraaijeveld, Ken; Stielow, Benjamin; Geml, József; Richardson, James E; Bakker, Freek T

2013-01-01

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In today's next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22-82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4-97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2-71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.

Estimating population diversity with CatchAll

PubMed Central

Bunge, John; Woodard, Linda; Böhning, Dankmar; Foster, James A.; Connolly, Sean; Allen, Heather K.

2012-01-01

Motivation: The massive data produced by next-generation sequencing require advanced statistical tools. We address estimating the total diversity or species richness in a population. To date, only relatively simple methods have been implemented in available software. There is a need for software employing modern, computationally intensive statistical analyses including error, goodness-of-fit and robustness assessments. Results: We present CatchAll, a fast, easy-to-use, platform-independent program that computes maximum likelihood estimates for finite-mixture models, weighted linear regression-based analyses and coverage-based non-parametric methods, along with outlier diagnostics. Given sample ‘frequency count’ data, CatchAll computes 12 different diversity estimates and applies a model-selection algorithm. CatchAll also derives discounted diversity estimates to adjust for possibly uncertain low-frequency counts. It is accompanied by an Excel-based graphics program. Availability: Free executable downloads for Linux, Windows and Mac OS, with manual and source code, at www.northeastern.edu/catchall. Contact: jab18@cornell.edu PMID:22333246

Comparative immunogenomics of molluscs.

PubMed

Schultz, Jonathan H; Adema, Coen M

2017-10-01

Comparative immunology, studying both vertebrates and invertebrates, provided the earliest descriptions of phagocytosis as a general immune mechanism. However, the large scale of animal diversity challenges all-inclusive investigations and the field of immunology has developed by mostly emphasizing study of a few vertebrate species. In addressing the lack of comprehensive understanding of animal immunity, especially that of invertebrates, comparative immunology helps toward management of invertebrates that are food sources, agricultural pests, pathogens, or transmit diseases, and helps interpret the evolution of animal immunity. Initial studies showed that the Mollusca (second largest animal phylum), and invertebrates in general, possess innate defenses but lack the lymphocytic immune system that characterizes vertebrate immunology. Recognizing the reality of both common and taxon-specific immune features, and applying up-to-date cell and molecular research capabilities, in-depth studies of a select number of bivalve and gastropod species continue to reveal novel aspects of molluscan immunity. The genomics era heralded a new stage of comparative immunology; large-scale efforts yielded an initial set of full molluscan genome sequences that is available for analyses of full complements of immune genes and regulatory sequences. Next-generation sequencing (NGS), due to lower cost and effort required, allows individual researchers to generate large sequence datasets for growing numbers of molluscs. RNAseq provides expression profiles that enable discovery of immune genes and genome sequences reveal distribution and diversity of immune factors across molluscan phylogeny. Although computational de novo sequence assembly will benefit from continued development and automated annotation may require some experimental validation, NGS is a powerful tool for comparative immunology, especially increasing coverage of the extensive molluscan diversity. To date, immunogenomics revealed new levels of complexity of molluscan defense by indicating sequence heterogeneity in individual snails and bivalves, and members of expanded immune gene families are expressed differentially to generate pathogen-specific defense responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cultivable Anaerobic Microbiota of Severe Early Childhood Caries▿¶

PubMed Central

Tanner, A. C. R.; Mathney, J. M. J.; Kent, R. L.; Chalmers, N. I.; Hughes, C. V.; Loo, C. Y.; Pradhan, N.; Kanasi, E.; Hwang, J.; Dahlan, M. A.; Papadopolou, E.; Dewhirst, F. E.

2011-01-01

Severe early childhood caries (ECC), while strongly associated with Streptococcus mutans using selective detection (culture, PCR), has also been associated with a widely diverse microbiota using molecular cloning approaches. The aim of this study was to evaluate the microbiota of severe ECC using anaerobic culture. The microbial composition of dental plaque from 42 severe ECC children was compared with that of 40 caries-free children. Bacterial samples were cultured anaerobically on blood and acid (pH 5) agars. Isolates were purified, and partial sequences for the 16S rRNA gene were obtained from 5,608 isolates. Sequence-based analysis of the 16S rRNA isolate libraries from blood and acid agars of severe ECC and caries-free children had >90% population coverage, with greater diversity occurring in the blood isolate library. Isolate sequences were compared with taxon sequences in the Human Oral Microbiome Database (HOMD), and 198 HOMD taxa were identified, including 45 previously uncultivated taxa, 29 extended HOMD taxa, and 45 potential novel groups. The major species associated with severe ECC included Streptococcus mutans, Scardovia wiggsiae, Veillonella parvula, Streptococcus cristatus, and Actinomyces gerensceriae. S. wiggsiae was significantly associated with severe ECC children in the presence and absence of S. mutans detection. We conclude that anaerobic culture detected as wide a diversity of species in ECC as that observed using cloning approaches. Culture coupled with 16S rRNA identification identified over 74 isolates for human oral taxa without previously cultivated representatives. The major caries-associated species were S. mutans and S. wiggsiae, the latter of which is a candidate as a newly recognized caries pathogen. PMID:21289150

[Annotation of the mobilomes of nine teleost species].

PubMed

Gao, Bo; Shen, Dan; Chen, Cai; Wang, Saisai; Yang, Kunlun; Chen, Wei; Wang, Wei; Zhang, Li; Song, Chengyi

2018-01-25

In this study, the mobilomes of nine teleost species were annotated by bioinformatics methods. Both of the mobilome size and constitute displayed a significant difference in 9 species of teleost fishes. The species of mobilome content ranking from high to low were zebrafish, medaka, tilapia, coelacanth, platyfish, cod, stickleback, tetradon and fugu. Mobilome content and genome size were positively correlated. The DNA transposons displayed higher diversity and larger variation in teleost (0.50% to 38.37%), was a major determinant of differences in teleost mobilomes, and hAT and Tc/Mariner superfamily were the major DNA transposons in teleost. RNA transposons also exhibited high diversity in teleost, LINE transposons accounted for 0.53% to 5.75% teleost genomic sequences, and 14 superfamilies were detected. L1, L2, RTE and Rex retrotransposons obtained significant amplification. While LTR displayed low amplification in most teleost with less than 2% of genome coverages, except in zebrafish and stickleback, where LTR reachs 5.58% and 2.51% of genome coverages respectively. And 6 LTR superfamilies (Copia, DIRS, ERV, Gypsy, Ngaro and Pao) were detected in the teleost, and Gypsy exhibits obvious amplication among them. While the SINE represents the weakest ampification types in teleost, only within zebrafish and coelacanth, it represents 3.28% and 5.64% of genome coverages, in the other 7 teleost, it occupies less than 1% of genomes, and tRNA, 5S and MIR families of SINE have a certain degree of amplification in some teleosts. This study shows that the teleost display high diversity and large variation of mobilome, there is a strong correlation with the size variations of genomes and mobilome contents in teleost, mobilome is an important factor in determining the teleost genome size.

Sequencing of Seven Haloarchaeal Genomes Reveals Patterns of Genomic Flux

PubMed Central

Lynch, Erin A.; Langille, Morgan G. I.; Darling, Aaron; Wilbanks, Elizabeth G.; Haltiner, Caitlin; Shao, Katie S. Y.; Starr, Michael O.; Teiling, Clotilde; Harkins, Timothy T.; Edwards, Robert A.; Eisen, Jonathan A.; Facciotti, Marc T.

2012-01-01

We report the sequencing of seven genomes from two haloarchaeal genera, Haloferax and Haloarcula. Ease of cultivation and the existence of well-developed genetic and biochemical tools for several diverse haloarchaeal species make haloarchaea a model group for the study of archaeal biology. The unique physiological properties of these organisms also make them good candidates for novel enzyme discovery for biotechnological applications. Seven genomes were sequenced to ∼20×coverage and assembled to an average of 50 contigs (range 5 scaffolds - 168 contigs). Comparisons of protein-coding gene compliments revealed large-scale differences in COG functional group enrichment between these genera. Analysis of genes encoding machinery for DNA metabolism reveals genera-specific expansions of the general transcription factor TATA binding protein as well as a history of extensive duplication and horizontal transfer of the proliferating cell nuclear antigen. Insights gained from this study emphasize the importance of haloarchaea for investigation of archaeal biology. PMID:22848480

A protocol for isolating insect mitochondrial genomes: a case study of NUMT in Melipona flavolineata (Hymenoptera: Apidae).

PubMed

Françoso, Elaine; Gomes, Fernando; Arias, Maria Cristina

2016-07-01

Nuclear mitochondrial DNA insertions (NUMTs) are mitochondrial DNA sequences that have been transferred into the nucleus and are recognized by the presence of indels and stop codons. Although NUMTs have been identified in a diverse range of species, their discovery was frequently accidental. Here, our initial goal was to develop and standardize a simple method for isolating NUMTs from the nuclear genome of a single bee. Subsequently, we tested our new protocol by determining whether the indels and stop codons of the cytochrome c oxidase subunit I (COI) sequence of Melipona flavolineata are of nuclear origin. The new protocol successfully demonstrated the presence of a COI NUMT. In addition to NUMT investigations, the protocol described here will also be very useful for studying mitochondrial mutations related to diseases and for sequencing complete mitochondrial genomes with high read coverage by Next-Generation technology.

Novel methods to optimize genotypic imputation for low-coverage, next-generation sequence data in crop plants

USDA-ARS?s Scientific Manuscript database

Next-generation sequencing technology such as genotyping-by-sequencing (GBS) made low-cost, but often low-coverage, whole-genome sequencing widely available. Extensive inbreeding in crop plants provides an untapped, high quality source of phased haplotypes for imputing missing genotypes. We introduc...

Modeling genome coverage in single-cell sequencing

PubMed Central

Daley, Timothy; Smith, Andrew D.

2014-01-01

Motivation: Single-cell DNA sequencing is necessary for examining genetic variation at the cellular level, which remains hidden in bulk sequencing experiments. But because they begin with such small amounts of starting material, the amount of information that is obtained from single-cell sequencing experiment is highly sensitive to the choice of protocol employed and variability in library preparation. In particular, the fraction of the genome represented in single-cell sequencing libraries exhibits extreme variability due to quantitative biases in amplification and loss of genetic material. Results: We propose a method to predict the genome coverage of a deep sequencing experiment using information from an initial shallow sequencing experiment mapped to a reference genome. The observed coverage statistics are used in a non-parametric empirical Bayes Poisson model to estimate the gain in coverage from deeper sequencing. This approach allows researchers to know statistical features of deep sequencing experiments without actually sequencing deeply, providing a basis for optimizing and comparing single-cell sequencing protocols or screening libraries. Availability and implementation: The method is available as part of the preseq software package. Source code is available at http://smithlabresearch.org/preseq. Contact: andrewds@usc.edu Supplementary information: Supplementary material is available at Bioinformatics online. PMID:25107873

Analysis of the distal gut bacterial community by 454-pyrosequencing in captive giraffes (Giraffa camelopardalis).

PubMed

AlZahal, Ousama; Valdes, Eduardo V; McBride, Brian W

2016-01-01

The objective of this study was to characterize the structure of the fecal bacterial community of five giraffes (Giraffa camelopardalis) at Disney's Animal Kingdom, FL. Fecal genomic DNA was extracted and variable regions 1-3 of the 16S rRNA gene was PCR-amplified and then sequenced. The MOTHUR software-program was used for sequence processing, diversity analysis, and classification. A total of 181,689 non-chimeric bacterial sequences were obtained, and average number of sequences per sample was 36,338 -± 8,818. Sequences were assigned to 8,284 operational taxonomic units (OTU) with 95% of genetic similarity, which included 2,942 singletons (36%). Number of OTUs per sample was 2,554 ± 264. Samples were normalized and alpha (intra-sample) diversity indices; Chao1, Inverse Simpson, Shannon, and coverage were estimated as 3,712 ± 430, 116 -± 70, 6.1 ± 0.4, and 96 ± 1%, respectively. Thirteen phyla were detected and Firmicutes, Bacteroidetes, and Spirochaetes were the most dominant phyla (more than 2% of total sequences), and constituted 92% of the classified sequences, 66% of total sequences, and 43% of total OTUs. Our computation predicted that three OTUs were likely to be present in at least three of the five samples at greater than 1% dominance rate. These OTUs were Treponema, an unidentified OTU belonging to the order Bacteroidales, and Ruminococcus. This report was the first to characterize the bacterial community of the distal gut in giraffes utilizing fecal samples, and it demonstrated that the distal gut of giraffes is likely a potential reservoir for a number of undocumented species of bacteria. © 2015 Wiley Periodicals, Inc.

Estimating the Population Mutation Rate from a de novo Assembled Bactrian Camel Genome and Cross-Species Comparison with Dromedary ESTs

PubMed Central

2014-01-01

The Bactrian camel (Camelus bactrianus) and the dromedary (Camelus dromedarius) are among the last species that have been domesticated around 3000–6000 years ago. During domestication, strong artificial (anthropogenic) selection has shaped the livestock, creating a huge amount of phenotypes and breeds. Hence, domestic animals represent a unique resource to understand the genetic basis of phenotypic variation and adaptation. Similar to its late domestication history, the Bactrian camel is also among the last livestock animals to have its genome sequenced and deciphered. As no genomic data have been available until recently, we generated a de novo assembly by shotgun sequencing of a single male Bactrian camel. We obtained 1.6 Gb genomic sequences, which correspond to more than half of the Bactrian camel’s genome. The aim of this study was to identify heterozygous single-nucleotide polymorphisms (SNPs) and to estimate population parameters and nucleotide diversity based on an individual camel. With an average 6.6-fold coverage, we detected over 116 000 heterozygous SNPs and recorded a genome-wide nucleotide diversity similar to that of other domesticated ungulates. More than 20 000 (85%) dromedary expressed sequence tags successfully aligned to our genomic draft. Our results provide a template for future association studies targeting economically relevant traits and to identify changes underlying the process of camel domestication and environmental adaptation. PMID:23454912

GenoCore: A simple and fast algorithm for core subset selection from large genotype datasets.

PubMed

Jeong, Seongmun; Kim, Jae-Yoon; Jeong, Soon-Chun; Kang, Sung-Taeg; Moon, Jung-Kyung; Kim, Namshin

2017-01-01

Selecting core subsets from plant genotype datasets is important for enhancing cost-effectiveness and to shorten the time required for analyses of genome-wide association studies (GWAS), and genomics-assisted breeding of crop species, etc. Recently, a large number of genetic markers (>100,000 single nucleotide polymorphisms) have been identified from high-density single nucleotide polymorphism (SNP) arrays and next-generation sequencing (NGS) data. However, there is no software available for picking out the efficient and consistent core subset from such a huge dataset. It is necessary to develop software that can extract genetically important samples in a population with coherence. We here present a new program, GenoCore, which can find quickly and efficiently the core subset representing the entire population. We introduce simple measures of coverage and diversity scores, which reflect genotype errors and genetic variations, and can help to select a sample rapidly and accurately for crop genotype dataset. Comparison of our method to other core collection software using example datasets are performed to validate the performance according to genetic distance, diversity, coverage, required system resources, and the number of selected samples. GenoCore selects the smallest, most consistent, and most representative core collection from all samples, using less memory with more efficient scores, and shows greater genetic coverage compared to the other software tested. GenoCore was written in R language, and can be accessed online with an example dataset and test results at https://github.com/lovemun/Genocore.

msgbsR: An R package for analysing methylation-sensitive restriction enzyme sequencing data.

PubMed

Mayne, Benjamin T; Leemaqz, Shalem Y; Buckberry, Sam; Rodriguez Lopez, Carlos M; Roberts, Claire T; Bianco-Miotto, Tina; Breen, James

2018-02-01

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( https://bioconductor.org/packages/release/bioc/html/msgbsR.html ).

BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction.

PubMed

Townsley, Brad T; Covington, Michael F; Ichihashi, Yasunori; Zumstein, Kristina; Sinha, Neelima R

2015-01-01

Next Generation Sequencing (NGS) is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq) has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing the terminal breathing of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq) reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE) libraries and can easily extend to full transcript coverage shotgun (SHO) type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

A multiple-alignment based primer design algorithm for genetically highly variable DNA targets

PubMed Central

2013-01-01

Background Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design. Results Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations. Conclusions PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next-generation sequencing, and other experimental protocols targeting highly variable DNA samples. PMID:23965160

GFam: a platform for automatic annotation of gene families.

PubMed

Sasidharan, Rajkumar; Nepusz, Tamás; Swarbreck, David; Huala, Eva; Paccanaro, Alberto

2012-10-01

We have developed GFam, a platform for automatic annotation of gene/protein families. GFam provides a framework for genome initiatives and model organism resources to build domain-based families, derive meaningful functional labels and offers a seamless approach to propagate functional annotation across periodic genome updates. GFam is a hybrid approach that uses a greedy algorithm to chain component domains from InterPro annotation provided by its 12 member resources followed by a sequence-based connected component analysis of un-annotated sequence regions to derive consensus domain architecture for each sequence and subsequently generate families based on common architectures. Our integrated approach increases sequence coverage by 7.2 percentage points and residue coverage by 14.6 percentage points higher than the coverage relative to the best single-constituent database within InterPro for the proteome of Arabidopsis. The true power of GFam lies in maximizing annotation provided by the different InterPro data sources that offer resource-specific coverage for different regions of a sequence. GFam's capability to capture higher sequence and residue coverage can be useful for genome annotation, comparative genomics and functional studies. GFam is a general-purpose software and can be used for any collection of protein sequences. The software is open source and can be obtained from http://www.paccanarolab.org/software/gfam/.

Complete mitochondrial genome of the versicoloured emerald hummingbird Amazilia versicolor, a polymorphic species.

PubMed

Prosdocimi, Francisco; Souto, Helena Magarinos; Ruschi, Piero Angeli; Furtado, Carolina; Jennings, W Bryan

2016-09-01

The genome of the versicoloured emerald hummingbird (Amazilia versicolor) was partially sequenced in one-sixth of an Illumina HiSeq lane. The mitochondrial genome was assembled using MIRA and MITObim software, yielding a circular molecule of 16,861 bp in length and deposited in GenBank under the accession number KF624601. The mitogenome contained 13 protein-coding genes, 22 transfer tRNAs, 2 ribosomal RNAs and 1 non-coding control region. The molecule was assembled using 21,927 sequencing reads of 100 bp each, resulting in ∼130 × coverage of uniformly distributed reads along the genome. This is the forth mitochondrial genome described for this highly diverse family of birds and may benefit further phylogenetic, phylogeographic, population genetic and species delimitation studies of hummingbirds.

Microeukaryote Community Patterns along an O2/H2S Gradient in a Supersulfidic Anoxic Fjord (Framvaren, Norway)†

PubMed Central

Behnke, Anke; Bunge, John; Barger, Kathryn; Breiner, Hans-Werner; Alla, Victoria; Stoeck, Thorsten

2006-01-01

To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O2/H2S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O2/H2S gradient. PMID:16672511

The need for high-quality whole-genome sequence databases in microbial forensics.

PubMed

Sjödin, Andreas; Broman, Tina; Melefors, Öjar; Andersson, Gunnar; Rasmusson, Birgitta; Knutsson, Rickard; Forsman, Mats

2013-09-01

Microbial forensics is an important part of a strengthened capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. The goal of a microbial forensic investigation is to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon--that is, the pathogen. The recent development of next-generation sequencing (NGS) technologies has greatly increased the resolution that can be achieved in microbial forensic analyses. It is now possible to identify, quickly and in an unbiased manner, previously undetectable genome differences between closely related isolates. This development is particularly relevant for the most deadly bacterial diseases that are caused by bacterial lineages with extremely low levels of genetic diversity. Whole-genome analysis of pathogens is envisaged to be increasingly essential for this purpose. In a microbial forensic context, whole-genome sequence analysis is the ultimate method for strain comparisons as it is informative during identification, characterization, and attribution--all 3 major stages of the investigation--and at all levels of microbial strain identity resolution (ie, it resolves the full spectrum from family to isolate). Given these capabilities, one bottleneck in microbial forensics investigations is the availability of high-quality reference databases of bacterial whole-genome sequences. To be of high quality, databases need to be curated and accurate in terms of sequences, metadata, and genetic diversity coverage. The development of whole-genome sequence databases will be instrumental in successfully tracing pathogens in the future.

Rate of de novo mutations and the importance of father's age to disease risk.

PubMed

Kong, Augustine; Frigge, Michael L; Masson, Gisli; Besenbacher, Soren; Sulem, Patrick; Magnusson, Gisli; Gudjonsson, Sigurjon A; Sigurdsson, Asgeir; Jonasdottir, Aslaug; Jonasdottir, Adalbjorg; Wong, Wendy S W; Sigurdsson, Gunnar; Walters, G Bragi; Steinberg, Stacy; Helgason, Hannes; Thorleifsson, Gudmar; Gudbjartsson, Daniel F; Helgason, Agnar; Magnusson, Olafur Th; Thorsteinsdottir, Unnur; Stefansson, Kari

2012-08-23

Mutations generate sequence diversity and provide a substrate for selection. The rate of de novo mutations is therefore of major importance to evolution. Here we conduct a study of genome-wide mutation rates by sequencing the entire genomes of 78 Icelandic parent-offspring trios at high coverage. We show that in our samples, with an average father's age of 29.7, the average de novo mutation rate is 1.20 × 10(-8) per nucleotide per generation. Most notably, the diversity in mutation rate of single nucleotide polymorphisms is dominated by the age of the father at conception of the child. The effect is an increase of about two mutations per year. An exponential model estimates paternal mutations doubling every 16.5 years. After accounting for random Poisson variation, father's age is estimated to explain nearly all of the remaining variation in the de novo mutation counts. These observations shed light on the importance of the father's age on the risk of diseases such as schizophrenia and autism.

Extending CATH: increasing coverage of the protein structure universe and linking structure with function

PubMed Central

Cuff, Alison L.; Sillitoe, Ian; Lewis, Tony; Clegg, Andrew B.; Rentzsch, Robert; Furnham, Nicholas; Pellegrini-Calace, Marialuisa; Jones, David; Thornton, Janet; Orengo, Christine A.

2011-01-01

CATH version 3.3 (class, architecture, topology, homology) contains 128 688 domains, 2386 homologous superfamilies and 1233 fold groups, and reflects a major focus on classifying structural genomics (SG) structures and transmembrane proteins, both of which are likely to add structural novelty to the database and therefore increase the coverage of protein fold space within CATH. For CATH version 3.4 we have significantly improved the presentation of sequence information and associated functional information for CATH superfamilies. The CATH superfamily pages now reflect both the functional and structural diversity within the superfamily and include structural alignments of close and distant relatives within the superfamily, annotated with functional information and details of conserved residues. A significantly more efficient search function for CATH has been established by implementing the search server Solr (http://lucene.apache.org/solr/). The CATH v3.4 webpages have been built using the Catalyst web framework. PMID:21097779

Cultivating a Professional Ethic in Covering Marginalized Populations: Learning about the Poor through Service-Learning

ERIC Educational Resources Information Center

Motley, Phillip; Sturgill, Amanda

2014-01-01

As communications students learn to tell stories, the curriculum should teach them to cover diverse groups accurately. Scholars have studied coverage of diversity in gender, nationality, ethnicity, and race. One area that has seen less attention is economic diversity, in particular, coverage of the poor. This paper examines how service-learning…

From chromatogram to analyte to metabolite. How to pick horses for courses from the massive web resources for mass spectral plant metabolomics

PubMed Central

Perez de Souza, Leonardo; Naake, Thomas; Tohge, Takayuki; Fernie, Alisdair R

2017-01-01

Abstract The grand challenge currently facing metabolomics is the expansion of the coverage of the metabolome from a minor percentage of the metabolic complement of the cell toward the level of coverage afforded by other post-genomic technologies such as transcriptomics and proteomics. In plants, this problem is exacerbated by the sheer diversity of chemicals that constitute the metabolome, with the number of metabolites in the plant kingdom generally considered to be in excess of 200 000. In this review, we focus on web resources that can be exploited in order to improve analyte and ultimately metabolite identification and quantification. There is a wide range of available software that not only aids in this but also in the related area of peak alignment; however, for the uninitiated, choosing which program to use is a daunting task. For this reason, we provide an overview of the pros and cons of the software as well as comments regarding the level of programing skills required to effectively exploit their basic functions. In addition, the torrent of available genome and transcriptome sequences that followed the advent of next-generation sequencing has opened up further valuable resources for metabolite identification. All things considered, we posit that only via a continued communal sharing of information such as that deposited in the databases described within the article are we likely to be able to make significant headway toward improving our coverage of the plant metabolome. PMID:28520864

An Enumerative Combinatorics Model for Fragmentation Patterns in RNA Sequencing Provides Insights into Nonuniformity of the Expected Fragment Starting-Point and Coverage Profile.

PubMed

Prakash, Celine; Haeseler, Arndt Von

2017-03-01

RNA sequencing (RNA-seq) has emerged as the method of choice for measuring the expression of RNAs in a given cell population. In most RNA-seq technologies, sequencing the full length of RNA molecules requires fragmentation into smaller pieces. Unfortunately, the issue of nonuniform sequencing coverage across a genomic feature has been a concern in RNA-seq and is attributed to biases for certain fragments in RNA-seq library preparation and sequencing. To investigate the expected coverage obtained from fragmentation, we develop a simple fragmentation model that is independent of bias from the experimental method and is not specific to the transcript sequence. Essentially, we enumerate all configurations for maximal placement of a given fragment length, F, on transcript length, T, to represent every possible fragmentation pattern, from which we compute the expected coverage profile across a transcript. We extend this model to incorporate general empirical attributes such as read length, fragment length distribution, and number of molecules of the transcript. We further introduce the fragment starting-point, fragment coverage, and read coverage profiles. We find that the expected profiles are not uniform and that factors such as fragment length to transcript length ratio, read length to fragment length ratio, fragment length distribution, and number of molecules influence the variability of coverage across a transcript. Finally, we explore a potential application of the model where, with simulations, we show that it is possible to correctly estimate the transcript copy number for any transcript in the RNA-seq experiment.

An Enumerative Combinatorics Model for Fragmentation Patterns in RNA Sequencing Provides Insights into Nonuniformity of the Expected Fragment Starting-Point and Coverage Profile

PubMed Central

Haeseler, Arndt Von

2017-01-01

Abstract RNA sequencing (RNA-seq) has emerged as the method of choice for measuring the expression of RNAs in a given cell population. In most RNA-seq technologies, sequencing the full length of RNA molecules requires fragmentation into smaller pieces. Unfortunately, the issue of nonuniform sequencing coverage across a genomic feature has been a concern in RNA-seq and is attributed to biases for certain fragments in RNA-seq library preparation and sequencing. To investigate the expected coverage obtained from fragmentation, we develop a simple fragmentation model that is independent of bias from the experimental method and is not specific to the transcript sequence. Essentially, we enumerate all configurations for maximal placement of a given fragment length, F, on transcript length, T, to represent every possible fragmentation pattern, from which we compute the expected coverage profile across a transcript. We extend this model to incorporate general empirical attributes such as read length, fragment length distribution, and number of molecules of the transcript. We further introduce the fragment starting-point, fragment coverage, and read coverage profiles. We find that the expected profiles are not uniform and that factors such as fragment length to transcript length ratio, read length to fragment length ratio, fragment length distribution, and number of molecules influence the variability of coverage across a transcript. Finally, we explore a potential application of the model where, with simulations, we show that it is possible to correctly estimate the transcript copy number for any transcript in the RNA-seq experiment. PMID:27661099

Metagenomic study of bacterial microbiota in persistent endodontic infections using Next-generation sequencing.

PubMed

Sánchez-Sanhueza, G; Bello-Toledo, H; González-Rocha, G; Gonçalves, A T; Valenzuela, V; Gallardo-Escárate, C

2018-05-22

To determine the bacterial microbiota in root canals associated with persistent apical periodontitis and their relationship with the clinical characteristics of patients using next-generation sequencing (NGS). Bacterial samples from root canals associated with teeth having persistent apical periodontitis were taken from 24 patients undergoing root canal retreatment. Bacterial DNA was extracted, and V3-V4 variable regions of the 16S rRNA gene were amplified. The amplification was deep sequenced by Illumina technology to establish the metagenetic relationships among the bacterial species identified. The composition and diversity of microbial communities in the root canal and their relationships with clinical features were analysed. Parametric and nonparametric tests were used to analyse differences between patient characteristics and microbial data. A total of 86 different operational taxonomic units (OTUs) were identified and Good's nonparametric coverage estimator method indicated that 99.9 ± 0.00001% diversity was recovered per sample. The largest number of bacteria belonged to the phylum Proteobacteria. According to the medical history from the American Society of Anesthesiologists (ASA) Classification System, ASA II-III had higher richness estimates and distinct phylogenetic relationships compared to ASA I individuals (P < 0.05). Periapical index (PAI) score 5 was associated with increased microbiota diversity in comparison to PAI score 4, and this index was reduced in symptomatic patients. Based on the findings of this study, it is possible to suggest a close relationship between several clinical features and greater microbiota diversity with persistent endodontic infections. This work provides a better understanding on how microbial communities interact with their host and vice versa. © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.

SVM-Prot 2016: A Web-Server for Machine Learning Prediction of Protein Functional Families from Sequence Irrespective of Similarity.

PubMed

Li, Ying Hong; Xu, Jing Yu; Tao, Lin; Li, Xiao Feng; Li, Shuang; Zeng, Xian; Chen, Shang Ying; Zhang, Peng; Qin, Chu; Zhang, Cheng; Chen, Zhe; Zhu, Feng; Chen, Yu Zong

2016-01-01

Knowledge of protein function is important for biological, medical and therapeutic studies, but many proteins are still unknown in function. There is a need for more improved functional prediction methods. Our SVM-Prot web-server employed a machine learning method for predicting protein functional families from protein sequences irrespective of similarity, which complemented those similarity-based and other methods in predicting diverse classes of proteins including the distantly-related proteins and homologous proteins of different functions. Since its publication in 2003, we made major improvements to SVM-Prot with (1) expanded coverage from 54 to 192 functional families, (2) more diverse protein descriptors protein representation, (3) improved predictive performances due to the use of more enriched training datasets and more variety of protein descriptors, (4) newly integrated BLAST analysis option for assessing proteins in the SVM-Prot predicted functional families that were similar in sequence to a query protein, and (5) newly added batch submission option for supporting the classification of multiple proteins. Moreover, 2 more machine learning approaches, K nearest neighbor and probabilistic neural networks, were added for facilitating collective assessment of protein functions by multiple methods. SVM-Prot can be accessed at http://bidd2.nus.edu.sg/cgi-bin/svmprot/svmprot.cgi.

HLA Diversity in the 1000 Genomes Dataset

PubMed Central

Gourraud, Pierre-Antoine; Khankhanian, Pouya; Cereb, Nezih; Yang, Soo Young; Feolo, Michael; Maiers, Martin; D. Rioux, John; Hauser, Stephen; Oksenberg, Jorge

2014-01-01

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation by sequencing at a level that should allow the genome-wide detection of most variants with frequencies as low as 1%. However, in the major histocompatibility complex (MHC), only the top 10 most frequent haplotypes are in the 1% frequency range whereas thousands of haplotypes are present at lower frequencies. Given the limitation of both the coverage and the read length of the sequences generated by the 1000 Genomes Project, the highly variable positions that define HLA alleles may be difficult to identify. We used classical Sanger sequencing techniques to type the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 genes in the available 1000 Genomes samples and combined the results with the 103,310 variants in the MHC region genotyped by the 1000 Genomes Project. Using pairwise identity-by-descent distances between individuals and principal component analysis, we established the relationship between ancestry and genetic diversity in the MHC region. As expected, both the MHC variants and the HLA phenotype can identify the major ancestry lineage, informed mainly by the most frequent HLA haplotypes. To some extent, regions of the genome with similar genetic or similar recombination rate have similar properties. An MHC-centric analysis underlines departures between the ancestral background of the MHC and the genome-wide picture. Our analysis of linkage disequilibrium (LD) decay in these samples suggests that overestimation of pairwise LD occurs due to a limited sampling of the MHC diversity. This collection of HLA-specific MHC variants, available on the dbMHC portal, is a valuable resource for future analyses of the role of MHC in population and disease studies. PMID:24988075

HLA diversity in the 1000 genomes dataset.

PubMed

Gourraud, Pierre-Antoine; Khankhanian, Pouya; Cereb, Nezih; Yang, Soo Young; Feolo, Michael; Maiers, Martin; Rioux, John D; Hauser, Stephen; Oksenberg, Jorge

2014-01-01

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation by sequencing at a level that should allow the genome-wide detection of most variants with frequencies as low as 1%. However, in the major histocompatibility complex (MHC), only the top 10 most frequent haplotypes are in the 1% frequency range whereas thousands of haplotypes are present at lower frequencies. Given the limitation of both the coverage and the read length of the sequences generated by the 1000 Genomes Project, the highly variable positions that define HLA alleles may be difficult to identify. We used classical Sanger sequencing techniques to type the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 genes in the available 1000 Genomes samples and combined the results with the 103,310 variants in the MHC region genotyped by the 1000 Genomes Project. Using pairwise identity-by-descent distances between individuals and principal component analysis, we established the relationship between ancestry and genetic diversity in the MHC region. As expected, both the MHC variants and the HLA phenotype can identify the major ancestry lineage, informed mainly by the most frequent HLA haplotypes. To some extent, regions of the genome with similar genetic or similar recombination rate have similar properties. An MHC-centric analysis underlines departures between the ancestral background of the MHC and the genome-wide picture. Our analysis of linkage disequilibrium (LD) decay in these samples suggests that overestimation of pairwise LD occurs due to a limited sampling of the MHC diversity. This collection of HLA-specific MHC variants, available on the dbMHC portal, is a valuable resource for future analyses of the role of MHC in population and disease studies.

A High-Coverage Yersinia pestis Genome from a Sixth-Century Justinianic Plague Victim.

PubMed

Feldman, Michal; Harbeck, Michaela; Keller, Marcel; Spyrou, Maria A; Rott, Andreas; Trautmann, Bernd; Scholz, Holger C; Päffgen, Bernd; Peters, Joris; McCormick, Michael; Bos, Kirsten; Herbig, Alexander; Krause, Johannes

2016-11-01

The Justinianic Plague, which started in the sixth century and lasted to the mid eighth century, is thought to be the first of three historically documented plague pandemics causing massive casualties. Historical accounts and molecular data suggest the bacterium Yersinia pestis as its etiological agent. Here we present a new high-coverage (17.9-fold) Y. pestis genome obtained from a sixth-century skeleton recovered from a southern German burial site close to Munich. The reconstructed genome enabled the detection of 30 unique substitutions as well as structural differences that have not been previously described. We report indels affecting a lacl family transcription regulator gene as well as nonsynonymous substitutions in the nrdE, fadJ, and pcp genes, that have been suggested as plague virulence determinants or have been shown to be upregulated in different models of plague infection. In addition, we identify 19 false positive substitutions in a previously published lower-coverage Y. pestis genome from another archaeological site of the same time period and geographical region that is otherwise genetically identical to the high-coverage genome sequence reported here, suggesting low-genetic diversity of the plague during the sixth century in rural southern Germany. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

An Integrated Tool to Study MHC Region: Accurate SNV Detection and HLA Genes Typing in Human MHC Region Using Targeted High-Throughput Sequencing

PubMed Central

Liu, Xiao; Xu, Yinyin; Liang, Dequan; Gao, Peng; Sun, Yepeng; Gifford, Benjamin; D’Ascenzo, Mark; Liu, Xiaomin; Tellier, Laurent C. A. M.; Yang, Fang; Tong, Xin; Chen, Dan; Zheng, Jing; Li, Weiyang; Richmond, Todd; Xu, Xun; Wang, Jun; Li, Yingrui

2013-01-01

The major histocompatibility complex (MHC) is one of the most variable and gene-dense regions of the human genome. Most studies of the MHC, and associated regions, focus on minor variants and HLA typing, many of which have been demonstrated to be associated with human disease susceptibility and metabolic pathways. However, the detection of variants in the MHC region, and diagnostic HLA typing, still lacks a coherent, standardized, cost effective and high coverage protocol of clinical quality and reliability. In this paper, we presented such a method for the accurate detection of minor variants and HLA types in the human MHC region, using high-throughput, high-coverage sequencing of target regions. A probe set was designed to template upon the 8 annotated human MHC haplotypes, and to encompass the 5 megabases (Mb) of the extended MHC region. We deployed our probes upon three, genetically diverse human samples for probe set evaluation, and sequencing data show that ∼97% of the MHC region, and over 99% of the genes in MHC region, are covered with sufficient depth and good evenness. 98% of genotypes called by this capture sequencing prove consistent with established HapMap genotypes. We have concurrently developed a one-step pipeline for calling any HLA type referenced in the IMGT/HLA database from this target capture sequencing data, which shows over 96% typing accuracy when deployed at 4 digital resolution. This cost-effective and highly accurate approach for variant detection and HLA typing in the MHC region may lend further insight into immune-mediated diseases studies, and may find clinical utility in transplantation medicine research. This one-step pipeline is released for general evaluation and use by the scientific community. PMID:23894464

Global assessment of genomic variation in cattle by genome resequencing and high-throughput genotyping

PubMed Central

2011-01-01

Background Integration of genomic variation with phenotypic information is an effective approach for uncovering genotype-phenotype associations. This requires an accurate identification of the different types of variation in individual genomes. Results We report the integration of the whole genome sequence of a single Holstein Friesian bull with data from single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) array technologies to determine a comprehensive spectrum of genomic variation. The performance of resequencing SNP detection was assessed by combining SNPs that were identified to be either in identity by descent (IBD) or in copy number variation (CNV) with results from SNP array genotyping. Coding insertions and deletions (indels) were found to be enriched for size in multiples of 3 and were located near the N- and C-termini of proteins. For larger indels, a combination of split-read and read-pair approaches proved to be complementary in finding different signatures. CNVs were identified on the basis of the depth of sequenced reads, and by using SNP and CGH arrays. Conclusions Our results provide high resolution mapping of diverse classes of genomic variation in an individual bovine genome and demonstrate that structural variation surpasses sequence variation as the main component of genomic variability. Better accuracy of SNP detection was achieved with little loss of sensitivity when algorithms that implemented mapping quality were used. IBD regions were found to be instrumental for calculating resequencing SNP accuracy, while SNP detection within CNVs tended to be less reliable. CNV discovery was affected dramatically by platform resolution and coverage biases. The combined data for this study showed that at a moderate level of sequencing coverage, an ensemble of platforms and tools can be applied together to maximize the accurate detection of sequence and structural variants. PMID:22082336

Biocrusts role on nitrogen cycle and microbial communities from underlying soils in drylands

NASA Astrophysics Data System (ADS)

Anguita-Maeso, Manuel; Miralles*, Isabel; van Wesemael, Bas; Lázaro, Roberto; Ortega, Raúl; Garcia-Salcedo, José Antonio; Soriano**, Miguel

2017-04-01

Biocrusts are distributed in arid areas widely covering most of the soil surface and playing an essential role in the functioning of nitrogen cycle. The absence of biocrust coverage might affect the soil nitrogen content and the quantity and diversity of microbial communities in underlying biocrust soils. To analyse this mater, we have collected three underlying soils biocrusts samples dominated by the lichen Diploschistes diacapsis and Squamarina lentigera from Tabernas desert (southeast of Spain) at two extremes of its spatial distribution range: one with a high percentage of biocrust coverage and other with a huge degradation and low percentage of biocrust coverage in order to determine differences on the total nitrogen content and microbial communities from these underlying soils. DNA from these samples was isolated though a commercial kit and it was used as template for metagenomic analysis. We accomplished a sequencing of the amplicons V4-V5 of the 16S rRNA gene with Next-Generation Sequencing (NGS) Illumina MiSeq platform and a relative quantity of bacteria (rRNA 16S) and fungi (ITS1-5.8S) were conducted by quantitative qPCR. Total nitrogen was measured by the Kjeldahl method. Statistical analyses were based on ANOVAs, heatmap and Generalized Linear Models (GLM). The results showed 1.89E+09 bacteria per gram of soil in the high biocrust coverage position while 6.98E+08 microorganisms per gram of soil were found in the less favourable position according to the lower percentage of biocrust coverage. Similarly, 1.19E+12 was the amount of fungi per gram of soil located in the favourable position with higher biocrust coverage and 7.62E+11 was found in the unfavourable position. Furthermore, the soil under high percentage of biocrust coverage showed the greatest total nitrogen content (1.1 g kg-1) whereas the soil sampled under depressed percentage of biocrust coverage displayed the fewest quantity of total nitrogen content (0.9 g kg-1). Metagenomic and statistical analysis exhibited different bacteria communities according to underlying soils with unlike percentage of biocrust coverage. Opitutus and Adhaeribacter predominated in soil under high biocrust coverage percentage whereas Chelatococcus was found as prevalent bacteria community in soils under low biocrust coverage percentage. Our data illustrate that the percentage of biocrust coverage influence the total nitrogen content in underlying biocrust soils and also affects the amount and the variety of bacteria communities in these underlying soils. (*) Financial support by Marie Curie Intra-European Fellowship (FP7-577 PEOPLE-2013-IEF, Proposal n° 623393) and (**) by the Ministerio de Economía y Competitividad (MINECO) cofinanced with FEDER funds (project CGL2015-71709-R) is acknowledged.

Utilization of sequence on relatives to improve analysis of individuals' low-coverage NGS data

USDA-ARS?s Scientific Manuscript database

Low-coverage sequence data is expected to have low call rates under the prevailing paradigm that genotypes are first “called” from sequence data of each individual independently and subsequent analyses (including determination of haplotypes) are dependent on those called genotypes. However, provide...

A comparison of sequencing platforms and bioinformatics pipelines for compositional analysis of the gut microbiome.

PubMed

Allali, Imane; Arnold, Jason W; Roach, Jeffrey; Cadenas, Maria Belen; Butz, Natasha; Hassan, Hosni M; Koci, Matthew; Ballou, Anne; Mendoza, Mary; Ali, Rizwana; Azcarate-Peril, M Andrea

2017-09-13

Advancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines. Chicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150-199 for GS FLX+ and bases 90-99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME. The three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.

High-resolution community profiling of arbuscular mycorrhizal fungi.

PubMed

Schlaeppi, Klaus; Bender, S Franz; Mascher, Fabio; Russo, Giancarlo; Patrignani, Andrea; Camenzind, Tessa; Hempel, Stefan; Rillig, Matthias C; van der Heijden, Marcel G A

2016-11-01

Community analyses of arbuscular mycorrhizal fungi (AMF) using ribosomal small subunit (SSU) or internal transcribed spacer (ITS) DNA sequences often suffer from low resolution or coverage. We developed a novel sequencing based approach for a highly resolving and specific profiling of AMF communities. We took advantage of previously established AMF-specific PCR primers that amplify a c. 1.5-kb long fragment covering parts of SSU, ITS and parts of the large ribosomal subunit (LSU), and we sequenced the resulting amplicons with single molecule real-time (SMRT) sequencing. The method was applicable to soil and root samples, detected all major AMF families and successfully discriminated closely related AMF species, which would not be discernible using SSU sequences. In inoculation tests we could trace the introduced AMF inoculum at the molecular level. One of the introduced strains almost replaced the local strain(s), revealing that AMF inoculation can have a profound impact on the native community. The methodology presented offers researchers a powerful new tool for AMF community analysis because it unifies improved specificity and enhanced resolution, whereas the drawback of medium sequencing throughput appears of lesser importance for low-diversity groups such as AMF. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Evolutionary dynamics of retrotransposons assessed by high-throughput sequencing in wild relatives of wheat.

PubMed

Senerchia, Natacha; Wicker, Thomas; Felber, François; Parisod, Christian

2013-01-01

Transposable elements (TEs) represent a major fraction of plant genomes and drive their evolution. An improved understanding of genome evolution requires the dynamics of a large number of TE families to be considered. We put forward an approach bypassing the required step of a complete reference genome to assess the evolutionary trajectories of high copy number TE families from genome snapshot with high-throughput sequencing. Low coverage sequencing of the complex genomes of Aegilops cylindrica and Ae. geniculata using 454 identified more than 70% of the sequences as known TEs, mainly long terminal repeat (LTR) retrotransposons. Comparing the abundance of reads as well as patterns of sequence diversity and divergence within and among genomes assessed the dynamics of 44 major LTR retrotransposon families of the 165 identified. In particular, molecular population genetics on individual TE copies distinguished recently active from quiescent families and highlighted different evolutionary trajectories of retrotransposons among related species. This work presents a suite of tools suitable for current sequencing data, allowing to address the genome-wide evolutionary dynamics of TEs at the family level and advancing our understanding of the evolution of nonmodel genomes.

Sequencing degraded DNA from non-destructively sampled museum specimens for RAD-tagging and low-coverage shotgun phylogenetics.

PubMed

Tin, Mandy Man-Ying; Economo, Evan Philip; Mikheyev, Alexander Sergeyevich

2014-01-01

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37 ± 0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

Lessons learned from the initial sequencing of the pig genome: comparative analysis of an 8 Mb region of pig chromosome 17

PubMed Central

Hart, Elizabeth A; Caccamo, Mario; Harrow, Jennifer L; Humphray, Sean J; Gilbert, James GR; Trevanion, Steve; Hubbard, Tim; Rogers, Jane; Rothschild, Max F

2007-01-01

Background We describe here the sequencing, annotation and comparative analysis of an 8 Mb region of pig chromosome 17, which provides a useful test region to assess coverage and quality for the pig genome sequencing project. We report our findings comparing the annotation of draft sequence assembled at different depths of coverage. Results Within this region we annotated 71 loci, of which 53 are orthologous to human known coding genes. When compared to the syntenic regions in human (20q13.13-q13.33) and mouse (chromosome 2, 167.5 Mb-178.3 Mb), this region was found to be highly conserved with respect to gene order. The most notable difference between the three species is the presence of a large expansion of zinc finger coding genes and pseudogenes on mouse chromosome 2 between Edn3 and Phactr3 that is absent from pig and human. All of our annotation has been made publicly available in the Vertebrate Genome Annotation browser, VEGA. We assessed the impact of coverage on sequence assembly across this region and found, as expected, that increased sequence depth resulted in fewer, longer contigs. One-third of our annotated loci could not be fully re-aligned back to the low coverage version of the sequence, principally because the transcripts are fragmented over several contigs. Conclusion We have demonstrated the considerable advantages of sequencing at increased read depths and discuss the implications that lower coverage sequence may have on subsequent comparative and functional studies, particularly those involving complex loci such as GNAS. PMID:17705864

Streptococcus pneumoniae Supragenome Hybridization Arrays for Profiling of Genetic Content and Gene Expression.

PubMed

Kadam, Anagha; Janto, Benjamin; Eutsey, Rory; Earl, Joshua P; Powell, Evan; Dahlgren, Margaret E; Hu, Fen Z; Ehrlich, Garth D; Hiller, N Luisa

2015-02-02

There is extensive genomic diversity among Streptococcus pneumoniae isolates. Approximately half of the comprehensive set of genes in the species (the supragenome or pangenome) is present in all the isolates (core set), and the remaining is unevenly distributed among strains (distributed set). The Streptococcus pneumoniae Supragenome Hybridization (SpSGH) array provides coverage for an extensive set of genes and polymorphisms encountered within this species, capturing this genomic diversity. Further, the capture is quantitative. In this manner, the SpSGH array allows for both genomic and transcriptomic analyses of diverse S. pneumoniae isolates on a single platform. In this unit, we present the SpSGH array, and describe in detail its design and implementation for both genomic and transcriptomic analyses. The methodology can be applied to construction and modification of SpSGH array platforms, as well to other bacterial species as long as multiple whole-genome sequences are available that collectively capture the vast majority of the species supragenome. Copyright © 2015 John Wiley & Sons, Inc.

A recent bottleneck of Y chromosome diversity coincides with a global change in culture.

PubMed

Karmin, Monika; Saag, Lauri; Vicente, Mário; Wilson Sayres, Melissa A; Järve, Mari; Talas, Ulvi Gerst; Rootsi, Siiri; Ilumäe, Anne-Mai; Mägi, Reedik; Mitt, Mario; Pagani, Luca; Puurand, Tarmo; Faltyskova, Zuzana; Clemente, Florian; Cardona, Alexia; Metspalu, Ene; Sahakyan, Hovhannes; Yunusbayev, Bayazit; Hudjashov, Georgi; DeGiorgio, Michael; Loogväli, Eva-Liis; Eichstaedt, Christina; Eelmets, Mikk; Chaubey, Gyaneshwer; Tambets, Kristiina; Litvinov, Sergei; Mormina, Maru; Xue, Yali; Ayub, Qasim; Zoraqi, Grigor; Korneliussen, Thorfinn Sand; Akhatova, Farida; Lachance, Joseph; Tishkoff, Sarah; Momynaliev, Kuvat; Ricaut, François-Xavier; Kusuma, Pradiptajati; Razafindrazaka, Harilanto; Pierron, Denis; Cox, Murray P; Sultana, Gazi Nurun Nahar; Willerslev, Rane; Muller, Craig; Westaway, Michael; Lambert, David; Skaro, Vedrana; Kovačevic, Lejla; Turdikulova, Shahlo; Dalimova, Dilbar; Khusainova, Rita; Trofimova, Natalya; Akhmetova, Vita; Khidiyatova, Irina; Lichman, Daria V; Isakova, Jainagul; Pocheshkhova, Elvira; Sabitov, Zhaxylyk; Barashkov, Nikolay A; Nymadawa, Pagbajabyn; Mihailov, Evelin; Seng, Joseph Wee Tien; Evseeva, Irina; Migliano, Andrea Bamberg; Abdullah, Syafiq; Andriadze, George; Primorac, Dragan; Atramentova, Lubov; Utevska, Olga; Yepiskoposyan, Levon; Marjanovic, Damir; Kushniarevich, Alena; Behar, Doron M; Gilissen, Christian; Vissers, Lisenka; Veltman, Joris A; Balanovska, Elena; Derenko, Miroslava; Malyarchuk, Boris; Metspalu, Andres; Fedorova, Sardana; Eriksson, Anders; Manica, Andrea; Mendez, Fernando L; Karafet, Tatiana M; Veeramah, Krishna R; Bradman, Neil; Hammer, Michael F; Osipova, Ludmila P; Balanovsky, Oleg; Khusnutdinova, Elza K; Johnsen, Knut; Remm, Maido; Thomas, Mark G; Tyler-Smith, Chris; Underhill, Peter A; Willerslev, Eske; Nielsen, Rasmus; Metspalu, Mait; Villems, Richard; Kivisild, Toomas

2015-04-01

It is commonly thought that human genetic diversity in non-African populations was shaped primarily by an out-of-Africa dispersal 50-100 thousand yr ago (kya). Here, we present a study of 456 geographically diverse high-coverage Y chromosome sequences, including 299 newly reported samples. Applying ancient DNA calibration, we date the Y-chromosomal most recent common ancestor (MRCA) in Africa at 254 (95% CI 192-307) kya and detect a cluster of major non-African founder haplogroups in a narrow time interval at 47-52 kya, consistent with a rapid initial colonization model of Eurasia and Oceania after the out-of-Africa bottleneck. In contrast to demographic reconstructions based on mtDNA, we infer a second strong bottleneck in Y-chromosome lineages dating to the last 10 ky. We hypothesize that this bottleneck is caused by cultural changes affecting variance of reproductive success among males. © 2015 Karmin et al.; Published by Cold Spring Harbor Laboratory Press.

Diversity and population-genetic properties of copy number variations and multicopy genes in cattle

PubMed Central

Bickhart, Derek M.; Xu, Lingyang; Hutchison, Jana L.; Cole, John B.; Null, Daniel J.; Schroeder, Steven G.; Song, Jiuzhou; Garcia, Jose Fernando; Sonstegard, Tad S.; Van Tassell, Curtis P.; Schnabel, Robert D.; Taylor, Jeremy F.; Lewin, Harris A.; Liu, George E.

2016-01-01

The diversity and population genetics of copy number variation (CNV) in domesticated animals are not well understood. In this study, we analysed 75 genomes of major taurine and indicine cattle breeds (including Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, and Romagnola), sequenced to 11-fold coverage to identify 1,853 non-redundant CNV regions. Supported by high validation rates in array comparative genomic hybridization (CGH) and qPCR experiments, these CNV regions accounted for 3.1% (87.5 Mb) of the cattle reference genome, representing a significant increase over previous estimates of the area of the genome that is copy number variable (∼2%). Further population genetics and evolutionary genomics analyses based on these CNVs revealed the population structures of the cattle taurine and indicine breeds and uncovered potential diversely selected CNVs near important functional genes, including AOX1, ASZ1, GAT, GLYAT, and KRTAP9-1. Additionally, 121 CNV gene regions were found to be either breed specific or differentially variable across breeds, such as RICTOR in dairy breeds and PNPLA3 in beef breeds. In contrast, clusters of the PRP and PAG genes were found to be duplicated in all sequenced animals, suggesting that subfunctionalization, neofunctionalization, or overdominance play roles in diversifying those fertility-related genes. These CNV results provide a new glimpse into the diverse selection histories of cattle breeds and a basis for correlating structural variation with complex traits in the future. PMID:27085184

A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.

PubMed

Quail, Michael A; Smith, Miriam; Coupland, Paul; Otto, Thomas D; Harris, Simon R; Connor, Thomas R; Bertoni, Anna; Swerdlow, Harold P; Gu, Yong

2012-07-24

Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent's PGM, Pacific Biosciences' RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.

Multi-Objective Optimization of Spacecraft Trajectories for Small-Body Coverage Missions

NASA Technical Reports Server (NTRS)

Hinckley, David, Jr.; Englander, Jacob; Hitt, Darren

2017-01-01

Visual coverage of surface elements of a small-body object requires multiple images to be taken that meet many requirements on their viewing angles, illumination angles, times of day, and combinations thereof. Designing trajectories capable of maximizing total possible coverage may not be useful since the image target sequence and the feasibility of said sequence given the rotation-rate limitations of the spacecraft are not taken into account. This work presents a means of optimizing, in a multi-objective manner, surface target sequences that account for such limitations.

Targeted Sequencing of Venom Genes from Cone Snail Genomes Improves Understanding of Conotoxin Molecular Evolution

PubMed Central

Mahardika, Gusti N

2018-01-01

Abstract To expand our capacity to discover venom sequences from the genomes of venomous organisms, we applied targeted sequencing techniques to selectively recover venom gene superfamilies and nontoxin loci from the genomes of 32 cone snail species (family, Conidae), a diverse group of marine gastropods that capture their prey using a cocktail of neurotoxic peptides (conotoxins). We were able to successfully recover conotoxin gene superfamilies across all species with high confidence (> 100× coverage) and used these data to provide new insights into conotoxin evolution. First, we found that conotoxin gene superfamilies are composed of one to six exons and are typically short in length (mean = ∼85 bp). Second, we expanded our understanding of the following genetic features of conotoxin evolution: 1) positive selection, where exons coding the mature toxin region were often three times more divergent than their adjacent noncoding regions, 2) expression regulation, with comparisons to transcriptome data showing that cone snails only express a fraction of the genes available in their genome (24–63%), and 3) extensive gene turnover, where Conidae species varied from 120 to 859 conotoxin gene copies. Finally, using comparative phylogenetic methods, we found that while diet specificity did not predict patterns of conotoxin evolution, dietary breadth was positively correlated with total conotoxin gene diversity. Overall, the targeted sequencing technique demonstrated here has the potential to radically increase the pace at which venom gene families are sequenced and studied, reshaping our ability to understand the impact of genetic changes on ecologically relevant phenotypes and subsequent diversification. PMID:29514313

Soup to Tree: The Phylogeny of Beetles Inferred by Mitochondrial Metagenomics of a Bornean Rainforest Sample

PubMed Central

Crampton-Platt, Alex; Timmermans, Martijn J.T.N.; Gimmel, Matthew L.; Kutty, Sujatha Narayanan; Cockerill, Timothy D.; Vun Khen, Chey; Vogler, Alfried P.

2015-01-01

In spite of the growth of molecular ecology, systematics and next-generation sequencing, the discovery and analysis of diversity is not currently integrated with building the tree-of-life. Tropical arthropod ecologists are well placed to accelerate this process if all specimens obtained through mass-trapping, many of which will be new species, could be incorporated routinely into phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures through mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from approximately 500 beetle specimens, originating from a single rainforest canopy fogging sample from Borneo, was pooled and shotgun sequenced, followed by de novo assembly of complete and partial mitogenomes for 175 species. The phylogenetic tree obtained from this local sample was highly similar to that from existing mitogenomes selected for global coverage of major lineages of Coleoptera. When all sequences were combined only minor topological changes were induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, while the ecological sample expanded the tip-level representation of several lineages. Robust trees generated from ecological samples now enable an evolutionary framework for ecology. Meanwhile, the inclusion of uncharacterized samples in the tree-of-life rapidly expands taxon and biogeographic representation of lineages without morphological identification. Mitogenomes from shotgun sequencing of unsorted environmental samples and their associated metadata, placed robustly into the phylogenetic tree, constitute novel DNA “superbarcodes” for testing hypotheses regarding global patterns of diversity. PMID:25957318

snpAD: An ancient DNA genotype caller.

PubMed

Prüfer, Kay

2018-06-21

The study of ancient genomes can elucidate the evolutionary past. However, analyses are complicated by base-modifications in ancient DNA molecules that result in errors in DNA sequences. These errors are particularly common near the ends of sequences and pose a challenge for genotype calling. I describe an iterative method that estimates genotype frequencies and errors along sequences to allow for accurate genotype calling from ancient sequences. The implementation of this method, called snpAD, performs well on high-coverage ancient data, as shown by simulations and by subsampling the data of a high-coverage Neandertal genome. Although estimates for low-coverage genomes are less accurate, I am able to derive approximate estimates of heterozygosity from several low-coverage Neandertals. These estimates show that low heterozygosity, compared to modern humans, was common among Neandertals. The C ++ code of snpAD is freely available at http://bioinf.eva.mpg.de/snpAD/. Supplementary data are available at Bioinformatics online.

Using Tablet for visual exploration of second-generation sequencing data.

PubMed

Milne, Iain; Stephen, Gordon; Bayer, Micha; Cock, Peter J A; Pritchard, Leighton; Cardle, Linda; Shaw, Paul D; Marshall, David

2013-03-01

The advent of second-generation sequencing (2GS) has provided a range of significant new challenges for the visualization of sequence assemblies. These include the large volume of data being generated, short-read lengths and different data types and data formats associated with the diversity of new sequencing technologies. This article illustrates how Tablet-a high-performance graphical viewer for visualization of 2GS assemblies and read mappings-plays an important role in the analysis of these data. We present Tablet, and through a selection of use cases, demonstrate its value in quality assurance and scientific discovery, through features such as whole-reference coverage overviews, variant highlighting, paired-end read mark-up, GFF3-based feature tracks and protein translations. We discuss the computing and visualization techniques utilized to provide a rich and responsive graphical environment that enables users to view a range of file formats with ease. Tablet installers can be freely downloaded from http://bioinf.hutton.ac.uk/tablet in 32 or 64-bit versions for Windows, OS X, Linux or Solaris. For further details on the Tablet, contact tablet@hutton.ac.uk.

Biogeography and Metabolic Potential of Soil Microbial Communities Across Local Environmental Gradients Illuminated Through Metagenomics

NASA Astrophysics Data System (ADS)

Sharrar, A.; Diamond, S.; Butterfield, C.; Starr, E.; Thomas, B. C.; Banfield, J. F.

2016-12-01

Soils are extremely heterogeneous and diverse microbial habitats. The distribution of microbes and their metabolic functions in soil is important for carbon and nitrogen cycling and overall ecosystem functioning. Little is known about biogeographical patterns of microbes in soil and how they relate to different environmental gradients. To address this topic, we have obtained metagenomes from 86 soil samples taken at the Eel River Critical Zone Observatory and a nearby meadow in the northern California Angelo Coast Range Reserve over a period of two years. These samples span a variety of environmental parameters, including depth, relief, time since last rainfall, and proximity to root carbon inputs. Phylogenetic diversity and community overlap between these samples was analyzed using ribosomal protein sequence identity and scaffold coverage. Independent of genomic bins, de-replicated ribosomal protein sequences were used to identify thousands of unique organisms between datasets. Despite environmental differences, a subset of these organisms were found in every sample. The dominant phyla in this cosmopolitan subset were Proteobacteria, Actinobacteria, Gemmatimonadetes, Verrucomicrobia, Acidobacteria, and Rokubacteria. Archaea and Nitrospirae were particularly prevalent at depths below 30cm. Patterns of ribosomal protein scaffold coverage across samples was used to compare the similarity of the microbial communities. We found that location was the greatest predictor of community similarity, followed by depth. In a subset of 60 samples taken in the same meadow, sampling plot was a greater predictor of community similarity than time since last rainfall in the same year. In addition, genome-independent analyses revealed differences in the metabolic potential for functions involved in nitrogen and carbon metabolism across environmental gradients. This study will further our understanding of soil biogeography and its relationship to overall biogeochemical cycling.

Genomic analyses inform on migration events during the peopling of Eurasia

NASA Astrophysics Data System (ADS)

Pagani, Luca; Lawson, Daniel John; Jagoda, Evelyn; Mörseburg, Alexander; Eriksson, Anders; Mitt, Mario; Clemente, Florian; Hudjashov, Georgi; Degiorgio, Michael; Saag, Lauri; Wall, Jeffrey D.; Cardona, Alexia; Mägi, Reedik; Sayres, Melissa A. Wilson; Kaewert, Sarah; Inchley, Charlotte; Scheib, Christiana L.; Järve, Mari; Karmin, Monika; Jacobs, Guy S.; Antao, Tiago; Iliescu, Florin Mircea; Kushniarevich, Alena; Ayub, Qasim; Tyler-Smith, Chris; Xue, Yali; Yunusbayev, Bayazit; Tambets, Kristiina; Mallick, Chandana Basu; Saag, Lehti; Pocheshkhova, Elvira; Andriadze, George; Muller, Craig; Westaway, Michael C.; Lambert, David M.; Zoraqi, Grigor; Turdikulova, Shahlo; Dalimova, Dilbar; Sabitov, Zhaxylyk; Sultana, Gazi Nurun Nahar; Lachance, Joseph; Tishkoff, Sarah; Momynaliev, Kuvat; Isakova, Jainagul; Damba, Larisa D.; Gubina, Marina; Nymadawa, Pagbajabyn; Evseeva, Irina; Atramentova, Lubov; Utevska, Olga; Ricaut, François-Xavier; Brucato, Nicolas; Sudoyo, Herawati; Letellier, Thierry; Cox, Murray P.; Barashkov, Nikolay A.; Škaro, Vedrana; Mulaha´, Lejla; Primorac, Dragan; Sahakyan, Hovhannes; Mormina, Maru; Eichstaedt, Christina A.; Lichman, Daria V.; Abdullah, Syafiq; Chaubey, Gyaneshwer; Wee, Joseph T. S.; Mihailov, Evelin; Karunas, Alexandra; Litvinov, Sergei; Khusainova, Rita; Ekomasova, Natalya; Akhmetova, Vita; Khidiyatova, Irina; Marjanović, Damir; Yepiskoposyan, Levon; Behar, Doron M.; Balanovska, Elena; Metspalu, Andres; Derenko, Miroslava; Malyarchuk, Boris; Voevoda, Mikhail; Fedorova, Sardana A.; Osipova, Ludmila P.; Lahr, Marta Mirazón; Gerbault, Pascale; Leavesley, Matthew; Migliano, Andrea Bamberg; Petraglia, Michael; Balanovsky, Oleg; Khusnutdinova, Elza K.; Metspalu, Ene; Thomas, Mark G.; Manica, Andrea; Nielsen, Rasmus; Villems, Richard; Willerslev, Eske; Kivisild, Toomas; Metspalu, Mait

2016-10-01

High-coverage whole-genome sequence studies have so far focused on a limited number of geographically restricted populations, or been targeted at specific diseases, such as cancer. Nevertheless, the availability of high-resolution genomic data has led to the development of new methodologies for inferring population history and refuelled the debate on the mutation rate in humans. Here we present the Estonian Biocentre Human Genome Diversity Panel (EGDP), a dataset of 483 high-coverage human genomes from 148 populations worldwide, including 379 new genomes from 125 populations, which we group into diversity and selection sets. We analyse this dataset to refine estimates of continent-wide patterns of heterozygosity, long- and short-distance gene flow, archaic admixture, and changes in effective population size through time as well as for signals of positive or balancing selection. We find a genetic signature in present-day Papuans that suggests that at least 2% of their genome originates from an early and largely extinct expansion of anatomically modern humans (AMHs) out of Africa. Together with evidence from the western Asian fossil record, and admixture between AMHs and Neanderthals predating the main Eurasian expansion, our results contribute to the mounting evidence for the presence of AMHs out of Africa earlier than 75,000 years ago.

Genomic analyses inform on migration events during the peopling of Eurasia.

PubMed

Pagani, Luca; Lawson, Daniel John; Jagoda, Evelyn; Mörseburg, Alexander; Eriksson, Anders; Mitt, Mario; Clemente, Florian; Hudjashov, Georgi; DeGiorgio, Michael; Saag, Lauri; Wall, Jeffrey D; Cardona, Alexia; Mägi, Reedik; Wilson Sayres, Melissa A; Kaewert, Sarah; Inchley, Charlotte; Scheib, Christiana L; Järve, Mari; Karmin, Monika; Jacobs, Guy S; Antao, Tiago; Iliescu, Florin Mircea; Kushniarevich, Alena; Ayub, Qasim; Tyler-Smith, Chris; Xue, Yali; Yunusbayev, Bayazit; Tambets, Kristiina; Mallick, Chandana Basu; Saag, Lehti; Pocheshkhova, Elvira; Andriadze, George; Muller, Craig; Westaway, Michael C; Lambert, David M; Zoraqi, Grigor; Turdikulova, Shahlo; Dalimova, Dilbar; Sabitov, Zhaxylyk; Sultana, Gazi Nurun Nahar; Lachance, Joseph; Tishkoff, Sarah; Momynaliev, Kuvat; Isakova, Jainagul; Damba, Larisa D; Gubina, Marina; Nymadawa, Pagbajabyn; Evseeva, Irina; Atramentova, Lubov; Utevska, Olga; Ricaut, François-Xavier; Brucato, Nicolas; Sudoyo, Herawati; Letellier, Thierry; Cox, Murray P; Barashkov, Nikolay A; Skaro, Vedrana; Mulahasanovic, Lejla; Primorac, Dragan; Sahakyan, Hovhannes; Mormina, Maru; Eichstaedt, Christina A; Lichman, Daria V; Abdullah, Syafiq; Chaubey, Gyaneshwer; Wee, Joseph T S; Mihailov, Evelin; Karunas, Alexandra; Litvinov, Sergei; Khusainova, Rita; Ekomasova, Natalya; Akhmetova, Vita; Khidiyatova, Irina; Marjanović, Damir; Yepiskoposyan, Levon; Behar, Doron M; Balanovska, Elena; Metspalu, Andres; Derenko, Miroslava; Malyarchuk, Boris; Voevoda, Mikhail; Fedorova, Sardana A; Osipova, Ludmila P; Lahr, Marta Mirazón; Gerbault, Pascale; Leavesley, Matthew; Migliano, Andrea Bamberg; Petraglia, Michael; Balanovsky, Oleg; Khusnutdinova, Elza K; Metspalu, Ene; Thomas, Mark G; Manica, Andrea; Nielsen, Rasmus; Villems, Richard; Willerslev, Eske; Kivisild, Toomas; Metspalu, Mait

2016-10-13

High-coverage whole-genome sequence studies have so far focused on a limited number of geographically restricted populations, or been targeted at specific diseases, such as cancer. Nevertheless, the availability of high-resolution genomic data has led to the development of new methodologies for inferring population history and refuelled the debate on the mutation rate in humans. Here we present the Estonian Biocentre Human Genome Diversity Panel (EGDP), a dataset of 483 high-coverage human genomes from 148 populations worldwide, including 379 new genomes from 125 populations, which we group into diversity and selection sets. We analyse this dataset to refine estimates of continent-wide patterns of heterozygosity, long- and short-distance gene flow, archaic admixture, and changes in effective population size through time as well as for signals of positive or balancing selection. We find a genetic signature in present-day Papuans that suggests that at least 2% of their genome originates from an early and largely extinct expansion of anatomically modern humans (AMHs) out of Africa. Together with evidence from the western Asian fossil record, and admixture between AMHs and Neanderthals predating the main Eurasian expansion, our results contribute to the mounting evidence for the presence of AMHs out of Africa earlier than 75,000 years ago.

Indexcov: fast coverage quality control for whole-genome sequencing.

PubMed

Pedersen, Brent S; Collins, Ryan L; Talkowski, Michael E; Quinlan, Aaron R

2017-11-01

The BAM and CRAM formats provide a supplementary linear index that facilitates rapid access to sequence alignments in arbitrary genomic regions. Comparing consecutive entries in a BAM or CRAM index allows one to infer the number of alignment records per genomic region for use as an effective proxy of sequence depth in each genomic region. Based on these properties, we have developed indexcov, an efficient estimator of whole-genome sequencing coverage to rapidly identify samples with aberrant coverage profiles, reveal large-scale chromosomal anomalies, recognize potential batch effects, and infer the sex of a sample. Indexcov is available at https://github.com/brentp/goleft under the MIT license. © The Authors 2017. Published by Oxford University Press.

Assessing the Relationship of Ancient and Modern Populations

PubMed Central

Schraiber, Joshua G.

2018-01-01

Genetic material sequenced from ancient samples is revolutionizing our understanding of the recent evolutionary past. However, ancient DNA is often degraded, resulting in low coverage, error-prone sequencing. Several solutions exist to this problem, ranging from simple approach, such as selecting a read at random for each site, to more complicated approaches involving genotype likelihoods. In this work, we present a novel method for assessing the relationship of an ancient sample with a modern population, while accounting for sequencing error and postmortem damage by analyzing raw reads from multiple ancient individuals simultaneously. We show that, when analyzing SNP data, it is better to sequence more ancient samples to low coverage: two samples sequenced to 0.5× coverage provide better resolution than a single sample sequenced to 2× coverage. We also examined the power to detect whether an ancient sample is directly ancestral to a modern population, finding that, with even a few high coverage individuals, even ancient samples that are very slightly diverged from the modern population can be detected with ease. When we applied our approach to European samples, we found that no ancient samples represent direct ancestors of modern Europeans. We also found that, as shown previously, the most ancient Europeans appear to have had the smallest effective population sizes, indicating a role for agriculture in modern population growth. PMID:29167200

From chromatogram to analyte to metabolite. How to pick horses for courses from the massive web resources for mass spectral plant metabolomics.

PubMed

Perez de Souza, Leonardo; Naake, Thomas; Tohge, Takayuki; Fernie, Alisdair R

2017-07-01

The grand challenge currently facing metabolomics is the expansion of the coverage of the metabolome from a minor percentage of the metabolic complement of the cell toward the level of coverage afforded by other post-genomic technologies such as transcriptomics and proteomics. In plants, this problem is exacerbated by the sheer diversity of chemicals that constitute the metabolome, with the number of metabolites in the plant kingdom generally considered to be in excess of 200 000. In this review, we focus on web resources that can be exploited in order to improve analyte and ultimately metabolite identification and quantification. There is a wide range of available software that not only aids in this but also in the related area of peak alignment; however, for the uninitiated, choosing which program to use is a daunting task. For this reason, we provide an overview of the pros and cons of the software as well as comments regarding the level of programing skills required to effectively exploit their basic functions. In addition, the torrent of available genome and transcriptome sequences that followed the advent of next-generation sequencing has opened up further valuable resources for metabolite identification. All things considered, we posit that only via a continued communal sharing of information such as that deposited in the databases described within the article are we likely to be able to make significant headway toward improving our coverage of the plant metabolome. © The Authors 2017. Published by Oxford University Press.

PolyA_DB 3 catalogs cleavage and polyadenylation sites identified by deep sequencing in multiple genomes

PubMed Central

Wang, Ruijia; Nambiar, Ram; Zheng, Dinghai

2018-01-01

Abstract PolyA_DB is a database cataloging cleavage and polyadenylation sites (PASs) in several genomes. Previous versions were based mainly on expressed sequence tags (ESTs), which had a limited amount and could lead to inaccurate PAS identification due to the presence of internal A-rich sequences in transcripts. Here, we present an updated version of the database based solely on deep sequencing data. First, PASs are mapped by the 3′ region extraction and deep sequencing (3′READS) method, ensuring unequivocal PAS identification. Second, a large volume of data based on diverse biological samples increases PAS coverage by 3.5-fold over the EST-based version and provides PAS usage information. Third, strand-specific RNA-seq data are used to extend annotated 3′ ends of genes to obtain more thorough annotations of alternative polyadenylation (APA) sites. Fourth, conservation information of PAS across mammals sheds light on significance of APA sites. The database (URL: http://www.polya-db.org/v3) currently holds PASs in human, mouse, rat and chicken, and has links to the UCSC genome browser for further visualization and for integration with other genomic data. PMID:29069441

Development of Genomic Microsatellite Markers in Carthamus tinctorius L. (Safflower) Using Next Generation Sequencing and Assessment of Their Cross-Species Transferability and Utility for Diversity Analysis

PubMed Central

Variath, Murali Tottekkad; Joshi, Gopal; Bali, Sapinder; Agarwal, Manu; Kumar, Amar; Jagannath, Arun; Goel, Shailendra

2015-01-01

Background Safflower (Carthamus tinctorius L.), an Asteraceae member, yields high quality edible oil rich in unsaturated fatty acids and is resilient to dry conditions. The crop holds tremendous potential for improvement through concerted molecular breeding programs due to the availability of significant genetic and phenotypic diversity. Genomic resources that could facilitate such breeding programs remain largely underdeveloped in the crop. The present study was initiated to develop a large set of novel microsatellite markers for safflower using next generation sequencing. Principal Findings Low throughput genome sequencing of safflower was performed using Illumina paired end technology providing ~3.5X coverage of the genome. Analysis of sequencing data allowed identification of 23,067 regions harboring perfect microsatellite loci. The safflower genome was found to be rich in dinucleotide repeats followed by tri-, tetra-, penta- and hexa-nucleotides. Primer pairs were designed for 5,716 novel microsatellite sequences with repeat length ≥ 20 bases and optimal flanking regions. A subset of 325 microsatellite loci was tested for amplification, of which 294 loci produced robust amplification. The validated primers were used for assessment of 23 safflower accessions belonging to diverse agro-climatic zones of the world leading to identification of 93 polymorphic primers (31.6%). The numbers of observed alleles at each locus ranged from two to four and mean polymorphism information content was found to be 0.3075. The polymorphic primers were tested for cross-species transferability on nine wild relatives of cultivated safflower. All primers except one showed amplification in at least two wild species while 25 primers amplified across all the nine species. The UPGMA dendrogram clustered C. tinctorius accessions and wild species separately into two major groups. The proposed progenitor species of safflower, C. oxyacantha and C. palaestinus were genetically closer to cultivated safflower and formed a distinct cluster. The cluster analysis also distinguished diploid and tetraploid wild species of safflower. Conclusion Next generation sequencing of safflower genome generated a large set of microsatellite markers. The novel markers developed in this study will add to the existing repertoire of markers and can be used for diversity analysis, synteny studies, construction of linkage maps and marker-assisted selection. PMID:26287743

Launch mission summary and sequence of events Telesat-F(anik-D1)/Delta-164

NASA Technical Reports Server (NTRS)

1982-01-01

The launch vehicle, spacecraft, and mission are summarized. Launch window information, vehicle telemetry coverage, real time data flow, telemetry coverage by station, selected trajectory information, and a brief sequence of flight events are included.

Almost 20 years of Neanderthal palaeogenetics: adaptation, admixture, diversity, demography and extinction

PubMed Central

Sánchez-Quinto, Federico; Lalueza-Fox, Carles

2015-01-01

Nearly two decades since the first retrieval of Neanderthal DNA, recent advances in next-generation sequencing technologies have allowed the generation of high-coverage genomes from two archaic hominins, a Neanderthal and a Denisovan, as well as a complete mitochondrial genome from remains which probably represent early members of the Neanderthal lineage. This genomic information, coupled with diversity exome data from several Neanderthal specimens is shedding new light on evolutionary processes such as the genetic basis of Neanderthal and modern human-specific adaptations—including morphological and behavioural traits—as well as the extent and nature of the admixture events between them. An emerging picture is that Neanderthals had a long-term small population size, lived in small and isolated groups and probably practised inbreeding at times. Deleterious genetic effects associated with these demographic factors could have played a role in their extinction. The analysis of DNA from further remains making use of new large-scale hybridization-capture-based methods as well as of new approaches to discriminate contaminant DNA sequences will provide genetic information in spatial and temporal scales that could help clarify the Neanderthal's—and our very own—evolutionary history. PMID:25487326

De novo protein sequencing by combining top-down and bottom-up tandem mass spectra.

PubMed

Liu, Xiaowen; Dekker, Lennard J M; Wu, Si; Vanduijn, Martijn M; Luider, Theo M; Tolić, Nikola; Kou, Qiang; Dvorkin, Mikhail; Alexandrova, Sonya; Vyatkina, Kira; Paša-Tolić, Ljiljana; Pevzner, Pavel A

2014-07-03

There are two approaches for de novo protein sequencing: Edman degradation and mass spectrometry (MS). Existing MS-based methods characterize a novel protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the protein. Because each tandem mass spectrum covers only a short peptide of the target protein, the key to high coverage protein sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resolution mass spectrometers have become accessible to many laboratories. These mass spectrometers are capable of analyzing molecules of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a protein. We propose an algorithm, TBNovo, for de novo protein sequencing by combining top-down and bottom-up MS. In TBNovo, a top-down tandem mass spectrum is utilized as a scaffold, and bottom-up tandem mass spectra are aligned to the scaffold to increase sequence coverage. Experiments on data sets of two proteins showed that TBNovo achieved high sequence coverage and high sequence accuracy.

Enhanced sequencing coverage with digital droplet multiple displacement amplification

PubMed Central

Sidore, Angus M.; Lan, Freeman; Lim, Shaun W.; Abate, Adam R.

2016-01-01

Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing. PMID:26704978

Advancing Genomics through the Global Invertebrate Genomics Alliance (GIGA)

PubMed Central

Voolstra, Christian R.; Wörheide, Gert; Lopez, Jose V.

2017-01-01

The Global Invertebrate Genomics Alliance (GIGA), a collaborative network of diverse scientists, marked its second anniversary with a workshop in Munich, Germany, where international attendees focused on discussing current progress, milestones and bioinformatics resources. The community determined the recruitment and training talented researchers as one of the most pressing future needs and identified opportunities for network funding. GIGA also promotes future research efforts to prioritize taxonomic diversity and create new synergies. Here, we announce the generation of a central and simple data repository portal with a wide coverage of available sequence data, via the compagen platform, in parallel with more focused and specialized organism databases to globally advance invertebrate genomics. Therefore this article serves the objectives of GIGA by disseminating current progress and future prospects in the science of invertebrate genomics with the aim of promotion and facilitation of interdisciplinary and international research. PMID:28603454

Advancing Genomics through the Global Invertebrate Genomics Alliance (GIGA).

PubMed

Voolstra, Christian R; Wörheide, Gert; Lopez, Jose V

2017-03-01

The Global Invertebrate Genomics Alliance (GIGA), a collaborative network of diverse scientists, marked its second anniversary with a workshop in Munich, Germany, where international attendees focused on discussing current progress, milestones and bioinformatics resources. The community determined the recruitment and training talented researchers as one of the most pressing future needs and identified opportunities for network funding. GIGA also promotes future research efforts to prioritize taxonomic diversity and create new synergies. Here, we announce the generation of a central and simple data repository portal with a wide coverage of available sequence data, via the compagen platform, in parallel with more focused and specialized organism databases to globally advance invertebrate genomics. Therefore this article serves the objectives of GIGA by disseminating current progress and future prospects in the science of invertebrate genomics with the aim of promotion and facilitation of interdisciplinary and international research.

Pre-Vaccination Nasopharyngeal Pneumococcal Carriage in a Nigerian Population: Epidemiology and Population Biology

PubMed Central

Adetifa, Ifedayo M. O.; Antonio, Martin; Okoromah, Christy A. N.; Ebruke, Chinelo; Inem, Victor; Nsekpong, David; Bojang, Abdoulie; Adegbola, Richard A.

2012-01-01

Background Introduction of pneumococcal vaccines in Nigeria is a priority as part of the Accelerated Vaccine Introduction Initiative (AVI) of the Global Alliance for Vaccines and Immunisation (GAVI). However, country data on the burden of pneumococcal disease (IPD) is limited and coverage by available conjugate vaccines is unknown. This study was carried out to describe the pre vaccination epidemiology and population biology of pneumococcal carriage in Nigeria. Methods This was a cross sectional survey. Nasopharyngeal swabs (NPS) were obtained from a population sample in 14 contiguous peri-urban Nigerian communities. Data on demographic characteristics and risk factor for carriage were obtained from all study participants. Pneumococci isolated from NPS were characterised by serotyping, antimicrobial susceptibility and Multi Locus Sequencing Typing (MLST). Results The prevalence of pneumococcal carriage was 52.5%. Carriage was higher in children compared to adults (67.4% vs. 26%), highest (≈90%) in infants aged <9 months and reduced significantly with increasing age (P<0.001). Serotypes 19F (18.6%) and 6A (14.4%) were most predominant. Potential vaccine coverage was 43.8%, 45.0% and 62% for PCV-7, PCV-10 and PCV-13 respectively. There were 16 novel alleles, 72 different sequence types (STs) from the isolates and 3 Sequence Types (280, 310 and 5543) were associated with isolates of more than one serotype indicative of serotype switching. Antimicrobial resistance was high for cotrimoxazole (93%) and tetracycline (84%), a third of isolates had intermediate resistance to penicillin. Young age was the only risk factor significantly associated with carriage. Conclusions Pneumococcal carriage and serotype diversity is highly prevalent in Nigeria especially in infants. Based on the coverage of serotypes in this study, PCV-13 is the obvious choice to reduce disease burden and prevalence of drug resistant pneumococci. However, its use will require careful monitoring. Our findings provide sound baseline data for impact assessment following vaccine introduction in Nigeria. PMID:22291984

Pre-vaccination nasopharyngeal pneumococcal carriage in a Nigerian population: epidemiology and population biology.

PubMed

Adetifa, Ifedayo M O; Antonio, Martin; Okoromah, Christy A N; Ebruke, Chinelo; Inem, Victor; Nsekpong, David; Bojang, Abdoulie; Adegbola, Richard A

2012-01-01

Introduction of pneumococcal vaccines in Nigeria is a priority as part of the Accelerated Vaccine Introduction Initiative (AVI) of the Global Alliance for Vaccines and Immunisation (GAVI). However, country data on the burden of pneumococcal disease (IPD) is limited and coverage by available conjugate vaccines is unknown. This study was carried out to describe the pre vaccination epidemiology and population biology of pneumococcal carriage in Nigeria. This was a cross sectional survey. Nasopharyngeal swabs (NPS) were obtained from a population sample in 14 contiguous peri-urban Nigerian communities. Data on demographic characteristics and risk factor for carriage were obtained from all study participants. Pneumococci isolated from NPS were characterised by serotyping, antimicrobial susceptibility and Multi Locus Sequencing Typing (MLST). The prevalence of pneumococcal carriage was 52.5%. Carriage was higher in children compared to adults (67.4% vs. 26%), highest (≈90%) in infants aged <9 months and reduced significantly with increasing age (P<0.001). Serotypes 19F (18.6%) and 6A (14.4%) were most predominant. Potential vaccine coverage was 43.8%, 45.0% and 62% for PCV-7, PCV-10 and PCV-13 respectively. There were 16 novel alleles, 72 different sequence types (STs) from the isolates and 3 Sequence Types (280, 310 and 5543) were associated with isolates of more than one serotype indicative of serotype switching. Antimicrobial resistance was high for cotrimoxazole (93%) and tetracycline (84%), a third of isolates had intermediate resistance to penicillin. Young age was the only risk factor significantly associated with carriage. Pneumococcal carriage and serotype diversity is highly prevalent in Nigeria especially in infants. Based on the coverage of serotypes in this study, PCV-13 is the obvious choice to reduce disease burden and prevalence of drug resistant pneumococci. However, its use will require careful monitoring. Our findings provide sound baseline data for impact assessment following vaccine introduction in Nigeria.

Estimating genotype error rates from high-coverage next-generation sequence data.

PubMed

Wall, Jeffrey D; Tang, Ling Fung; Zerbe, Brandon; Kvale, Mark N; Kwok, Pui-Yan; Schaefer, Catherine; Risch, Neil

2014-11-01

Exome and whole-genome sequencing studies are becoming increasingly common, but little is known about the accuracy of the genotype calls made by the commonly used platforms. Here we use replicate high-coverage sequencing of blood and saliva DNA samples from four European-American individuals to estimate lower bounds on the error rates of Complete Genomics and Illumina HiSeq whole-genome and whole-exome sequencing. Error rates for nonreference genotype calls range from 0.1% to 0.6%, depending on the platform and the depth of coverage. Additionally, we found (1) no difference in the error profiles or rates between blood and saliva samples; (2) Complete Genomics sequences had substantially higher error rates than Illumina sequences had; (3) error rates were higher (up to 6%) for rare or unique variants; (4) error rates generally declined with genotype quality (GQ) score, but in a nonlinear fashion for the Illumina data, likely due to loss of specificity of GQ scores greater than 60; and (5) error rates increased with increasing depth of coverage for the Illumina data. These findings, especially (3)-(5), suggest that caution should be taken in interpreting the results of next-generation sequencing-based association studies, and even more so in clinical application of this technology in the absence of validation by other more robust sequencing or genotyping methods. © 2014 Wall et al.; Published by Cold Spring Harbor Laboratory Press.

Electron-Transfer Ion/Ion Reactions of Doubly Protonated Peptides: Effect of Elevated Bath Gas Temperature

PubMed Central

Pitteri, Sharon J.; Chrisman, Paul A.; McLuckey, Scott A.

2005-01-01

In this study, the electron-transfer dissociation (ETD) behavior of cations derived from 27 different peptides (22 of which are tryptic peptides) has been studied in a 3D quadrupole ion trap mass spectrometer. Ion/ion reactions between peptide cations and nitrobenzene anions have been examined at both room temperature and in an elevated temperature bath gas environment to form ETD product ions. From the peptides studied, the ETD sequence coverage tends to be inversely related to peptide size. At room temperature, very high sequence coverage (~100%) was observed for small peptides (≤7 amino acids). For medium-sized peptides composed of 8–11 amino acids, the average sequence coverage was 46%. Larger peptides with 14 or more amino acids yielded an average sequence coverage of 23%. Elevated-temperature ETD provided increased sequence coverage over room-temperature experiments for the peptides of greater than 7 residues, giving an average of 67% for medium-sized peptides and 63% for larger peptides. Percent ETD, a measure of the extent of electron transfer, has also been calculated for the peptides and also shows an inverse relation with peptide size. Bath gas temperature does not have a consistent effect on percent ETD, however. For the tryptic peptides, fragmentation is localized at the ends of the peptides suggesting that the distribution of charge within the peptide may play an important role in determining fragmentation sites. A triply protonated peptide has also been studied and shows behavior similar to the doubly charged peptides. These preliminary results suggest that for a given charge state there is a maximum size for which high sequence coverage is obtained and that increasing the bath gas temperature can increase this maximum. PMID:16131079

Maintaining and Enhancing Diversity of Sampled Protein Conformations in Robotics-Inspired Methods.

PubMed

Abella, Jayvee R; Moll, Mark; Kavraki, Lydia E

2018-01-01

The ability to efficiently sample structurally diverse protein conformations allows one to gain a high-level view of a protein's energy landscape. Algorithms from robot motion planning have been used for conformational sampling, and several of these algorithms promote diversity by keeping track of "coverage" in conformational space based on the local sampling density. However, large proteins present special challenges. In particular, larger systems require running many concurrent instances of these algorithms, but these algorithms can quickly become memory intensive because they typically keep previously sampled conformations in memory to maintain coverage estimates. In addition, robotics-inspired algorithms depend on defining useful perturbation strategies for exploring the conformational space, which is a difficult task for large proteins because such systems are typically more constrained and exhibit complex motions. In this article, we introduce two methodologies for maintaining and enhancing diversity in robotics-inspired conformational sampling. The first method addresses algorithms based on coverage estimates and leverages the use of a low-dimensional projection to define a global coverage grid that maintains coverage across concurrent runs of sampling. The second method is an automatic definition of a perturbation strategy through readily available flexibility information derived from B-factors, secondary structure, and rigidity analysis. Our results show a significant increase in the diversity of the conformations sampled for proteins consisting of up to 500 residues when applied to a specific robotics-inspired algorithm for conformational sampling. The methodologies presented in this article may be vital components for the scalability of robotics-inspired approaches.

A Comparative Metagenome Survey of the Fecal Microbiota of a Breast- and a Plant-Fed Asian Elephant Reveals an Unexpectedly High Diversity of Glycoside Hydrolase Family Enzymes

PubMed Central

Ilmberger, Nele; Güllert, Simon; Dannenberg, Joana; Rabausch, Ulrich; Torres, Jeremy; Wemheuer, Bernd; Alawi, Malik; Poehlein, Anja; Chow, Jennifer; Turaev, Dimitrij; Rattei, Thomas; Schmeisser, Christel; Salomon, Jesper; Olsen, Peter B.; Daniel, Rolf; Grundhoff, Adam; Borchert, Martin S.; Streit, Wolfgang R.

2014-01-01

A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant) was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs) were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH) genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs), which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals. PMID:25208077

PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection

PubMed Central

O’Halloran, Damien M.

2016-01-01

Primer design represents a widely employed gambit in diverse molecular applications including PCR, sequencing, and probe hybridization. Variations of PCR, including primer walking, allele-specific PCR, and nested PCR provide specialized validation and detection protocols for molecular analyses that often require screening large numbers of DNA fragments. In these cases, automated sequence retrieval and processing become important features, and furthermore, a graphic that provides the user with a visual guide to the distribution of designed primers across targets is most helpful in quickly ascertaining primer coverage. To this end, I describe here, PrimerMapper, which provides a comprehensive graphical user interface that designs robust primers from any number of inputted sequences while providing the user with both, graphical maps of primer distribution for each inputted sequence, and also a global assembled map of all inputted sequences with designed primers. PrimerMapper also enables the visualization of graphical maps within a browser and allows the user to draw new primers directly onto the webpage. Other features of PrimerMapper include allele-specific design features for SNP genotyping, a remote BLAST window to NCBI databases, and remote sequence retrieval from GenBank and dbSNP. PrimerMapper is hosted at GitHub and freely available without restriction. PMID:26853558

Power structure in Chilean news media

PubMed Central

Bahamonde, Jorge; Bollen, Johan; Ferres, Leo; Poblete, Barbara

2018-01-01

Even democracies endowed with the most active free press struggle to maintain diversity of news coverage. Consolidation and market forces may cause only a few dominant players to control the news cycle. Editorial policies may be biased by corporate ownership relations, narrowing news coverage and focus. To an increasing degree this problem also applies to social media news distribution, since it is subject to the same socio-economic drivers. To study the effects of consolidation and ownership on news diversity, we model the diversity of Chilean coverage on the basis of ownership records and social media data. We create similarity networks of news outlets on the basis of their ownership and the topics they cover. We then examine the relationships between the topology of ownership networks and content similarity to characterize how ownership affects news coverage. A network analysis reveals that Chilean media is highly concentrated both in terms of ownership as well as in terms of topics covered. Our method can be used to determine which groups of outlets and ownership exert the greatest influence on news coverage. PMID:29874241

Power structure in Chilean news media.

PubMed

Bahamonde, Jorge; Bollen, Johan; Elejalde, Erick; Ferres, Leo; Poblete, Barbara

2018-01-01

Even democracies endowed with the most active free press struggle to maintain diversity of news coverage. Consolidation and market forces may cause only a few dominant players to control the news cycle. Editorial policies may be biased by corporate ownership relations, narrowing news coverage and focus. To an increasing degree this problem also applies to social media news distribution, since it is subject to the same socio-economic drivers. To study the effects of consolidation and ownership on news diversity, we model the diversity of Chilean coverage on the basis of ownership records and social media data. We create similarity networks of news outlets on the basis of their ownership and the topics they cover. We then examine the relationships between the topology of ownership networks and content similarity to characterize how ownership affects news coverage. A network analysis reveals that Chilean media is highly concentrated both in terms of ownership as well as in terms of topics covered. Our method can be used to determine which groups of outlets and ownership exert the greatest influence on news coverage.

The repetitive landscape of the chicken genome.

PubMed

Wicker, Thomas; Robertson, Jon S; Schulze, Stefan R; Feltus, F Alex; Magrini, Vincent; Morrison, Jason A; Mardis, Elaine R; Wilson, Richard K; Peterson, Daniel G; Paterson, Andrew H; Ivarie, Robert

2005-01-01

Cot-based cloning and sequencing (CBCS) is a powerful tool for isolating and characterizing the various repetitive components of any genome, combining the established principles of DNA reassociation kinetics with high-throughput sequencing. CBCS was used to generate sequence libraries representing the high, middle, and low-copy fractions of the chicken genome. Sequencing high-copy DNA of chicken to about 2.7 x coverage of its estimated sequence complexity led to the initial identification of several new repeat families, which were then used for a survey of the newly released first draft of the complete chicken genome. The analysis provided insight into the diversity and biology of known repeat structures such as CR1 and CNM, for which only limited sequence data had previously been available. Cot sequence data also resulted in the identification of four novel repeats (Birddawg, Hitchcock, Kronos, and Soprano), two new subfamilies of CR1 repeats, and many elements absent from the chicken genome assembly. Multiple autonomous elements were found for a novel Mariner-like transposon, Galluhop, in addition to nonautonomous deletion derivatives. Phylogenetic analysis of the high-copy repeats CR1, Galluhop, and Birddawg provided insight into two distinct genome dispersion strategies. This study also exemplifies the power of the CBCS method to create representative databases for the repetitive fractions of genomes for which only limited sequence data is available.

The repetitive landscape of the chicken genome

PubMed Central

Wicker, Thomas; Robertson, Jon S.; Schulze, Stefan R.; Feltus, F. Alex; Magrini, Vincent; Morrison, Jason A.; Mardis, Elaine R.; Wilson, Richard K.; Peterson, Daniel G.; Paterson, Andrew H.; Ivarie, Robert

2005-01-01

Cot-based cloning and sequencing (CBCS) is a powerful tool for isolating and characterizing the various repetitive components of any genome, combining the established principles of DNA reassociation kinetics with high-throughput sequencing. CBCS was used to generate sequence libraries representing the high, middle, and low-copy fractions of the chicken genome. Sequencing high-copy DNA of chicken to about 2.7× coverage of its estimated sequence complexity led to the initial identification of several new repeat families, which were then used for a survey of the newly released first draft of the complete chicken genome. The analysis provided insight into the diversity and biology of known repeat structures such as CR1 and CNM, for which only limited sequence data had previously been available. Cot sequence data also resulted in the identification of four novel repeats (Birddawg, Hitchcock, Kronos, and Soprano), two new subfamilies of CR1 repeats, and many elements absent from the chicken genome assembly. Multiple autonomous elements were found for a novel Mariner-like transposon, Galluhop, in addition to nonautonomous deletion derivatives. Phylogenetic analysis of the high-copy repeats CR1, Galluhop, and Birddawg provided insight into two distinct genome dispersion strategies. This study also exemplifies the power of the CBCS method to create representative databases for the repetitive fractions of genomes for which only limited sequence data is available. PMID:15256510

The microbiomes of blowflies and houseflies as bacterial transmission reservoirs.

PubMed

Junqueira, Ana Carolina M; Ratan, Aakrosh; Acerbi, Enzo; Drautz-Moses, Daniela I; Premkrishnan, Balakrishnan N V; Costea, Paul I; Linz, Bodo; Purbojati, Rikky W; Paulo, Daniel F; Gaultier, Nicolas E; Subramanian, Poorani; Hasan, Nur A; Colwell, Rita R; Bork, Peer; Azeredo-Espin, Ana Maria L; Bryant, Donald A; Schuster, Stephan C

2017-11-24

Blowflies and houseflies are mechanical vectors inhabiting synanthropic environments around the world. They feed and breed in fecal and decaying organic matter, but the microbiome they harbour and transport is largely uncharacterized. We sampled 116 individual houseflies and blowflies from varying habitats on three continents and subjected them to high-coverage, whole-genome shotgun sequencing. This allowed for genomic and metagenomic analyses of the host-associated microbiome at the species level. Both fly host species segregate based on principal coordinate analysis of their microbial communities, but they also show an overlapping core microbiome. Legs and wings displayed the largest microbial diversity and were shown to be an important route for microbial dispersion. The environmental sequencing approach presented here detected a stochastic distribution of human pathogens, such as Helicobacter pylori, thereby demonstrating the potential of flies as proxies for environmental and public health surveillance.

De novo gene mutations highlight patterns of genetic and neural complexity in schizophrenia

PubMed Central

Xu, Bin; Ionita-Laza, Iuliana; Roos, J. Louw; Boone, Braden; Woodrick, Scarlet; Sun, Yan; Levy, Shawn; Gogos, Joseph A.; Karayiorgou, Maria

2013-01-01

To evaluate evidence for de novo etiologies in schizophrenia, we sequenced at high coverage the exomes of families recruited from two populations with distinct demographic structure and history. We sequenced a total of 795 exomes from 231 parent-proband trios enriched for sporadic schizophrenia cases, as well as 34 unaffected trios. We observed in cases an excess of non-synonymous single nucleotide variants as well as a higher prevalence of gene-disruptive de novo mutations. We found four genes (LAMA2, DPYD, TRRAP and VPS39) affected by recurrent de novo events within or across the two populations, a finding unlikely to have occurred by chance. We show that de novo mutations affect genes with diverse functions and developmental profiles but we also find a substantial contribution of mutations in genes with higher expression in early fetal life. Our results help define the pattern of genomic and neural architecture of schizophrenia. PMID:23042115

Impervious Surfaces Alter Soil Bacterial Communities in Urban Areas: A Case Study in Beijing, China

PubMed Central

Hu, Yinhong; Dou, Xiaolin; Li, Juanyong; Li, Feng

2018-01-01

The rapid expansion of urbanization has caused land cover change, especially the increasing area of impervious surfaces. Such alterations have significant effects on the soil ecosystem by impeding the exchange of gasses, water, and materials between soil and the atmosphere. It is unclear whether impervious surfaces have any effects on soil bacterial diversity and community composition. In the present study, we conducted an investigation of bacterial communities across five typical land cover types, including impervious surfaces (concrete), permeable pavement (bricks with round holes), shrub coverage (Buxus megistophylla Levl.), lawns (Festuca elata Keng ex E. Alexeev), and roadside trees (Sophora japonica Linn.) in Beijing, to explore the response of bacteria to impervious surfaces. The soil bacterial communities were addressed by high-throughput sequencing of the bacterial 16S rRNA gene. We found that Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, and Firmicutes were the predominant phyla in urban soils. Soil from impervious surfaces presented a lower bacterial diversity, and differed greatly from other types of land cover. Soil bacterial diversity was predominantly affected by Zn, dissolved organic carbon (DOC), and soil moisture content (SMC). The composition of the bacterial community was similar under shrub coverage, roadside trees, and lawns, but different from beneath impervious surfaces and permeable pavement. Variance partitioning analysis showed that edaphic properties contributed to 12% of the bacterial community variation, heavy metal pollution explained 3.6% of the variation, and interaction between the two explained 33% of the variance. Together, our data indicate that impervious surfaces induced changes in bacterial community composition and decrease of bacterial diversity. Interactions between edaphic properties and heavy metals were here found to change the composition of the bacterial community and diversity across areas with different types of land cover, and soil properties play a more important role than heavy metals. PMID:29545776

Links between Soil Fungal Diversity and Plant and Soil Properties on the Loess Plateau

PubMed Central

Yang, Yang; Dou, Yanxing; Huang, Yimei; An, Shaoshan

2017-01-01

Previous studies have revealed inconsistent correlations between fungal diversity and plant/soil properties from local to global scales. Here, we investigated the internal relationships between soil fungal diversity and plant/soil properties on the Loess Plateau following vegetation restoration, using Illumina sequencing of the internal transcribed spacer 2 (ITS2) region for fungal identification. We found significant effects of land use types (Af, Artificial forest; Ns, Natural shrub; Ag, Artificial grassland; Ng, Natural grassland; Sc, slope cropland) on soil fungal communities composition, and the dominant phyla were Ascomycota, Basidiomycota, and Zygomycota, which transitioned from Basidiomycota-dominant to Ascomycota-dominant community due to vegetation restoration. The Chao1 richness, Shannon’s diversity and ACE indices were significantly influenced by land use types with the order of Ns > Af > Ng > Ag > Sc, and the total number of OTUs varied widely. In contrast, Good’s coverage and Simpson’s diversity indicated no significant difference among land use types (p > 0.05). Correlation analysis showed that plant and soil properties were closely related to fungal diversity regardless of land use types. In addition, soil organic carbon (SOC) and Hplant (plant richness, Shannon-Wiener index) were strong driving factors that explained fungal diversity. As revealed by the structural equation model (SEM) and generalized additive models (GAMs), fungal diversity was directly and indirectly affected by soil and plant properties, respectively, providing evidence for strong links between soil fungal diversity and plant and soil properties on the Loess Plateau. PMID:29163460

Links between Soil Fungal Diversity and Plant and Soil Properties on the Loess Plateau.

PubMed

Yang, Yang; Dou, Yanxing; Huang, Yimei; An, Shaoshan

2017-01-01

Previous studies have revealed inconsistent correlations between fungal diversity and plant/soil properties from local to global scales. Here, we investigated the internal relationships between soil fungal diversity and plant/soil properties on the Loess Plateau following vegetation restoration, using Illumina sequencing of the internal transcribed spacer 2 (ITS2) region for fungal identification. We found significant effects of land use types (Af, Artificial forest; Ns, Natural shrub; Ag, Artificial grassland; Ng, Natural grassland; Sc, slope cropland) on soil fungal communities composition, and the dominant phyla were Ascomycota, Basidiomycota , and Zygomycota , which transitioned from Basidiomycota -dominant to Ascomycota -dominant community due to vegetation restoration. The Chao1 richness, Shannon's diversity and ACE indices were significantly influenced by land use types with the order of Ns > Af > Ng > Ag > Sc, and the total number of OTUs varied widely. In contrast, Good's coverage and Simpson's diversity indicated no significant difference among land use types ( p > 0.05). Correlation analysis showed that plant and soil properties were closely related to fungal diversity regardless of land use types. In addition, soil organic carbon (SOC) and H plant (plant richness, Shannon-Wiener index) were strong driving factors that explained fungal diversity. As revealed by the structural equation model (SEM) and generalized additive models (GAMs), fungal diversity was directly and indirectly affected by soil and plant properties, respectively, providing evidence for strong links between soil fungal diversity and plant and soil properties on the Loess Plateau.

Phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi)sulfite reductases

PubMed Central

Müller, Albert Leopold; Kjeldsen, Kasper Urup; Rattei, Thomas; Pester, Michael; Loy, Alexander

2015-01-01

The energy metabolism of essential microbial guilds in the biogeochemical sulfur cycle is based on a DsrAB-type dissimilatory (bi)sulfite reductase that either catalyzes the reduction of sulfite to sulfide during anaerobic respiration of sulfate, sulfite and organosulfonates, or acts in reverse during sulfur oxidation. Common use of dsrAB as a functional marker showed that dsrAB richness in many environments is dominated by novel sequence variants and collectively represents an extensive, largely uncharted sequence assemblage. Here, we established a comprehensive, manually curated dsrAB/DsrAB database and used it to categorize the known dsrAB diversity, reanalyze the evolutionary history of dsrAB and evaluate the coverage of published dsrAB-targeted primers. Based on a DsrAB consensus phylogeny, we introduce an operational classification system for environmental dsrAB sequences that integrates established taxonomic groups with operational taxonomic units (OTUs) at multiple phylogenetic levels, ranging from DsrAB enzyme families that reflect reductive or oxidative DsrAB types of bacterial or archaeal origin, superclusters, uncultured family-level lineages to species-level OTUs. Environmental dsrAB sequences constituted at least 13 stable family-level lineages without any cultivated representatives, suggesting that major taxa of sulfite/sulfate-reducing microorganisms have not yet been identified. Three of these uncultured lineages occur mainly in marine environments, while specific habitat preferences are not evident for members of the other 10 uncultured lineages. In summary, our publically available dsrAB/DsrAB database, the phylogenetic framework, the multilevel classification system and a set of recommended primers provide a necessary foundation for large-scale dsrAB ecology studies with next-generation sequencing methods. PMID:25343514

Soup to Tree: The Phylogeny of Beetles Inferred by Mitochondrial Metagenomics of a Bornean Rainforest Sample.

PubMed

Crampton-Platt, Alex; Timmermans, Martijn J T N; Gimmel, Matthew L; Kutty, Sujatha Narayanan; Cockerill, Timothy D; Vun Khen, Chey; Vogler, Alfried P

2015-09-01

In spite of the growth of molecular ecology, systematics and next-generation sequencing, the discovery and analysis of diversity is not currently integrated with building the tree-of-life. Tropical arthropod ecologists are well placed to accelerate this process if all specimens obtained through mass-trapping, many of which will be new species, could be incorporated routinely into phylogeny reconstruction. Here we test a shotgun sequencing approach, whereby mitochondrial genomes are assembled from complex ecological mixtures through mitochondrial metagenomics, and demonstrate how the approach overcomes many of the taxonomic impediments to the study of biodiversity. DNA from approximately 500 beetle specimens, originating from a single rainforest canopy fogging sample from Borneo, was pooled and shotgun sequenced, followed by de novo assembly of complete and partial mitogenomes for 175 species. The phylogenetic tree obtained from this local sample was highly similar to that from existing mitogenomes selected for global coverage of major lineages of Coleoptera. When all sequences were combined only minor topological changes were induced against this reference set, indicating an increasingly stable estimate of coleopteran phylogeny, while the ecological sample expanded the tip-level representation of several lineages. Robust trees generated from ecological samples now enable an evolutionary framework for ecology. Meanwhile, the inclusion of uncharacterized samples in the tree-of-life rapidly expands taxon and biogeographic representation of lineages without morphological identification. Mitogenomes from shotgun sequencing of unsorted environmental samples and their associated metadata, placed robustly into the phylogenetic tree, constitute novel DNA "superbarcodes" for testing hypotheses regarding global patterns of diversity. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Hybrid-denovo: a de novo OTU-picking pipeline integrating single-end and paired-end 16S sequence tags.

PubMed

Chen, Xianfeng; Johnson, Stephen; Jeraldo, Patricio; Wang, Junwen; Chia, Nicholas; Kocher, Jean-Pierre A; Chen, Jun

2018-03-01

Illumina paired-end sequencing has been increasingly popular for 16S rRNA gene-based microbiota profiling. It provides higher phylogenetic resolution than single-end reads due to a longer read length. However, the reverse read (R2) often has significant low base quality, and a large proportion of R2s will be discarded after quality control, resulting in a mixture of paired-end and single-end reads. A typical 16S analysis pipeline usually processes either paired-end or single-end reads but not a mixture. Thus, the quantification accuracy and statistical power will be reduced due to the loss of a large amount of reads. As a result, rare taxa may not be detectable with the paired-end approach, or low taxonomic resolution will result in a single-end approach. To have both the higher phylogenetic resolution provided by paired-end reads and the higher sequence coverage by single-end reads, we propose a novel OTU-picking pipeline, hybrid-denovo, that can process a hybrid of single-end and paired-end reads. Using high-quality paired-end reads as a gold standard, we show that hybrid-denovo achieved the highest correlation with the gold standard and performed better than the approaches based on paired-end or single-end reads in terms of quantifying the microbial diversity and taxonomic abundances. By applying our method to a rheumatoid arthritis (RA) data set, we demonstrated that hybrid-denovo captured more microbial diversity and identified more RA-associated taxa than a paired-end or single-end approach. Hybrid-denovo utilizes both paired-end and single-end 16S sequencing reads and is recommended for 16S rRNA gene targeted paired-end sequencing data.

Haplotype estimation using sequencing reads.

PubMed

Delaneau, Olivier; Howie, Bryan; Cox, Anthony J; Zagury, Jean-François; Marchini, Jonathan

2013-10-03

High-throughput sequencing technologies produce short sequence reads that can contain phase information if they span two or more heterozygote genotypes. This information is not routinely used by current methods that infer haplotypes from genotype data. We have extended the SHAPEIT2 method to use phase-informative sequencing reads to improve phasing accuracy. Our model incorporates the read information in a probabilistic model through base quality scores within each read. The method is primarily designed for high-coverage sequence data or data sets that already have genotypes called. One important application is phasing of single samples sequenced at high coverage for use in medical sequencing and studies of rare diseases. Our method can also use existing panels of reference haplotypes. We tested the method by using a mother-father-child trio sequenced at high-coverage by Illumina together with the low-coverage sequence data from the 1000 Genomes Project (1000GP). We found that use of phase-informative reads increases the mean distance between switch errors by 22% from 274.4 kb to 328.6 kb. We also used male chromosome X haplotypes from the 1000GP samples to simulate sequencing reads with varying insert size, read length, and base error rate. When using short 100 bp paired-end reads, we found that using mixtures of insert sizes produced the best results. When using longer reads with high error rates (5-20 kb read with 4%-15% error per base), phasing performance was substantially improved. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

CORNAS: coverage-dependent RNA-Seq analysis of gene expression data without biological replicates.

PubMed

Low, Joel Z B; Khang, Tsung Fei; Tammi, Martti T

2017-12-28

In current statistical methods for calling differentially expressed genes in RNA-Seq experiments, the assumption is that an adjusted observed gene count represents an unknown true gene count. This adjustment usually consists of a normalization step to account for heterogeneous sample library sizes, and then the resulting normalized gene counts are used as input for parametric or non-parametric differential gene expression tests. A distribution of true gene counts, each with a different probability, can result in the same observed gene count. Importantly, sequencing coverage information is currently not explicitly incorporated into any of the statistical models used for RNA-Seq analysis. We developed a fast Bayesian method which uses the sequencing coverage information determined from the concentration of an RNA sample to estimate the posterior distribution of a true gene count. Our method has better or comparable performance compared to NOISeq and GFOLD, according to the results from simulations and experiments with real unreplicated data. We incorporated a previously unused sequencing coverage parameter into a procedure for differential gene expression analysis with RNA-Seq data. Our results suggest that our method can be used to overcome analytical bottlenecks in experiments with limited number of replicates and low sequencing coverage. The method is implemented in CORNAS (Coverage-dependent RNA-Seq), and is available at https://github.com/joel-lzb/CORNAS .

Phenotypic Heterogeneity of Genomically-Diverse Isolates of Streptococcus mutans

PubMed Central

Palmer, Sara R.; Miller, James H.; Abranches, Jacqueline; Zeng, Lin; Lefebure, Tristan; Richards, Vincent P.; Lemos, José A.; Stanhope, Michael J.; Burne, Robert A.

2013-01-01

High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease. PMID:23613838

The normal vaginal and uterine bacterial microbiome in giant pandas (Ailuropoda melanoleuca).

PubMed

Yang, Xin; Cheng, Guangyang; Li, Caiwu; Yang, Jiang; Li, Jianan; Chen, Danyu; Zou, Wencheng; Jin, SenYan; Zhang, Hemin; Li, Desheng; He, Yongguo; Wang, Chengdong; Wang, Min; Wang, Hongning

2017-06-01

While the health effects of the colonization of the reproductive tracts of mammals by bacterial communities are widely known, there is a dearth of knowledge specifically in relation to giant panda microbiomes. In order to investigate the vaginal and uterine bacterial diversity of healthy giant pandas, we used high-throughput sequence analysis of portions of the 16S rRNA gene, based on samples taken from the vaginas (GPV group) and uteri (GPU group) of these animals. Results showed that the four most abundant phyla, which contained in excess of 98% of the total sequences, were Proteobacteria (59.2% for GPV and 51.4% for GPU), Firmicutes (34.4% for GPV and 23.3% for GPU), Actinobacteria (5.2% for GPV and 14.0% for GPU) and Bacteroidetes (0.3% for GPV and 10.3% for GPU). At the genus level, Escherichia was most abundant (11.0%) in the GPV, followed by Leuconostoc (8.7%), Pseudomonas (8.0%), Acinetobacter (7.3%), Streptococcus (6.3%) and Lactococcus (6.0%). In relation to the uterine samples, Janthinobacterium had the highest prevalence rate (20.2%), followed by Corynebacterium (13.2%), Streptococcus (19.6%), Psychrobacter (9.3%), Escherichia (7.5%) and Bacteroides (6.2%). Moreover, both Chao1 and abundance-based coverage estimator (ACE) species richness indices, which were operating at the same sequencing depth for each sample, demonstrated that GPV had more species richness than GPU, while Simpson and Shannon indices of diversity indicated that GPV had the higher bacterial diversity. These findings contribute to our understanding of the potential influence abnormal reproductive tract microbial communities have on negative pregnancy outcomes in giant pandas. Copyright © 2017 Elsevier GmbH. All rights reserved.

High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic.

PubMed

Sealfon, Rachel; Gire, Stephen; Ellis, Crystal; Calderwood, Stephen; Qadri, Firdausi; Hensley, Lisa; Kellis, Manolis; Ryan, Edward T; LaRocque, Regina C; Harris, Jason B; Sabeti, Pardis C

2012-09-11

Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.

Diversity of dsDNA Viruses in a South African Hot Spring Assessed by Metagenomics and Microscopy.

PubMed

Zablocki, Olivier; van Zyl, Leonardo Joaquim; Kirby, Bronwyn; Trindade, Marla

2017-11-18

The current view of virus diversity in terrestrial hot springs is limited to a few sampling sites. To expand our current understanding of hot spring viral community diversity, this study aimed to investigate the first African hot spring (Brandvlei hot spring; 60 °C, pH 5.7) by means of electron microscopy and sequencing of the virus fraction. Microscopy analysis revealed a mixture of regular- and 'jumbo'-sized tailed morphotypes ( Caudovirales ), lemon-shaped virions ( Fuselloviridae- like; salterprovirus-like) and pleiomorphic virus-like particles. Metavirome analysis corroborated the presence of His1-like viruses and has expanded the current clade of salterproviruses using a polymerase B gene phylogeny. The most represented viral contig was to a cyanophage genome fragment, which may underline basic ecosystem functioning provided by these viruses. Furthermore, a putative Gemmata -related phage was assembled with high coverage, a previously undocumented phage-host association. This study demonstrated that a moderately thermophilic spring environment contained a highly novel pool of viruses and should encourage future characterization of a wider temperature range of hot springs throughout the world.

Quasispecies Analyses of the HIV-1 Near-full-length Genome With Illumina MiSeq

PubMed Central

Ode, Hirotaka; Matsuda, Masakazu; Matsuoka, Kazuhiro; Hachiya, Atsuko; Hattori, Junko; Kito, Yumiko; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

2015-01-01

Human immunodeficiency virus type-1 (HIV-1) exhibits high between-host genetic diversity and within-host heterogeneity, recognized as quasispecies. Because HIV-1 quasispecies fluctuate in terms of multiple factors, such as antiretroviral exposure and host immunity, analyzing the HIV-1 genome is critical for selecting effective antiretroviral therapy and understanding within-host viral coevolution mechanisms. Here, to obtain HIV-1 genome sequence information that includes minority variants, we sought to develop a method for evaluating quasispecies throughout the HIV-1 near-full-length genome using the Illumina MiSeq benchtop deep sequencer. To ensure the reliability of minority mutation detection, we applied an analysis method of sequence read mapping onto a consensus sequence derived from de novo assembly followed by iterative mapping and subsequent unique error correction. Deep sequencing analyses of aHIV-1 clone showed that the analysis method reduced erroneous base prevalence below 1% in each sequence position and discarded only < 1% of all collected nucleotides, maximizing the usage of the collected genome sequences. Further, we designed primer sets to amplify the HIV-1 near-full-length genome from clinical plasma samples. Deep sequencing of 92 samples in combination with the primer sets and our analysis method provided sufficient coverage to identify >1%-frequency sequences throughout the genome. When we evaluated sequences of pol genes from 18 treatment-naïve patients' samples, the deep sequencing results were in agreement with Sanger sequencing and identified numerous additional minority mutations. The results suggest that our deep sequencing method would be suitable for identifying within-host viral population dynamics throughout the genome. PMID:26617593

Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

PubMed Central

Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia; Breitbart, Mya; Edwards, Robert A.

2015-01-01

Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set of publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. We propose adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution. PMID:26005436

Metasecretome-selective phage display approach for mining the functional potential of a rumen microbial community.

PubMed

Ciric, Milica; Moon, Christina D; Leahy, Sinead C; Creevey, Christopher J; Altermann, Eric; Attwood, Graeme T; Rakonjac, Jasna; Gagic, Dragana

2014-05-12

In silico, secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences. For metasecretome (collection of surface, secreted and transmembrane proteins from environmental microbial communities) this approach is impractical, considering that the metasecretome open reading frames (ORFs) comprise only 10% to 30% of total metagenome, and are poorly represented in the dataset due to overall low coverage of metagenomic gene pool, even in large-scale projects. By combining secretome-selective phage display and next-generation sequencing, we focused the sequence analysis of complex rumen microbial community on the metasecretome component of the metagenome. This approach achieved high enrichment (29 fold) of secreted fibrolytic enzymes from the plant-adherent microbial community of the bovine rumen. In particular, we identified hundreds of heretofore rare modules belonging to cellulosomes, cell-surface complexes specialised for recognition and degradation of the plant fibre. As a method, metasecretome phage display combined with next-generation sequencing has a power to sample the diversity of low-abundance surface and secreted proteins that would otherwise require exceptionally large metagenomic sequencing projects. As a resource, metasecretome display library backed by the dataset obtained by next-generation sequencing is ready for i) affinity selection by standard phage display methodology and ii) easy purification of displayed proteins as part of the virion for individual functional analysis.

Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

DOE PAGES

Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia; ...

2015-05-08

Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set ofmore » publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. By adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.« less

The Histone Database: an integrated resource for histones and histone fold-containing proteins

PubMed Central

Mariño-Ramírez, Leonardo; Levine, Kevin M.; Morales, Mario; Zhang, Suiyuan; Moreland, R. Travis; Baxevanis, Andreas D.; Landsman, David

2011-01-01

Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins. Database URL: The Histone Sequence Database is freely available and can be accessed at http://research.nhgri.nih.gov/histones/. PMID:22025671

Multidimensional metrics for estimating phage abundance, distribution, gene density, and sequence coverage in metagenomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aziz, Ramy K.; Dwivedi, Bhakti; Akhter, Sajia

Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set ofmore » publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. By adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.« less

Microbiota diversity and gene expression dynamics in human oral biofilms

PubMed Central

2014-01-01

Background Micro-organisms inhabiting teeth surfaces grow on biofilms where a specific and complex succession of bacteria has been described by co-aggregation tests and DNA-based studies. Although the composition of oral biofilms is well established, the active portion of the bacterial community and the patterns of gene expression in vivo have not been studied. Results Using RNA-sequencing technologies, we present the first metatranscriptomic study of human dental plaque, performed by two different approaches: (1) A short-reads, high-coverage approach by Illumina sequencing to characterize the gene activity repertoire of the microbial community during biofilm development; (2) A long-reads, lower-coverage approach by pyrosequencing to determine the taxonomic identity of the active microbiome before and after a meal ingestion. The high-coverage approach allowed us to analyze over 398 million reads, revealing that microbial communities are individual-specific and no bacterial species was detected as key player at any time during biofilm formation. We could identify some gene expression patterns characteristic for early and mature oral biofilms. The transcriptomic profile of several adhesion genes was confirmed through qPCR by measuring expression of fimbriae-associated genes. In addition to the specific set of gene functions overexpressed in early and mature oral biofilms, as detected through the short-reads dataset, the long-reads approach detected specific changes when comparing the metatranscriptome of the same individual before and after a meal, which can narrow down the list of organisms responsible for acid production and therefore potentially involved in dental caries. Conclusions The bacteria changing activity during biofilm formation and after meal ingestion were person-specific. Interestingly, some individuals showed extreme homeostasis with virtually no changes in the active bacterial population after food ingestion, suggesting the presence of a microbial community which could be associated to dental health. PMID:24767457

Microbiota diversity and gene expression dynamics in human oral biofilms.

PubMed

Benítez-Páez, Alfonso; Belda-Ferre, Pedro; Simón-Soro, Aurea; Mira, Alex

2014-04-27

Micro-organisms inhabiting teeth surfaces grow on biofilms where a specific and complex succession of bacteria has been described by co-aggregation tests and DNA-based studies. Although the composition of oral biofilms is well established, the active portion of the bacterial community and the patterns of gene expression in vivo have not been studied. Using RNA-sequencing technologies, we present the first metatranscriptomic study of human dental plaque, performed by two different approaches: (1) A short-reads, high-coverage approach by Illumina sequencing to characterize the gene activity repertoire of the microbial community during biofilm development; (2) A long-reads, lower-coverage approach by pyrosequencing to determine the taxonomic identity of the active microbiome before and after a meal ingestion. The high-coverage approach allowed us to analyze over 398 million reads, revealing that microbial communities are individual-specific and no bacterial species was detected as key player at any time during biofilm formation. We could identify some gene expression patterns characteristic for early and mature oral biofilms. The transcriptomic profile of several adhesion genes was confirmed through qPCR by measuring expression of fimbriae-associated genes. In addition to the specific set of gene functions overexpressed in early and mature oral biofilms, as detected through the short-reads dataset, the long-reads approach detected specific changes when comparing the metatranscriptome of the same individual before and after a meal, which can narrow down the list of organisms responsible for acid production and therefore potentially involved in dental caries. The bacteria changing activity during biofilm formation and after meal ingestion were person-specific. Interestingly, some individuals showed extreme homeostasis with virtually no changes in the active bacterial population after food ingestion, suggesting the presence of a microbial community which could be associated to dental health.

FY11 Report on Metagenome Analysis using Pathogen Marker Libraries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Shea N.; Allen, Jonathan E.; McLoughlin, Kevin S.

2011-06-02

A method, sequence library, and software suite was invented to rapidly assess whether any member of a pre-specified list of threat organisms or their near neighbors is present in a metagenome. The system was designed to handle mega- to giga-bases of FASTA-formatted raw sequence reads from short or long read next generation sequencing platforms. The approach is to pre-calculate a viral and a bacterial "Pathogen Marker Library" (PML) containing sub-sequences specific to pathogens or their near neighbors. A list of expected matches comparing every bacterial or viral genome against the PML sequences is also pre-calculated. To analyze a metagenome, readsmore » are compared to the PML, and observed PML-metagenome matches are compared to the expected PML-genome matches, and the ratio of observed relative to expected matches is reported. In other words, a 3-way comparison among the PML, metagenome, and existing genome sequences is used to quickly assess which (if any) species included in the PML is likely to be present in the metagenome, based on available sequence data. Our tests showed that the species with the most PML matches correctly indicated the organism sequenced for empirical metagenomes consisting of a cultured, relatively pure isolate. These runs completed in 1 minute to 3 hours on 12 CPU (1 thread/CPU), depending on the metagenome and PML. Using more threads on the same number of CPU resulted in speed improvements roughly proportional to the number of threads. Simulations indicated that detection sensitivity depends on both sequencing coverage levels for a species and the size of the PML: species were correctly detected even at ~0.003x coverage by the large PMLs, and at ~0.03x coverage by the smaller PMLs. Matches to true positive species were 3-4 orders of magnitude higher than to false positives. Simulations with short reads (36 nt and ~260 nt) showed that species were usually detected for metagenome coverage above 0.005x and coverage in the PML above 0.05x, and detection probability appears to be a function of both coverages. Multiple species could be detected simultaneously in a simulated low-coverage, complex metagenome, and the largest PML gave no false negative species and no false positive genera. The presence of multiple species was predicted in a complex metagenome from a human gut microbiome with 1.9 GB of short reads (75 nt); the species predicted were reasonable gut flora and no biothreat agents were detected, showing the feasibility of PML analysis of empirical complex metagenomes.« less

Association mapping identifies loci for canopy coverage in diverse soybean genotypes

USDA-ARS?s Scientific Manuscript database

Rapid establishment of canopy coverage decreases soil evaporation relative to transpiration (T), improves water use efficiency (WUE) and light interception, and increases soybean competitiveness against weeds. The objective of this study was to identify genomic loci associated with canopy coverage (...

Use of low-coverage, large-insert, short-read data for rapid and accurate generation of enhanced-quality draft Pseudomonas genome sequences.

PubMed

O'Brien, Heath E; Gong, Yunchen; Fung, Pauline; Wang, Pauline W; Guttman, David S

2011-01-01

Next-generation genomic technology has both greatly accelerated the pace of genome research as well as increased our reliance on draft genome sequences. While groups such as the Genomics Standards Consortium have made strong efforts to promote genome standards there is a still a general lack of uniformity among published draft genomes, leading to challenges for downstream comparative analyses. This lack of uniformity is a particular problem when using standard draft genomes that frequently have large numbers of low-quality sequencing tracts. Here we present a proposal for an "enhanced-quality draft" genome that identifies at least 95% of the coding sequences, thereby effectively providing a full accounting of the genic component of the genome. Enhanced-quality draft genomes are easily attainable through a combination of small- and large-insert next-generation, paired-end sequencing. We illustrate the generation of an enhanced-quality draft genome by re-sequencing the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola 1448A (Pph 1448A), which has a published, closed genome sequence of 5.93 Mbp. We use a combination of Illumina paired-end and mate-pair sequencing, and surprisingly find that de novo assemblies with 100x paired-end coverage and mate-pair sequencing with as low as low as 2-5x coverage are substantially better than assemblies based on higher coverage. The rapid and low-cost generation of large numbers of enhanced-quality draft genome sequences will be of particular value for microbial diagnostics and biosecurity, which rely on precise discrimination of potentially dangerous clones from closely related benign strains.

Long-term changes in abundance and diversity of macrophyte and waterfowl populations in an estuary with exotic macrophytes and improving water quality

USGS Publications Warehouse

Rybicki, N.B.; Landwehr, J.M.

2007-01-01

We assessed species-specific coverage (km2) of a submerged aquatic vegetation (SAV) community in the fresh and upper oligohaline Potomac Estuary from 1985 to 2001 using a method combining field observations of species-proportional coverage data with congruent remotely sensed coverage and density (percent canopy cover) data. Biomass (estimated by density-weighted coverage) of individual species was calculated. Under improving water quality conditions, exotic SAV species did not displace native SAV; rather, the percent of natives increased over time. While coverage-based diversity did fluctuate and increased, richness-based community turnover rates were not significantly different from zero. SAV diversity was negatively related to nitrogen concentration. Differences in functional traits, such as reproductive potential, between the dominant native and exotic species may explain some interannual patterns in SAV. Biomass of native, as well as exotic, SAV species varied with factors affecting water column light attenuation. We also show a positive response by a higher trophic level, waterfowl, to SAV communities dominated by exotic SAV from 1959 to 2001. ?? 2007, by the American Society of Limnology and Oceanography, Inc.

Breaking Lander-Waterman’s Coverage Bound

PubMed Central

Nashta-ali, Damoun; Motahari, Seyed Abolfazl; Hosseinkhalaj, Babak

2016-01-01

Lander-Waterman’s coverage bound establishes the total number of reads required to cover the whole genome of size G bases. In fact, their bound is a direct consequence of the well-known solution to the coupon collector’s problem which proves that for such genome, the total number of bases to be sequenced should be O(G ln G). Although the result leads to a tight bound, it is based on a tacit assumption that the set of reads are first collected through a sequencing process and then are processed through a computation process, i.e., there are two different machines: one for sequencing and one for processing. In this paper, we present a significant improvement compared to Lander-Waterman’s result and prove that by combining the sequencing and computing processes, one can re-sequence the whole genome with as low as O(G) sequenced bases in total. Our approach also dramatically reduces the required computational power for the combined process. Simulation results are performed on real genomes with different sequencing error rates. The results support our theory predicting the log G improvement on coverage bound and corresponding reduction in the total number of bases required to be sequenced. PMID:27806058

A Poisson hierarchical modelling approach to detecting copy number variation in sequence coverage data.

PubMed

Sepúlveda, Nuno; Campino, Susana G; Assefa, Samuel A; Sutherland, Colin J; Pain, Arnab; Clark, Taane G

2013-02-26

The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data.

Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes.

PubMed

Fu, Yong-Bi; Peterson, Gregory W; Dong, Yibo

2016-04-07

Genotyping-by-sequencing (GBS) has emerged as a useful genomic approach for exploring genome-wide genetic variation. However, GBS commonly samples a genome unevenly and can generate a substantial amount of missing data. These technical features would limit the power of various GBS-based genetic and genomic analyses. Here we present software called IgCoverage for in silico evaluation of genomic coverage through GBS with an individual or pair of restriction enzymes on one sequenced genome, and report a new set of 21 restriction enzyme combinations that can be applied to enhance GBS applications. These enzyme combinations were developed through an application of IgCoverage on 22 plant, animal, and fungus species with sequenced genomes, and some of them were empirically evaluated with different runs of Illumina MiSeq sequencing in 12 plant species. The in silico analysis of 22 organisms revealed up to eight times more genome coverage for the new combinations consisted of pairing four- or five-cutter restriction enzymes than the commonly used enzyme combination PstI + MspI. The empirical evaluation of the new enzyme combination (HinfI + HpyCH4IV) in 12 plant species showed 1.7-6 times more genome coverage than PstI + MspI, and 2.3 times more genome coverage in dicots than monocots. Also, the SNP genotyping in 12 Arabidopsis and 12 rice plants revealed that HinfI + HpyCH4IV generated 7 and 1.3 times more SNPs (with 0-16.7% missing observations) than PstI + MspI, respectively. These findings demonstrate that these novel enzyme combinations can be utilized to increase genome sampling and improve SNP genotyping in various GBS applications. Copyright © 2016 Fu et al.

Expanding proteome coverage with orthogonal-specificity α-Lytic proteases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer, Jesse G.; Kim, Sangtae; Maltby, David A.

2014-03-01

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage by cleavage at sequences complimentary to trypsin may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type alpha-lytic protease (WaLP), and an active site mutant of WaLP, M190A alpha-lytic protease (MaLP). We assess several relevant factors including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. Bymore » combining data from separate digestions with trypsin, LysC, WaLP, and MaLP, proteome coverage was increased 101% compared to trypsin digestion alone. To demonstrate how the gained sequence coverage can access additional PTM information, we show identification of a number of novel phosphorylation sites in the S. pombe proteome and include an illustrative example from the protein MPD2, wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.« less

Fully immunized child: coverage, timing and sequencing of routine immunization in an urban poor settlement in Nairobi, Kenya.

PubMed

Mutua, Martin Kavao; Kimani-Murage, Elizabeth; Ngomi, Nicholas; Ravn, Henrik; Mwaniki, Peter; Echoka, Elizabeth

2016-01-01

More efforts have been put in place to increase full immunization coverage rates in the last decade. Little is known about the levels and consequences of delaying or vaccinating children in different schedules. Vaccine effectiveness depends on the timing of its administration, and it is not optimal if given early, delayed or not given as recommended. Evidence of non-specific effects of vaccines is well documented and could be linked to timing and sequencing of immunization. This paper documents the levels of coverage, timing and sequencing of routine childhood vaccines. The study was conducted between 2007 and 2014 in two informal urban settlements in Nairobi. A total of 3856 children, aged 12-23 months and having a vaccination card seen were included in analysis. Vaccination dates recorded from the cards seen were used to define full immunization coverage, timeliness and sequencing. Proportions, medians and Kaplan-Meier curves were used to assess and describe the levels of full immunization coverage, vaccination delays and sequencing. The findings indicate that 67 % of the children were fully immunized by 12 months of age. Missing measles and third doses of polio and pentavalent vaccine were the main reason for not being fully immunized. Delays were highest for third doses of polio and pentavalent and measles. About 22 % of fully immunized children had vaccines in an out-of-sequence manner with 18 % not receiving pentavalent together with polio vaccine as recommended. Results show higher levels of missed opportunities and low coverage of routine childhood vaccinations given at later ages. New strategies are needed to enable health care providers and parents/guardians to work together to increase the levels of completion of all required vaccinations. In particular, more focus is needed on vaccines given in multiple doses (polio, pentavalent and pneumococcal conjugate vaccines).

Development of simple sequence repeat (SSR) markers from a genome survey of Chinese bayberry (Myrica rubra)

PubMed Central

2012-01-01

Background Chinese bayberry (Myrica rubra Sieb. and Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species. Results The whole genome shotgun survey of Chinese bayberry produced 9.01Gb of sequence data, about 26x coverage of the estimated genome size of 323 Mb. The genome sequences were highly heterozygous, but with little duplication. From the initial assembled scaffold covering 255 Mb sequence data, 28,602 SSRs (≥5 repeats) were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica species: 91.14%, 89.87% and 46.84% SSRs could be used in Myrica adenophora, Myrica nana and Myrica cerifera, respectively. The UPGMA dendrogram tree showed that cultivated Myrica rubra is closely related to Myrica adenophora and Myrica nana, originating in southwest China, and very distantly related to Myrica cerifera, originating in America. These markers can be used in the construction of a linkage map and for genetic diversity studies in Myrica species. Conclusion Myrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species. PMID:22621340

Discovery of rare, diagnostic AluYb8/9 elements in diverse human populations.

PubMed

Feusier, Julie; Witherspoon, David J; Scott Watkins, W; Goubert, Clément; Sasani, Thomas A; Jorde, Lynn B

2017-01-01

Polymorphic human Alu elements are excellent tools for assessing population structure, and new retrotransposition events can contribute to disease. Next-generation sequencing has greatly increased the potential to discover Alu elements in human populations, and various sequencing and bioinformatics methods have been designed to tackle the problem of detecting these highly repetitive elements. However, current techniques for Alu discovery may miss rare, polymorphic Alu elements. Combining multiple discovery approaches may provide a better profile of the polymorphic Alu mobilome. Alu Yb8/9 elements have been a focus of our recent studies as they are young subfamilies (~2.3 million years old) that contribute ~30% of recent polymorphic Alu retrotransposition events. Here, we update our ME-Scan methods for detecting Alu elements and apply these methods to discover new insertions in a large set of individuals with diverse ancestral backgrounds. We identified 5,288 putative Alu insertion events, including several hundred novel Alu Yb8/9 elements from 213 individuals from 18 diverse human populations. Hundreds of these loci were specific to continental populations, and 23 non-reference population-specific loci were validated by PCR. We provide high-quality sequence information for 68 rare Alu Yb8/9 elements, of which 11 have hallmarks of an active source element. Our subfamily distribution of rare Alu Yb8/9 elements is consistent with previous datasets, and may be representative of rare loci. We also find that while ME-Scan and low-coverage, whole-genome sequencing (WGS) detect different Alu elements in 41 1000 Genomes individuals, the two methods yield similar population structure results. Current in-silico methods for Alu discovery may miss rare, polymorphic Alu elements. Therefore, using multiple techniques can provide a more accurate profile of Alu elements in individuals and populations. We improved our false-negative rate as an indicator of sample quality for future ME-Scan experiments. In conclusion, we demonstrate that ME-Scan is a good supplement for next-generation sequencing methods and is well-suited for population-level analyses.

Binning of shallowly sampled metagenomic sequence fragments reveals that low abundance bacteria play important roles in sulfur cycling and degradation of complex organic polymers in an acid mine drainage community

NASA Astrophysics Data System (ADS)

Dick, G. J.; Andersson, A.; Banfield, J. F.

2007-12-01

Our understanding of environmental microbiology has been greatly enhanced by community genome sequencing of DNA recovered directly the environment. Community genomics provides insights into the diversity, community structure, metabolic function, and evolution of natural populations of uncultivated microbes, thereby revealing dynamics of how microorganisms interact with each other and their environment. Recent studies have demonstrated the potential for reconstructing near-complete genomes from natural environments while highlighting the challenges of analyzing community genomic sequence, especially from diverse environments. A major challenge of shotgun community genome sequencing is identification of DNA fragments from minor community members for which only low coverage of genomic sequence is present. We analyzed community genome sequence retrieved from biofilms in an acid mine drainage (AMD) system in the Richmond Mine at Iron Mountain, CA, with an emphasis on identification and assembly of DNA fragments from low-abundance community members. The Richmond mine hosts an extensive, relatively low diversity subterranean chemolithoautotrophic community that is sustained entirely by oxidative dissolution of pyrite. The activity of these microorganisms greatly accelerates the generation of AMD. Previous and ongoing work in our laboratory has focused on reconstrucing genomes of dominant community members, including several bacteria and archaea. We binned contigs from several samples (including one new sample and two that had been previously analyzed) by tetranucleotide frequency with clustering by Self-Organizing Maps (SOM). The binning, evaluated by comparison with information from the manually curated assembly of the dominant organisms, was found to be very effective: fragments were correctly assigned with 95% accuracy. Improperly assigned fragments often contained sequences that are either evolutionarily constrained (e.g. 16S rRNA genes) or mobile elements that are not expected to reflect the tetranucleotide frequency signature of the host genome. Four unknown tetranucleotide frequency clusters with significant sequence (6 Mb total) were noted and analyzed further. Based on phylogenetic markers and BLAST results, these clusters represent low abundance bacteria including Acintobacteria, Firmicutes, and Proteobacteria. Functional analysis of these clusters revealved that the low- abundance bacteria harbor genes that could potentially encode important ecosystem functions such as sulfur utilization (e.g. polysulfide reductase) and polymer degradation (e.g. chitinase and glycoside hydrolase). We conclude that ESOM clustering of tetranucleotide frequency patterns is an effective method for rapidly binning shotgun community genomic sequences and a valuable tool for analyzing minor community members, which despite their low abundance may play crucial ecological roles.

Low-coverage MiSeq next generation sequencing reveals the mitochondrial genome of the Eastern Rock Lobster, Sagmariasus verreauxi.

PubMed

Doyle, Stephen R; Griffith, Ian S; Murphy, Nick P; Strugnell, Jan M

2015-01-01

The complete mitochondrial genome of the Eastern Rock lobster, Sagmariasus verreauxi, is reported for the first time. Using low-coverage, long read MiSeq next generation sequencing, we constructed and determined the mtDNA genome organization of the 15,470 bp sequence from two isolates from Eastern Tasmania, Australia and Northern New Zealand, and identified 46 polymorphic nucleotides between the two sequences. This genome sequence and its genetic polymorphisms will likely be useful in understanding the distribution and population connectivity of the Eastern Rock Lobster, and in the fisheries management of this commercially important species.

Comparing de novo genome assembly: the long and short of it.

PubMed

Narzisi, Giuseppe; Mishra, Bud

2011-04-29

Recent advances in DNA sequencing technology and their focal role in Genome Wide Association Studies (GWAS) have rekindled a growing interest in the whole-genome sequence assembly (WGSA) problem, thereby, inundating the field with a plethora of new formalizations, algorithms, heuristics and implementations. And yet, scant attention has been paid to comparative assessments of these assemblers' quality and accuracy. No commonly accepted and standardized method for comparison exists yet. Even worse, widely used metrics to compare the assembled sequences emphasize only size, poorly capturing the contig quality and accuracy. This paper addresses these concerns: it highlights common anomalies in assembly accuracy through a rigorous study of several assemblers, compared under both standard metrics (N50, coverage, contig sizes, etc.) as well as a more comprehensive metric (Feature-Response Curves, FRC) that is introduced here; FRC transparently captures the trade-offs between contigs' quality against their sizes. For this purpose, most of the publicly available major sequence assemblers--both for low-coverage long (Sanger) and high-coverage short (Illumina) reads technologies--are compared. These assemblers are applied to microbial (Escherichia coli, Brucella, Wolbachia, Staphylococcus, Helicobacter) and partial human genome sequences (Chr. Y), using sequence reads of various read-lengths, coverages, accuracies, and with and without mate-pairs. It is hoped that, based on these evaluations, computational biologists will identify innovative sequence assembly paradigms, bioinformaticists will determine promising approaches for developing "next-generation" assemblers, and biotechnologists will formulate more meaningful design desiderata for sequencing technology platforms. A new software tool for computing the FRC metric has been developed and is available through the AMOS open-source consortium.

Effects of dietary supplementation of Ulva pertusa and non-starch polysaccharide enzymes on gut microbiota of Siganus canaliculatus

NASA Astrophysics Data System (ADS)

Zhang, Xinxu; Wu, Huijuan; Li, Zhongzhen; Li, Yuanyou; Wang, Shuqi; Zhu, Dashi; Wen, Xiaobo; Li, Shengkang

2018-03-01

Fishes represent the highest diversity of vertebrates; however, our understanding of the compositions and functions of their gut microbiota is limited. In this study, we provided the first insight into the gut microbiota of the herbivorous fish Siganus canaliculatus by using three molecular ecology techniques based on the 16S rRNA genes (denaturing gradient gel electrophoresis, clone library construction, and highthroughput Illumina sequencing), and the Illumina sequencing technique is suggested here due to its higher overall coverage of the total 16S rRNA genes. A core gut microbiota of 29 bacterial groups, covering >99.9% of the total bacterial community, was found to be dominated by Proteobacteria and Firmicutes in fish fed three different diets with/without the supplementation of Ulva pertusa and non-starch polysaccharide (NSP) enzymes (cellulase, xylanase, and β-glucanase). Diverse potential NSP-degrading bacteria and probiotics (e.g., Ruminococcus, Clostridium and Lachnospiraceae) were detected in the intestine of the fish fed U. pertusa, suggesting that these microorganisms likely participated in the degradation of NSPs derived from U. pertusa. This study supports our previous conclusion that U. pertusa-based diets are suitable for the production of S. canaliculatus with lower costs without compromising quality.

Effects of dietary supplementation of Ulva pertusa and non-starch polysaccharide enzymes on gut microbiota of Siganus canaliculatus

NASA Astrophysics Data System (ADS)

Zhang, Xinxu; Wu, Huijuan; Li, Zhongzhen; Li, Yuanyou; Wang, Shuqi; Zhu, Dashi; Wen, Xiaobo; Li, Shengkang

2017-05-01

Fishes represent the highest diversity of vertebrates; however, our understanding of the compositions and functions of their gut microbiota is limited. In this study, we provided the first insight into the gut microbiota of the herbivorous fish Siganus canaliculatus (S. canaliculatus) by using three molecular ecology techniques based on the 16S rRNA genes (denaturing gradient gel electrophoresis, clone library construction, and high-throughput Illumina sequencing), and the Illumina sequencing technique is suggested here due to its higher overall coverage of the total 16S rRNA genes. A core gut microbiota of 29 bacterial groups, covering >99.9% of the total bacterial community, was found to be dominated by Proteobacteria and Firmicutes in fish fed three different diets with/without the supplementation of Ulva pertusa (U. pertusa) and non-starch polysaccharide (NSP) enzymes (cellulase, xylanase, and β-glucanase). Diverse potential NSP-degrading bacteria and probiotics (e.g., Ruminococcus, Clostridium and Lachnospiraceae) were detected in the intestine of the fish fed U. pertusa, suggesting that these microorganisms likely participated in the degradation of NSPs derived from U. pertusa. This study supports our previous conclusion that U. pertusa-based diets are suitable for the production of S. canaliculatus with lower costs without compromising quality.

A polyvalent hybrid protein elicits antibodies against the diverse allelic types of block 2 in Plasmodium falciparum merozoite surface protein 1.

PubMed

Tetteh, Kevin K A; Conway, David J

2011-10-13

Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identifying tagging SNPs for African specific genetic variation from the African Diaspora Genome

PubMed Central

Johnston, Henry Richard; Hu, Yi-Juan; Gao, Jingjing; O’Connor, Timothy D.; Abecasis, Gonçalo R.; Wojcik, Genevieve L; Gignoux, Christopher R.; Gourraud, Pierre-Antoine; Lizee, Antoine; Hansen, Mark; Genuario, Rob; Bullis, Dave; Lawley, Cindy; Kenny, Eimear E.; Bustamante, Carlos; Beaty, Terri H.; Mathias, Rasika A.; Barnes, Kathleen C.; Qin, Zhaohui S.; Preethi Boorgula, Meher; Campbell, Monica; Chavan, Sameer; Ford, Jean G.; Foster, Cassandra; Gao, Li; Hansel, Nadia N.; Horowitz, Edward; Huang, Lili; Ortiz, Romina; Potee, Joseph; Rafaels, Nicholas; Ruczinski, Ingo; Scott, Alan F.; Taub, Margaret A.; Vergara, Candelaria; Levin, Albert M.; Padhukasahasram, Badri; Williams, L. Keoki; Dunston, Georgia M.; Faruque, Mezbah U.; Gietzen, Kimberly; Deshpande, Aniket; Grus, Wendy E.; Locke, Devin P.; Foreman, Marilyn G.; Avila, Pedro C.; Grammer, Leslie; Kim, Kwang-Youn A.; Kumar, Rajesh; Schleimer, Robert; De La Vega, Francisco M.; Shringarpure, Suyash S.; Musharoff, Shaila; Burchard, Esteban G.; Eng, Celeste; Hernandez, Ryan D.; Pino-Yanes, Maria; Torgerson, Dara G.; Szpiech, Zachary A.; Torres, Raul; Nicolae, Dan L.; Ober, Carole; Olopade, Christopher O; Olopade, Olufunmilayo; Oluwole, Oluwafemi; Arinola, Ganiyu; Song, Wei; Correa, Adolfo; Musani, Solomon; Wilson, James G.; Lange, Leslie A.; Akey, Joshua; Bamshad, Michael; Chong, Jessica; Fu, Wenqing; Nickerson, Deborah; Reiner, Alexander; Hartert, Tina; Ware, Lorraine B.; Bleecker, Eugene; Meyers, Deborah; Ortega, Victor E.; Maul, Pissamai; Maul, Trevor; Watson, Harold; Ilma Araujo, Maria; Riccio Oliveira, Ricardo; Caraballo, Luis; Marrugo, Javier; Martinez, Beatriz; Meza, Catherine; Ayestas, Gerardo; Francisco Herrera-Paz, Edwin; Landaverde-Torres, Pamela; Erazo, Said Omar Leiva; Martinez, Rosella; Mayorga, Alvaro; Mayorga, Luis F.; Mejia-Mejia, Delmy-Aracely; Ramos, Hector; Saenz, Allan; Varela, Gloria; Marina Vasquez, Olga; Ferguson, Trevor; Knight-Madden, Jennifer; Samms-Vaughan, Maureen; Wilks, Rainford J.; Adegnika, Akim; Ateba-Ngoa, Ulysse; Yazdanbakhsh, Maria

2017-01-01

A primary goal of The Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) is to develop an ‘African Diaspora Power Chip’ (ADPC), a genotyping array consisting of tagging SNPs, useful in comprehensively identifying African specific genetic variation. This array is designed based on the novel variation identified in 642 CAAPA samples of African ancestry with high coverage whole genome sequence data (~30× depth). This novel variation extends the pattern of variation catalogued in the 1000 Genomes and Exome Sequencing Projects to a spectrum of populations representing the wide range of West African genomic diversity. These individuals from CAAPA also comprise a large swath of the African Diaspora population and incorporate historical genetic diversity covering nearly the entire Atlantic coast of the Americas. Here we show the results of designing and producing such a microchip array. This novel array covers African specific variation far better than other commercially available arrays, and will enable better GWAS analyses for researchers with individuals of African descent in their study populations. A recent study cataloging variation in continental African populations suggests this type of African-specific genotyping array is both necessary and valuable for facilitating large-scale GWAS in populations of African ancestry. PMID:28429804

Vaccination coverage and out-of-sequence vaccinations in rural Guinea-Bissau: an observational cohort study

PubMed Central

Hornshøj, Linda; Benn, Christine Stabell; Fernandes, Manuel; Rodrigues, Amabelia; Aaby, Peter; Fisker, Ane Bærent

2012-01-01

Objective The WHO aims for 90% coverage of the Expanded Program on Immunization (EPI), which in Guinea-Bissau included BCG vaccine at birth, three doses of diphtheria−tetanus−pertussis vaccine (DTP) and oral polio vaccine (OPV) at 6, 10 and 14 weeks and measles vaccine (MV) at 9 months when this study was conducted. The WHO assesses coverage by 12 months of age. The sequence of vaccines may have an effect on child mortality, but is not considered in official statistics or assessments of programme performance. We assessed vaccination coverage and frequency of out-of-sequence vaccinations by 12 and 24 months of age. Design Observational cohort study. Setting and participants The Bandim Health Project's (BHP) rural Health and Demographic Surveillance site covers 258 randomly selected villages in all regions of Guinea-Bissau. Villages are visited biannually and vaccination cards inspected to ascertain vaccination status. Between 2003 and 2009 vaccination status by 12 months of age was assessed for 5806 children aged 12–23 months; vaccination status by 24 months of age was assessed for 3792 children aged 24–35 months. Outcome measures Coverage of EPI vaccinations and frequency of out-of-sequence vaccinations. Results Half of 12-month-old children and 65% of 24-month-old children had completed all EPI vaccinations. Many children received vaccines out of sequence: by 12 months of age 54% of BCG-vaccinated children had received DTP with or before BCG and 28% of measles-vaccinated children had received DTP with or after MV. By 24 months of age the proportion of out-of-sequence vaccinations was 58% and 35%, respectively, for BCG and MV. Conclusions In rural Guinea-Bissau vaccination coverage by 12 months of age was low, but continued to increase beyond 12 months of age. More than half of all children received vaccinations out of sequence. This highlights the need to improve vaccination services. PMID:23166127

Lessons for livestock genomics from genome and transcriptome sequencing in cattle and other mammals.

PubMed

Taylor, Jeremy F; Whitacre, Lynsey K; Hoff, Jesse L; Tizioto, Polyana C; Kim, JaeWoo; Decker, Jared E; Schnabel, Robert D

2016-08-17

Decreasing sequencing costs and development of new protocols for characterizing global methylation, gene expression patterns and regulatory regions have stimulated the generation of large livestock datasets. Here, we discuss experiences in the analysis of whole-genome and transcriptome sequence data. We analyzed whole-genome sequence (WGS) data from 132 individuals from five canid species (Canis familiaris, C. latrans, C. dingo, C. aureus and C. lupus) and 61 breeds, three bison (Bison bison), 64 water buffalo (Bubalus bubalis) and 297 bovines from 17 breeds. By individual, data vary in extent of reference genome depth of coverage from 4.9X to 64.0X. We have also analyzed RNA-seq data for 580 samples representing 159 Bos taurus and Rattus norvegicus animals and 98 tissues. By aligning reads to a reference assembly and calling variants, we assessed effects of average depth of coverage on the actual coverage and on the number of called variants. We examined the identity of unmapped reads by assembling them and querying produced contigs against the non-redundant nucleic acids database. By imputing high-density single nucleotide polymorphism data on 4010 US registered Angus animals to WGS using Run4 of the 1000 Bull Genomes Project and assessing the accuracy of imputation, we identified misassembled reference sequence regions. We estimate that a 24X depth of coverage is required to achieve 99.5 % coverage of the reference assembly and identify 95 % of the variants within an individual's genome. Genomes sequenced to low average coverage (e.g., <10X) may fail to cover 10 % of the reference genome and identify <75 % of variants. About 10 % of genomic DNA or transcriptome sequence reads fail to align to the reference assembly. These reads include loci missing from the reference assembly and misassembled genes and interesting symbionts, commensal and pathogenic organisms. Assembly errors and a lack of annotation of functional elements significantly limit the utility of the current draft livestock reference assemblies. The Functional Annotation of Animal Genomes initiative seeks to annotate functional elements, while a 70X Pac-Bio assembly for cow is underway and may result in a significantly improved reference assembly.

Genomic Characterization of DArT Markers Based on High-Density Linkage Analysis and Physical Mapping to the Eucalyptus Genome

PubMed Central

Petroli, César D.; Sansaloni, Carolina P.; Carling, Jason; Steane, Dorothy A.; Vaillancourt, René E.; Myburg, Alexander A.; da Silva, Orzenil Bonfim; Pappas, Georgios Joannis; Kilian, Andrzej; Grattapaglia, Dario

2012-01-01

Diversity Arrays Technology (DArT) provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for which no reference genome is yet available to allow such detailed characterization. PMID:22984541

Genome-wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNVs) are large insertions, deletions or duplications in the genome that vary between members of a species and are known to affect a wide variety of phenotypic traits. In this study, we identified CNVs in a population of bulls using low coverage next-generation sequence data....

Leaf Transcriptome Sequencing for Identifying Genic-SSR Markers and SNP Heterozygosity in Crossbred Mango Variety 'Amrapali' (Mangifera indica L.).

PubMed

Mahato, Ajay Kumar; Sharma, Nimisha; Singh, Akshay; Srivastav, Manish; Jaiprakash; Singh, Sanjay Kumar; Singh, Anand Kumar; Sharma, Tilak Raj; Singh, Nagendra Kumar

2016-01-01

Mango (Mangifera indica L.) is called "king of fruits" due to its sweetness, richness of taste, diversity, large production volume and a variety of end usage. Despite its huge economic importance genomic resources in mango are scarce and genetics of useful horticultural traits are poorly understood. Here we generated deep coverage leaf RNA sequence data for mango parental varieties 'Neelam', 'Dashehari' and their hybrid 'Amrapali' using next generation sequencing technologies. De-novo sequence assembly generated 27,528, 20,771 and 35,182 transcripts for the three genotypes, respectively. The transcripts were further assembled into a non-redundant set of 70,057 unigenes that were used for SSR and SNP identification and annotation. Total 5,465 SSR loci were identified in 4,912 unigenes with 288 type I SSR (n ≥ 20 bp). One hundred type I SSR markers were randomly selected of which 43 yielded PCR amplicons of expected size in the first round of validation and were designated as validated genic-SSR markers. Further, 22,306 SNPs were identified by aligning high quality sequence reads of the three mango varieties to the reference unigene set, revealing significantly enhanced SNP heterozygosity in the hybrid Amrapali. The present study on leaf RNA sequencing of mango varieties and their hybrid provides useful genomic resource for genetic improvement of mango.

Leaf Transcriptome Sequencing for Identifying Genic-SSR Markers and SNP Heterozygosity in Crossbred Mango Variety ‘Amrapali’ (Mangifera indica L.)

PubMed Central

Mahato, Ajay Kumar; Sharma, Nimisha; Singh, Akshay; Srivastav, Manish; Jaiprakash; Singh, Sanjay Kumar; Singh, Anand Kumar; Sharma, Tilak Raj; Singh, Nagendra Kumar

2016-01-01

Mango (Mangifera indica L.) is called “king of fruits” due to its sweetness, richness of taste, diversity, large production volume and a variety of end usage. Despite its huge economic importance genomic resources in mango are scarce and genetics of useful horticultural traits are poorly understood. Here we generated deep coverage leaf RNA sequence data for mango parental varieties ‘Neelam’, ‘Dashehari’ and their hybrid ‘Amrapali’ using next generation sequencing technologies. De-novo sequence assembly generated 27,528, 20,771 and 35,182 transcripts for the three genotypes, respectively. The transcripts were further assembled into a non-redundant set of 70,057 unigenes that were used for SSR and SNP identification and annotation. Total 5,465 SSR loci were identified in 4,912 unigenes with 288 type I SSR (n ≥ 20 bp). One hundred type I SSR markers were randomly selected of which 43 yielded PCR amplicons of expected size in the first round of validation and were designated as validated genic-SSR markers. Further, 22,306 SNPs were identified by aligning high quality sequence reads of the three mango varieties to the reference unigene set, revealing significantly enhanced SNP heterozygosity in the hybrid Amrapali. The present study on leaf RNA sequencing of mango varieties and their hybrid provides useful genomic resource for genetic improvement of mango. PMID:27736892

Exploring abundance, diversity and variation of a widespread antibiotic resistance gene in wastewater treatment plants.

PubMed

Wei, Ziyan; Feng, Kai; Li, Shuzhen; Zhang, Yu; Chen, Hongrui; Yin, Huaqun; Xu, Meiying; Deng, Ye

2018-05-09

An updated sul1 gene sequence database was constructed and new degenerate primers were designed to better investigate the abundance, diversity, and variation of a ubiquitous antibiotic resistance gene, sul1, with PCR-based methods in activated sludge from wastewater treatment plants (WWTPs). The newly designed degenerate primers showed high specificity and higher coverage in both in-silico evaluations and activated sludge samples compared to previous sul1 primers. Using the new primers, the abundance and diversity of sul1 gene, together with 16S rRNA gene, in activated sludge from five WWTPs in summer and winter were determined by quantitative PCR and MiSeq sequencing. The sul1 gene was found to be prevalent and displayed a comparable abundance (0.081 copies per bacterial cell in average) to the total bacteria across all samples. However, compared to the significant seasonal and geographical divergences in the quantity and diversity of bacterial communities in WWTPs, there were no significant seasonal or geographical variations of representative clusters of sul1 gene in most cases. Additionally, the representative sul1 clusters showed fairly close phylogeny and there was no obvious correlation between sul1 gene and the dominant bacterial genera, as well as the int1 gene, suggesting that bacterial hosts of sul1 gene is not stable, the sul1 gene may be carried by mobile genetic elements, sometimes integrated with class 1 integrons and sometimes not. Thus mobile genetic elements likely play a greater role than specific microbial taxa in determining the composition of sul1 gene in WWTPs. Copyright © 2018. Published by Elsevier Ltd.

Initial sequence and comparative analysis of the cat genome

PubMed Central

Pontius, Joan U.; Mullikin, James C.; Smith, Douglas R.; Lindblad-Toh, Kerstin; Gnerre, Sante; Clamp, Michele; Chang, Jean; Stephens, Robert; Neelam, Beena; Volfovsky, Natalia; Schäffer, Alejandro A.; Agarwala, Richa; Narfström, Kristina; Murphy, William J.; Giger, Urs; Roca, Alfred L.; Antunes, Agostinho; Menotti-Raymond, Marilyn; Yuhki, Naoya; Pecon-Slattery, Jill; Johnson, Warren E.; Bourque, Guillaume; Tesler, Glenn; O’Brien, Stephen J.

2007-01-01

The genome sequence (1.9-fold coverage) of an inbred Abyssinian domestic cat was assembled, mapped, and annotated with a comparative approach that involved cross-reference to annotated genome assemblies of six mammals (human, chimpanzee, mouse, rat, dog, and cow). The results resolved chromosomal positions for 663,480 contigs, 20,285 putative feline gene orthologs, and 133,499 conserved sequence blocks (CSBs). Additional annotated features include repetitive elements, endogenous retroviral sequences, nuclear mitochondrial (numt) sequences, micro-RNAs, and evolutionary breakpoints that suggest historic balancing of translocation and inversion incidences in distinct mammalian lineages. Large numbers of single nucleotide polymorphisms (SNPs), deletion insertion polymorphisms (DIPs), and short tandem repeats (STRs), suitable for linkage or association studies were characterized in the context of long stretches of chromosome homozygosity. In spite of the light coverage capturing ∼65% of euchromatin sequence from the cat genome, these comparative insights shed new light on the tempo and mode of gene/genome evolution in mammals, promise several research applications for the cat, and also illustrate that a comparative approach using more deeply covered mammals provides an informative, preliminary annotation of a light (1.9-fold) coverage mammal genome sequence. PMID:17975172

Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten.

PubMed

Nair, Shalima S; Luu, Phuc-Loi; Qu, Wenjia; Maddugoda, Madhavi; Huschtscha, Lily; Reddel, Roger; Chenevix-Trench, Georgia; Toso, Martina; Kench, James G; Horvath, Lisa G; Hayes, Vanessa M; Stricker, Phillip D; Hughes, Timothy P; White, Deborah L; Rasko, John E J; Wong, Justin J-L; Clark, Susan J

2018-05-28

Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing. In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×). Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.

RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing.

PubMed

Chang, Lun-Ching; Das, Biswajit; Lih, Chih-Jian; Si, Han; Camalier, Corinne E; McGregor, Paul M; Polley, Eric

2016-01-01

With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly (r = 0.96-0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearman's coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis.

Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics*

PubMed Central

Guo, Xiaofeng; Trudgian, David C.; Lemoff, Andrew; Yadavalli, Sivaramakrishna; Mirzaei, Hamid

2014-01-01

Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins determined that Asp-N yielded higher signal in five of eight cases. PMID:24696503

Effects of Re-vegetation on Herbaceous Species Composition and Biological Soil Crusts Development in a Coal Mine Dumping Site

NASA Astrophysics Data System (ADS)

Zhao, Yang; Zhang, Peng; Hu, Yigang; Huang, Lei

2016-02-01

Despite the critical roles of plant species' diversity and biological soil crusts (BSCs) in arid and semi-arid ecosystems, the restoration of the diversity of herbaceous species and BSCs are rarely discussed during the process of vegetation restoration of anthropogenically damaged areas in these regions. In this study, the herbaceous plant species composition, along with the BSCs coverage and thicknesses, was investigated at six different re-vegetation type sites, and the natural vegetation site of the Heidaigou open pit coal mine in China's Inner Mongolia Autonomous Region was used as a reference. The highest total species richness (16), as well as the species richness (4.4), occurred in the Tree and Herbaceous vegetation type site. The species composition similarities between the restored sites and the reference site were shown to be very low, and ranged from 0.09 to 0.42. Also, among the restored sites, the similarities of the species were fairly high and similar, and ranged from 0.45 to 0.93. The density and height of the re-vegetated woody plants were significantly correlated with the indexes of the diversity of the species. The Shrub vegetation type site showed the greatest total coverage (80 %) of BSCs and algae crust coverage (48 %). The Shrub and Herbaceous type had the greatest thicknesses of BSCs, with as much as 3.06 mm observed, which was followed by 2.64 mm for the Shrub type. There was a significant correlation observed between the coverage of the total BSCs, and the total vegetation and herbaceous vegetation coverage, as well as between the algae crust coverage and the herbaceous vegetation coverage. It has been suggested that the re-vegetated dwarf woody plant species (such as shrubs and semi-shrubs) should be chosen for the optimal methods of the restoration of herbaceous species diversity at dumping sites, and these should be planted with low density. Furthermore, the effects of vegetation coverage on the colonization and development the BSCs should be considered in order to reconstruct the vegetation in disturbed environments, such as mine dumpsites in arid areas.

Development, validation and application of specific primers for analyzing the clostridial diversity in dark fermentation pit mud by PCR-DGGE.

PubMed

Hu, Xiao-Long; Wang, Hai-Yan; Wu, Qun; Xu, Yan

2014-07-01

In this study, a Clostridia-specific primer set SJ-F and SJ-R, based on the available 16S rRNA genes sequences from database, was successfully designed and authenticated by theoretical and experimental evaluations. It targeted 19 clostridial families and unclassified_Clostridia with different coverage rates. The specificity and universality of novel primer set was tested again using the dark fermentation pit mud (FPM). It was demonstrated that a total of 13 closest relatives including 12 species were affiliated with 7 clostridial genera, respectively. Compared to the well-accepted bacterial universal primer pair P2/P3, five unexpected clostridial genera including Roseburia, Tissierella, Sporanaerobacter, Alkalibacter and Halothermothrix present in the FPM were also revealed. Therefore, this study could provide a good alternative to investigate the clostridial diversity and monitor their population dynamics rapidly and efficiently in various anaerobic environments and dark fermentation systems in future. Copyright © 2014 Elsevier Ltd. All rights reserved.

Human body epigenome maps reveal noncanonical DNA methylation variation.

PubMed

Schultz, Matthew D; He, Yupeng; Whitaker, John W; Hariharan, Manoj; Mukamel, Eran A; Leung, Danny; Rajagopal, Nisha; Nery, Joseph R; Urich, Mark A; Chen, Huaming; Lin, Shin; Lin, Yiing; Jung, Inkyung; Schmitt, Anthony D; Selvaraj, Siddarth; Ren, Bing; Sejnowski, Terrence J; Wang, Wei; Ecker, Joseph R

2015-07-09

Understanding the diversity of human tissues is fundamental to disease and requires linking genetic information, which is identical in most of an individual's cells, with epigenetic mechanisms that could have tissue-specific roles. Surveys of DNA methylation in human tissues have established a complex landscape including both tissue-specific and invariant methylation patterns. Here we report high coverage methylomes that catalogue cytosine methylation in all contexts for the major human organ systems, integrated with matched transcriptomes and genomic sequence. By combining these diverse data types with each individuals' phased genome, we identified widespread tissue-specific differential CG methylation (mCG), partially methylated domains, allele-specific methylation and transcription, and the unexpected presence of non-CG methylation (mCH) in almost all human tissues. mCH correlated with tissue-specific functions, and using this mark, we made novel predictions of genes that escape X-chromosome inactivation in specific tissues. Overall, DNA methylation in several genomic contexts varies substantially among human tissues.

Calibrating genomic and allelic coverage bias in single-cell sequencing.

PubMed

Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher

2015-04-16

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.

Calibrating genomic and allelic coverage bias in single-cell sequencing

PubMed Central

Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher

2016-01-01

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913

Mapping HLA-A2, -A3 and -B7 supertype-restricted T-cell epitopes in the ebolavirus proteome.

PubMed

Lim, Wan Ching; Khan, Asif M

2018-01-19

Ebolavirus (EBOV) is responsible for one of the most fatal diseases encountered by mankind. Cellular T-cell responses have been implicated to be important in providing protection against the virus. Antigenic variation can result in viral escape from immune recognition. Mapping targets of immune responses among the sequence of viral proteins is, thus, an important first step towards understanding the immune responses to viral variants and can aid in the identification of vaccine targets. Herein, we performed a large-scale, proteome-wide mapping and diversity analyses of putative HLA supertype-restricted T-cell epitopes of Zaire ebolavirus (ZEBOV), the most pathogenic species among the EBOV family. All publicly available ZEBOV sequences (14,098) for each of the nine viral proteins were retrieved, removed of irrelevant and duplicate sequences, and aligned. The overall proteome diversity of the non-redundant sequences was studied by use of Shannon's entropy. The sequences were predicted, by use of the NetCTLpan server, for HLA-A2, -A3, and -B7 supertype-restricted epitopes, which are relevant to African and other ethnicities and provide for large (~86%) population coverage. The predicted epitopes were mapped to the alignment of each protein for analyses of antigenic sequence diversity and relevance to structure and function. The putative epitopes were validated by comparison with experimentally confirmed epitopes. ZEBOV proteome was generally conserved, with an average entropy of 0.16. The 185 HLA supertype-restricted T-cell epitopes predicted (82 (A2), 37 (A3) and 66 (B7)) mapped to 125 alignment positions and covered ~24% of the proteome length. Many of the epitopes showed a propensity to co-localize at select positions of the alignment. Thirty (30) of the mapped positions were completely conserved and may be attractive for vaccine design. The remaining (95) positions had one or more epitopes, with or without non-epitope variants. A significant number (24) of the putative epitopes matched reported experimentally validated HLA ligands/T-cell epitopes of A2, A3 and/or B7 supertype representative allele restrictions. The epitopes generally corresponded to functional motifs/domains and there was no correlation to localization on the protein 3D structure. These data and the epitope map provide important insights into the interaction between EBOV and the host immune system.

Low-level APC mutational mosaicism is the underlying cause in a substantial fraction of unexplained colorectal adenomatous polyposis cases.

PubMed

Spier, Isabel; Drichel, Dmitriy; Kerick, Martin; Kirfel, Jutta; Horpaopan, Sukanya; Laner, Andreas; Holzapfel, Stefanie; Peters, Sophia; Adam, Ronja; Zhao, Bixiao; Becker, Tim; Lifton, Richard P; Perner, Sven; Hoffmann, Per; Kristiansen, Glen; Timmermann, Bernd; Nöthen, Markus M; Holinski-Feder, Elke; Schweiger, Michal R; Aretz, Stefan

2016-03-01

In 30-50% of patients with colorectal adenomatous polyposis, no germline mutation in the known genes APC, causing familial adenomatous polyposis, MUTYH, causing MUTYH-associated polyposis, or POLE or POLD1, causing polymerase-proofreading-associated polyposis can be identified, although a hereditary aetiology is likely. This study aimed to explore the impact of APC mutational mosaicism in unexplained polyposis. To comprehensively screen for somatic low-level APC mosaicism, high-coverage next-generation sequencing of the APC gene was performed using DNA from leucocytes and a total of 53 colorectal tumours from 20 unrelated patients with unexplained sporadic adenomatous polyposis. APC mosaicism was assumed if the same loss-of-function APC mutation was present in ≥ 2 anatomically separated colorectal adenomas/carcinomas per patient. All mutations were validated using diverse methods. In 25% (5/20) of patients, somatic mosaicism of a pathogenic APC mutation was identified as underlying cause of the disease. In 2/5 cases, the mosaic level in leucocyte DNA was slightly below the sensitivity threshold of Sanger sequencing; while in 3/5 cases, the allelic fraction was either very low (0.1-1%) or no mutations were detectable. The majority of mosaic mutations were located outside the somatic mutation cluster region of the gene. The present data indicate a high prevalence of pathogenic mosaic APC mutations below the detection thresholds of routine diagnostics in adenomatous polyposis, even if high-coverage sequencing of leucocyte DNA alone is taken into account. This has important implications for both routine work-up and strategies to identify new causative genes in this patient group. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree

PubMed Central

2013-01-01

Background With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. Results The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. Conclusions Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology. PMID:23497206

Mining and validation of pyrosequenced simple sequence repeats (SSRs) from American cranberry (Vaccinium macrocarpon Ait.).

PubMed

Zhu, H; Senalik, D; McCown, B H; Zeldin, E L; Speers, J; Hyman, J; Bassil, N; Hummer, K; Simon, P W; Zalapa, J E

2012-01-01

The American cranberry (Vaccinium macrocarpon Ait.) is a major commercial fruit crop in North America, but limited genetic resources have been developed for the species. Furthermore, the paucity of codominant DNA markers has hampered the advance of genetic research in cranberry and the Ericaceae family in general. Therefore, we used Roche 454 sequencing technology to perform low-coverage whole genome shotgun sequencing of the cranberry cultivar 'HyRed'. After de novo assembly, the obtained sequence covered 266.3 Mb of the estimated 540-590 Mb in cranberry genome. A total of 107,244 SSR loci were detected with an overall density across the genome of 403 SSR/Mb. The AG repeat was the most frequent motif in cranberry accounting for 35% of all SSRs and together with AAG and AAAT accounted for 46% of all loci discovered. To validate the SSR loci, we designed 96 primer-pairs using contig sequence data containing perfect SSR repeats, and studied the genetic diversity of 25 cranberry genotypes. We identified 48 polymorphic SSR loci with 2-15 alleles per locus for a total of 323 alleles in the 25 cranberry genotypes. Genetic clustering by principal coordinates and genetic structure analyzes confirmed the heterogeneous nature of cranberries. The parentage composition of several hybrid cultivars was evident from the structure analyzes. Whole genome shotgun 454 sequencing was a cost-effective and efficient way to identify numerous SSR repeats in the cranberry sequence for marker development.

Front-End Electron Transfer Dissociation Coupled to a 21 Tesla FT-ICR Mass Spectrometer for Intact Protein Sequence Analysis

NASA Astrophysics Data System (ADS)

Weisbrod, Chad R.; Kaiser, Nathan K.; Syka, John E. P.; Early, Lee; Mullen, Christopher; Dunyach, Jean-Jacques; English, A. Michelle; Anderson, Lissa C.; Blakney, Greg T.; Shabanowitz, Jeffrey; Hendrickson, Christopher L.; Marshall, Alan G.; Hunt, Donald F.

2017-09-01

High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., 60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. [Figure not available: see fulltext.

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations

PubMed Central

Zhou, Shuntai; Jones, Corbin; Mieczkowski, Piotr

2015-01-01

ABSTRACT Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS. IMPORTANCE Although next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set. PMID:26041299

Immobilized Pepsin Microreactor for Rapid Peptide Mapping with Nanoelectrospray Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Long, Ying; Wood, Troy D.

2015-01-01

Most enzymatic microreactors for protein digestion are based on trypsin, but proteins with hydrophobic segments may be difficult to digest because of the paucity of Arg and Lys residues. Microreactors based on pepsin, which is less specific than trypsin, can overcome this challenge. Here, an integrated immobilized pepsin microreactor (IPMR)/nanoelectrospray emitter is examined for its potential for peptide mapping. For myoglobin, equivalent sequence coverage is obtained in a thousandth the time of solution digestion with better sequence coverage. While sequence coverage of cytochrome c is lesser than solution in this short duration, more highly-charged peptic peptides are produced and a number of peaks are unidentified at low-resolution, suggesting that high-resolution mass spectrometry is needed to take full advantage of integrated IPMR/nanoelectrospray devices.

Mapping and analysis of Caenorhabditis elegans transcription factor sequence specificities

PubMed Central

Narasimhan, Kamesh; Lambert, Samuel A; Yang, Ally WH; Riddell, Jeremy; Mnaimneh, Sanie; Zheng, Hong; Albu, Mihai; Najafabadi, Hamed S; Reece-Hoyes, John S; Fuxman Bass, Juan I; Walhout, Albertha JM; Weirauch, Matthew T; Hughes, Timothy R

2015-01-01

Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been limited, as DNA-binding motifs are known for only 71 of the estimated 763 sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray experiments on representatives of canonical TF families in C. elegans, obtaining motifs for 129 TFs. Additionally, we predict motifs for many TFs that have DNA-binding domains similar to those already characterized, increasing coverage of binding specificities to 292 C. elegans TFs (∼40%). These data highlight the diversification of binding motifs for the nuclear hormone receptor and C2H2 zinc finger families and reveal unexpected diversity of motifs for T-box and DM families. Motif enrichment in promoters of functionally related genes is consistent with known biology and also identifies putative regulatory roles for unstudied TFs. DOI: http://dx.doi.org/10.7554/eLife.06967.001 PMID:25905672

Landscape of genomic diversity and trait discovery in soybean.

PubMed

Valliyodan, Babu; Dan Qiu; Patil, Gunvant; Zeng, Peng; Huang, Jiaying; Dai, Lu; Chen, Chengxuan; Li, Yanjun; Joshi, Trupti; Song, Li; Vuong, Tri D; Musket, Theresa A; Xu, Dong; Shannon, J Grover; Shifeng, Cheng; Liu, Xin; Nguyen, Henry T

2016-03-31

Cultivated soybean [Glycine max (L.) Merr.] is a primary source of vegetable oil and protein. We report a landscape analysis of genome-wide genetic variation and an association study of major domestication and agronomic traits in soybean. A total of 106 soybean genomes representing wild, landraces, and elite lines were re-sequenced at an average of 17x depth with a 97.5% coverage. Over 10 million high-quality SNPs were discovered, and 35.34% of these have not been previously reported. Additionally, 159 putative domestication sweeps were identified, which includes 54.34 Mbp (4.9%) and 4,414 genes; 146 regions were involved in artificial selection during domestication. A genome-wide association study of major traits including oil and protein content, salinity, and domestication traits resulted in the discovery of novel alleles. Genomic information from this study provides a valuable resource for understanding soybean genome structure and evolution, and can also facilitate trait dissection leading to sequencing-based molecular breeding.

Landscape of genomic diversity and trait discovery in soybean

PubMed Central

Valliyodan, Babu; Dan Qiu; Patil, Gunvant; Zeng, Peng; Huang, Jiaying; Dai, Lu; Chen, Chengxuan; Li, Yanjun; Joshi, Trupti; Song, Li; Vuong, Tri D.; Musket, Theresa A.; Xu, Dong; Shannon, J. Grover; Shifeng, Cheng; Liu, Xin; Nguyen, Henry T.

2016-01-01

Cultivated soybean [Glycine max (L.) Merr.] is a primary source of vegetable oil and protein. We report a landscape analysis of genome-wide genetic variation and an association study of major domestication and agronomic traits in soybean. A total of 106 soybean genomes representing wild, landraces, and elite lines were re-sequenced at an average of 17x depth with a 97.5% coverage. Over 10 million high-quality SNPs were discovered, and 35.34% of these have not been previously reported. Additionally, 159 putative domestication sweeps were identified, which includes 54.34 Mbp (4.9%) and 4,414 genes; 146 regions were involved in artificial selection during domestication. A genome-wide association study of major traits including oil and protein content, salinity, and domestication traits resulted in the discovery of novel alleles. Genomic information from this study provides a valuable resource for understanding soybean genome structure and evolution, and can also facilitate trait dissection leading to sequencing-based molecular breeding. PMID:27029319

Bacterial community dynamics during the early stages of biofilm formation in a chlorinated experimental drinking water distribution system: implications for drinking water discolouration.

PubMed

Douterelo, I; Sharpe, R; Boxall, J

2014-07-01

To characterize bacterial communities during the early stages of biofilm formation and their role in water discolouration in a fully representative, chlorinated, experimental drinking water distribution systems (DWDS). Biofilm development was monitored in an experimental DWDS over 28 days; subsequently the system was disturbed by raising hydraulic conditions to simulate pipe burst, cleaning or other system conditions. Biofilm cell cover was monitored by fluorescent microscopy and a fingerprinting technique used to assess changes in bacterial community. Selected samples were analysed by cloning and sequencing of the 16S rRNA gene. Fingerprinting analysis revealed significant changes in the bacterial community structure over time (P < 0·05). Cell coverage increased over time accompanied by an increase in bacterial richness and diversity. Shifts in the bacterial community structure were observed along with an increase in cell coverage, bacterial richness and diversity. Species related to Pseudomonas spp. and Janthinobacterium spp. dominated the process of initial attachment. Based on fingerprinting results, the hydraulic regimes did not affect the bacteriological composition of biofilms, but they did influence their mechanical stability. This study gives a better insight into the early stages of biofilm formation in DWDS and will contribute to the improvement of management strategies to control the formation of biofilms and the risk of discolouration. © 2014 The Authors. published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.

Bacterial community dynamics during the early stages of biofilm formation in a chlorinated experimental drinking water distribution system: implications for drinking water discolouration

PubMed Central

Douterelo, I; Sharpe, R; Boxall, J

2014-01-01

Aims To characterize bacterial communities during the early stages of biofilm formation and their role in water discolouration in a fully representative, chlorinated, experimental drinking water distribution systems (DWDS). Methods and Results Biofilm development was monitored in an experimental DWDS over 28 days; subsequently the system was disturbed by raising hydraulic conditions to simulate pipe burst, cleaning or other system conditions. Biofilm cell cover was monitored by fluorescent microscopy and a fingerprinting technique used to assess changes in bacterial community. Selected samples were analysed by cloning and sequencing of the 16S rRNA gene. Fingerprinting analysis revealed significant changes in the bacterial community structure over time (P < 0·05). Cell coverage increased over time accompanied by an increase in bacterial richness and diversity. Conclusions Shifts in the bacterial community structure were observed along with an increase in cell coverage, bacterial richness and diversity. Species related to Pseudomonas spp. and Janthinobacterium spp. dominated the process of initial attachment. Based on fingerprinting results, the hydraulic regimes did not affect the bacteriological composition of biofilms, but they did influence their mechanical stability. Significance and Importance of the Study This study gives a better insight into the early stages of biofilm formation in DWDS and will contribute to the improvement of management strategies to control the formation of biofilms and the risk of discolouration. PMID:24712449

Leadership and Diversity

ERIC Educational Resources Information Center

Coleman, Marianne

2012-01-01

As part of the special edition recognizing the 40th anniversary of "Educational Management Administration & Leadership" this article reviews the coverage of leadership and diversity issues in the journal. The majority of articles concerning diversity have focused on gender, with attention turning to the wider concept of diversity since the year…

Identification of an EMS-induced causal mutation in a gene required for boron-mediated root development by low-coverage genome re-sequencing in Arabidopsis

PubMed Central

Tabata, Ryo; Kamiya, Takehiro; Shigenobu, Shuji; Yamaguchi, Katsushi; Yamada, Masashi; Hasebe, Mitsuyasu; Fujiwara, Toru; Sawa, Shinichiro

2013-01-01

Next-generation sequencing (NGS) technologies enable the rapid production of an enormous quantity of sequence data. These powerful new technologies allow the identification of mutations by whole-genome sequencing. However, most reported NGS-based mapping methods, which are based on bulked segregant analysis, are costly and laborious. To address these limitations, we designed a versatile NGS-based mapping method that consists of a combination of low- to medium-coverage multiplex SOLiD (Sequencing by Oligonucleotide Ligation and Detection) and classical genetic rough mapping. Using only low to medium coverage reduces the SOLiD sequencing costs and, since just 10 to 20 mutant F2 plants are required for rough mapping, the operation is simple enough to handle in a laboratory with limited space and funding. As a proof of principle, we successfully applied this method to identify the CTR1, which is involved in boron-mediated root development, from among a population of high boron requiring Arabidopsis thaliana mutants. Our work demonstrates that this NGS-based mapping method is a moderately priced and versatile method that can readily be applied to other model organisms. PMID:23104114

A Poisson hierarchical modelling approach to detecting copy number variation in sequence coverage data

PubMed Central

2013-01-01

Background The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Results Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. Conclusions In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data. PMID:23442253

A global map of genetic diversity in Babesia microti reveals strong population structure and identifies variants associated with clinical relapse

PubMed Central

Lemieux, Jacob E.; Tran, Alice D.; Freimark, Lisa; Schaffner, Stephen F.; Goethert, Heidi; Andersen, Kristian G.; Bazner, Suzane; Li, Amy; McGrath, Graham; Sloan, Lynne; Vannier, Edouard; Milner, Dan; Pritt, Bobbi; Rosenberg, Eric; Telford, Sam; Bailey, Jeffrey A.; Sabeti, Pardis C.

2017-01-01

Human babesiosis caused by Babesia microti is an emerging tick-borne zoonosis of increasing importance due to rising incidence and expanding geographic range1. Infection with this organism, an intraerythrocytic parasite of the phylum Apicomplexa, causes a febrile syndrome similar to malaria2. Relapsing disease is common among immunocompromised and asplenic individuals3,4, and drug resistance has recently been reported5. To investigate the origin and genetic diversity of this parasite, we sequenced the complete genomes of 42 B. microti samples from around the world, including deep coverage of clinical infections at endemic sites in the continental United States. Samples from the continental US segregate into a Northeast lineage and a Midwest lineage, with subsequent divergence of subpopulations along geographic lines. We identify parasite variants that associate with relapsing disease, including amino acid substitutions in the atovaquone-binding regions of cytochrome b (cytb) and the azithromycin-binding region of ribosomal protein subunit L4 (rpl4). Our results shed light on the origin, diversity, and evolution of B. microti, suggest possible mechanisms for clinical relapse, and create the foundation for further research on this emerging pathogen. PMID:27572973

A global map of genetic diversity in Babesia microti reveals strong population structure and identifies variants associated with clinical relapse.

PubMed

Lemieux, Jacob E; Tran, Alice D; Freimark, Lisa; Schaffner, Stephen F; Goethert, Heidi; Andersen, Kristian G; Bazner, Suzane; Li, Amy; McGrath, Graham; Sloan, Lynne; Vannier, Edouard; Milner, Dan; Pritt, Bobbi; Rosenberg, Eric; Telford, Sam; Bailey, Jeffrey A; Sabeti, Pardis C

2016-06-13

Human babesiosis caused by Babesia microti is an emerging tick-borne zoonosis of increasing importance due to its rising incidence and expanding geographic range(1). Infection with this organism, an intraerythrocytic parasite of the phylum Apicomplexa, causes a febrile syndrome similar to malaria(2). Relapsing disease is common among immunocompromised and asplenic individuals(3,4) and drug resistance has recently been reported(5). To investigate the origin and genetic diversity of this parasite, we sequenced the complete genomes of 42 B. microti samples from around the world, including deep coverage of clinical infections at endemic sites in the continental USA. Samples from the continental USA segregate into a Northeast lineage and a Midwest lineage, with subsequent divergence of subpopulations along geographic lines. We identify parasite variants that associate with relapsing disease, including amino acid substitutions in the atovaquone-binding regions of cytochrome b (cytb) and the azithromycin-binding region of ribosomal protein subunit L4 (rpl4). Our results shed light on the origin, diversity and evolution of B. microti, suggest possible mechanisms for clinical relapse, and create the foundation for further research on this emerging pathogen.

Analysis of quality raw data of second generation sequencers with Quality Assessment Software.

PubMed

Ramos, Rommel Tj; Carneiro, Adriana R; Baumbach, Jan; Azevedo, Vasco; Schneider, Maria Pc; Silva, Artur

2011-04-18

Second generation technologies have advantages over Sanger; however, they have resulted in new challenges for the genome construction process, especially because of the small size of the reads, despite the high degree of coverage. Independent of the program chosen for the construction process, DNA sequences are superimposed, based on identity, to extend the reads, generating contigs; mismatches indicate a lack of homology and are not included. This process improves our confidence in the sequences that are generated. We developed Quality Assessment Software, with which one can review graphs showing the distribution of quality values from the sequencing reads. This software allow us to adopt more stringent quality standards for sequence data, based on quality-graph analysis and estimated coverage after applying the quality filter, providing acceptable sequence coverage for genome construction from short reads. Quality filtering is a fundamental step in the process of constructing genomes, as it reduces the frequency of incorrect alignments that are caused by measuring errors, which can occur during the construction process due to the size of the reads, provoking misassemblies. Application of quality filters to sequence data, using the software Quality Assessment, along with graphing analyses, provided greater precision in the definition of cutoff parameters, which increased the accuracy of genome construction.

Population genetic inference from personal genome data: impact of ancestry and admixture on human genomic variation.

PubMed

Kidd, Jeffrey M; Gravel, Simon; Byrnes, Jake; Moreno-Estrada, Andres; Musharoff, Shaila; Bryc, Katarzyna; Degenhardt, Jeremiah D; Brisbin, Abra; Sheth, Vrunda; Chen, Rong; McLaughlin, Stephen F; Peckham, Heather E; Omberg, Larsson; Bormann Chung, Christina A; Stanley, Sarah; Pearlstein, Kevin; Levandowsky, Elizabeth; Acevedo-Acevedo, Suehelay; Auton, Adam; Keinan, Alon; Acuña-Alonzo, Victor; Barquera-Lozano, Rodrigo; Canizales-Quinteros, Samuel; Eng, Celeste; Burchard, Esteban G; Russell, Archie; Reynolds, Andy; Clark, Andrew G; Reese, Martin G; Lincoln, Stephen E; Butte, Atul J; De La Vega, Francisco M; Bustamante, Carlos D

2012-10-05

Full sequencing of individual human genomes has greatly expanded our understanding of human genetic variation and population history. Here, we present a systematic analysis of 50 human genomes from 11 diverse global populations sequenced at high coverage. Our sample includes 12 individuals who have admixed ancestry and who have varying degrees of recent (within the last 500 years) African, Native American, and European ancestry. We found over 21 million single-nucleotide variants that contribute to a 1.75-fold range in nucleotide heterozygosity across diverse human genomes. This heterozygosity ranged from a high of one heterozygous site per kilobase in west African genomes to a low of 0.57 heterozygous sites per kilobase in segments inferred to have diploid Native American ancestry from the genomes of Mexican and Puerto Rican individuals. We show evidence of all three continental ancestries in the genomes of Mexican, Puerto Rican, and African American populations, and the genome-wide statistics are highly consistent across individuals from a population once ancestry proportions have been accounted for. Using a generalized linear model, we identified subtle variations across populations in the proportion of neutral versus deleterious variation and found that genome-wide statistics vary in admixed populations even once ancestry proportions have been factored in. We further infer that multiple periods of gene flow shaped the diversity of admixed populations in the Americas-70% of the European ancestry in today's African Americans dates back to European gene flow happening only 7-8 generations ago. Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Population Genetic Inference from Personal Genome Data: Impact of Ancestry and Admixture on Human Genomic Variation

PubMed Central

Kidd, Jeffrey M.; Gravel, Simon; Byrnes, Jake; Moreno-Estrada, Andres; Musharoff, Shaila; Bryc, Katarzyna; Degenhardt, Jeremiah D.; Brisbin, Abra; Sheth, Vrunda; Chen, Rong; McLaughlin, Stephen F.; Peckham, Heather E.; Omberg, Larsson; Bormann Chung, Christina A.; Stanley, Sarah; Pearlstein, Kevin; Levandowsky, Elizabeth; Acevedo-Acevedo, Suehelay; Auton, Adam; Keinan, Alon; Acuña-Alonzo, Victor; Barquera-Lozano, Rodrigo; Canizales-Quinteros, Samuel; Eng, Celeste; Burchard, Esteban G.; Russell, Archie; Reynolds, Andy; Clark, Andrew G.; Reese, Martin G.; Lincoln, Stephen E.; Butte, Atul J.; De La Vega, Francisco M.; Bustamante, Carlos D.

2012-01-01

Full sequencing of individual human genomes has greatly expanded our understanding of human genetic variation and population history. Here, we present a systematic analysis of 50 human genomes from 11 diverse global populations sequenced at high coverage. Our sample includes 12 individuals who have admixed ancestry and who have varying degrees of recent (within the last 500 years) African, Native American, and European ancestry. We found over 21 million single-nucleotide variants that contribute to a 1.75-fold range in nucleotide heterozygosity across diverse human genomes. This heterozygosity ranged from a high of one heterozygous site per kilobase in west African genomes to a low of 0.57 heterozygous sites per kilobase in segments inferred to have diploid Native American ancestry from the genomes of Mexican and Puerto Rican individuals. We show evidence of all three continental ancestries in the genomes of Mexican, Puerto Rican, and African American populations, and the genome-wide statistics are highly consistent across individuals from a population once ancestry proportions have been accounted for. Using a generalized linear model, we identified subtle variations across populations in the proportion of neutral versus deleterious variation and found that genome-wide statistics vary in admixed populations even once ancestry proportions have been factored in. We further infer that multiple periods of gene flow shaped the diversity of admixed populations in the Americas—70% of the European ancestry in today’s African Americans dates back to European gene flow happening only 7–8 generations ago. PMID:23040495

MCMC-ODPR: primer design optimization using Markov Chain Monte Carlo sampling.

PubMed

Kitchen, James L; Moore, Jonathan D; Palmer, Sarah A; Allaby, Robin G

2012-11-05

Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base.

MCMC-ODPR: Primer design optimization using Markov Chain Monte Carlo sampling

PubMed Central

2012-01-01

Background Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. Results After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. Conclusions MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base. PMID:23126469

Optimizing and evaluating the reconstruction of Metagenome-assembled microbial genomes.

PubMed

Papudeshi, Bhavya; Haggerty, J Matthew; Doane, Michael; Morris, Megan M; Walsh, Kevin; Beattie, Douglas T; Pande, Dnyanada; Zaeri, Parisa; Silva, Genivaldo G Z; Thompson, Fabiano; Edwards, Robert A; Dinsdale, Elizabeth A

2017-11-28

Microbiome/host interactions describe characteristics that affect the host's health. Shotgun metagenomics includes sequencing a random subset of the microbiome to analyze its taxonomic and metabolic potential. Reconstruction of DNA fragments into genomes from metagenomes (called metagenome-assembled genomes) assigns unknown fragments to taxa/function and facilitates discovery of novel organisms. Genome reconstruction incorporates sequence assembly and sorting of assembled sequences into bins, characteristic of a genome. However, the microbial community composition, including taxonomic and phylogenetic diversity may influence genome reconstruction. We determine the optimal reconstruction method for four microbiome projects that had variable sequencing platforms (IonTorrent and Illumina), diversity (high or low), and environment (coral reefs and kelp forests), using a set of parameters to select for optimal assembly and binning tools. We tested the effects of the assembly and binning processes on population genome reconstruction using 105 marine metagenomes from 4 projects. Reconstructed genomes were obtained from each project using 3 assemblers (IDBA, MetaVelvet, and SPAdes) and 2 binning tools (GroopM and MetaBat). We assessed the efficiency of assemblers using statistics that including contig continuity and contig chimerism and the effectiveness of binning tools using genome completeness and taxonomic identification. We concluded that SPAdes, assembled more contigs (143,718 ± 124 contigs) of longer length (N50 = 1632 ± 108 bp), and incorporated the most sequences (sequences-assembled = 19.65%). The microbial richness and evenness were maintained across the assembly, suggesting low contig chimeras. SPAdes assembly was responsive to the biological and technological variations within the project, compared with other assemblers. Among binning tools, we conclude that MetaBat produced bins with less variation in GC content (average standard deviation: 1.49), low species richness (4.91 ± 0.66), and higher genome completeness (40.92 ± 1.75) across all projects. MetaBat extracted 115 bins from the 4 projects of which 66 bins were identified as reconstructed metagenome-assembled genomes with sequences belonging to a specific genus. We identified 13 novel genomes, some of which were 100% complete, but show low similarity to genomes within databases. In conclusion, we present a set of biologically relevant parameters for evaluation to select for optimal assembly and binning tools. For the tools we tested, SPAdes assembler and MetaBat binning tools reconstructed quality metagenome-assembled genomes for the four projects. We also conclude that metagenomes from microbial communities that have high coverage of phylogenetically distinct, and low taxonomic diversity results in highest quality metagenome-assembled genomes.

Genomic resources and genetic diversity of captive lesser kudu (Tragelaphus imberbis).

PubMed

Bock, Friederike; Gallus, Susanne; Janke, Axel; Hailer, Frank; Steck, Beatrice L; Kumar, Vikas; Nilsson, Maria A

2014-01-01

The lesser kudu (Tragelaphus imberbis) is a spiral-horned antelope native to northeastern Africa. Individuals kept in zoological gardens are suspected to be highly inbred due to few founder individuals and a small breeding stock. A morphological study suggested two distinct subspecies of the lesser kudu. However, subspecies designation and population structure in zoological gardens has not been analyzed using molecular markers. We analyzed one mitochondrial marker and two nuclear intron loci (total: 2,239 nucleotides) in 52 lesser kudu individuals. Of these, 48 individuals were bred in captivity and sampled from seven different zoos. The four remaining individuals were recently captured in Somalia and are currently held in the Maktoum zoo. Maternally inherited mitochondrial sequences indicate substantial amounts of genetic variation in the zoo populations, while the biparentally inherited intron sequences are, as expected, less variable. The analyzed individuals show 10 mitochondrial haplotypes with a maximal distance of 10 mutational steps. No prominent subspecies structure is detectable in this study. For further studies of the lesser kudu population genetics, we present microsatellite markers from a low-coverage genome survey using 454 sequencing technology. © 2014 Wiley Periodicals, Inc.

The international Genome sample resource (IGSR): A worldwide collection of genome variation incorporating the 1000 Genomes Project data

PubMed Central

Clarke, Laura; Fairley, Susan; Zheng-Bradley, Xiangqun; Streeter, Ian; Perry, Emily; Lowy, Ernesto; Tassé, Anne-Marie; Flicek, Paul

2017-01-01

The International Genome Sample Resource (IGSR; http://www.internationalgenome.org) expands in data type and population diversity the resources from the 1000 Genomes Project. IGSR represents the largest open collection of human variation data and provides easy access to these resources. IGSR was established in 2015 to maintain and extend the 1000 Genomes Project data, which has been widely used as a reference set of human variation and by researchers developing analysis methods. IGSR has mapped all of the 1000 Genomes sequence to the newest human reference (GRCh38), and will release updated variant calls to ensure maximal usefulness of the existing data. IGSR is collecting new structural variation data on the 1000 Genomes samples from long read sequencing and other technologies, and will collect relevant functional data into a single comprehensive resource. IGSR is extending coverage with new populations sequenced by collaborating groups. Here, we present the new data and analysis that IGSR has made available. We have also introduced a new data portal that increases discoverability of our data—previously only browseable through our FTP site—by focusing on particular samples, populations or data sets of interest. PMID:27638885

High-Throughput Next-Generation Sequencing of Polioviruses

PubMed Central

Montmayeur, Anna M.; Schmidt, Alexander; Zhao, Kun; Magaña, Laura; Iber, Jane; Castro, Christina J.; Chen, Qi; Henderson, Elizabeth; Ramos, Edward; Shaw, Jing; Tatusov, Roman L.; Dybdahl-Sissoko, Naomi; Endegue-Zanga, Marie Claire; Adeniji, Johnson A.; Oberste, M. Steven; Burns, Cara C.

2016-01-01

ABSTRACT The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance. PMID:27927929

Genome survey of pistachio (Pistacia vera L.) by next generation sequencing: Development of novel SSR markers and genetic diversity in Pistacia species.

PubMed

Ziya Motalebipour, Elmira; Kafkas, Salih; Khodaeiaminjan, Mortaza; Çoban, Nergiz; Gözel, Hatice

2016-12-07

Pistachio (Pistacia vera L.) is one of the most important nut crops in the world. There are about 11 wild species in the genus Pistacia, and they have importance as rootstock seed sources for cultivated P. vera and forest trees. Published information on the pistachio genome is limited. Therefore, a genome survey is necessary to obtain knowledge on the genome structure of pistachio by next generation sequencing. Simple sequence repeat (SSR) markers are useful tools for germplasm characterization, genetic diversity analysis, and genetic linkage mapping, and may help to elucidate genetic relationships among pistachio cultivars and species. To explore the genome structure of pistachio, a genome survey was performed using the Illumina platform at approximately 40× coverage depth in the P. vera cv. Siirt. The K-mer analysis indicated that pistachio has a genome that is about 600 Mb in size and is highly heterozygous. The assembly of 26.77 Gb Illumina data produced 27,069 scaffolds at N50 = 3.4 kb with a total of 513.5 Mb. A total of 59,280 SSR motifs were detected with a frequency of 8.67 kb. A total of 206 SSRs were used to characterize 24 P. vera cultivars and 20 wild Pistacia genotypes (four genotypes from each five wild Pistacia species) belonging to P. atlantica, P. integerrima, P. chinenesis, P. terebinthus, and P. lentiscus genotypes. Overall 135 SSR loci amplified in all 44 cultivars and genotypes, 41 were polymorphic in six Pistacia species. The novel SSR loci developed from cultivated pistachio were highly transferable to wild Pistacia species. The results from a genome survey of pistachio suggest that the genome size of pistachio is about 600 Mb with a high heterozygosity rate. This information will help to design whole genome sequencing strategies for pistachio. The newly developed novel polymorphic SSRs in this study may help germplasm characterization, genetic diversity, and genetic linkage mapping studies in the genus Pistacia.

The importance of standardization for biodiversity comparisons: A case study using autonomous reef monitoring structures (ARMS) and metabarcoding to measure cryptic diversity on Mo’orea coral reefs, French Polynesia

PubMed Central

Geller, Jonathan B.; Timmers, Molly; Leray, Matthieu; Mahardini, Angka; Sembiring, Andrianus; Collins, Allen G.; Meyer, Christopher P.

2017-01-01

The advancement of metabarcoding techniques, declining costs of high-throughput sequencing and development of systematic sampling devices, such as autonomous reef monitoring structures (ARMS), have provided the means to gather a vast amount of diversity data from cryptic marine communities. However, such increased capability could also lead to analytical challenges if the methods used to examine these communities across local and global scales are not standardized. Here we compare and assess the underlying biases of four ARMS field processing methods, preservation media, and current bioinformatic pipelines in evaluating diversity from cytochrome c oxidase I metabarcoding data. Illustrating the ability of ARMS-based metabarcoding to capture a wide spectrum of biodiversity, 3,372 OTUs and twenty-eight phyla, including 17 of 33 marine metazoan phyla, were detected from 3 ARMS (2.607 m2 area) collected on coral reefs in Mo’orea, French Polynesia. Significant differences were found between processing and preservation methods, demonstrating the need to standardize methods for biodiversity comparisons. We recommend the use of a standardized protocol (NOAA method) combined with DMSO preservation of tissues for sessile macroorganisms because it gave a more accurate representation of the underlying communities, is cost effective and removes chemical restrictions associated with sample transportation. We found that sequences identified at ≥ 97% similarity increased more than 7-fold (5.1% to 38.6%) using a geographically local barcode inventory, highlighting the importance of local species inventories. Phylogenetic approaches that assign higher taxonomic ranks accrued phylum identification errors (9.7%) due to sparse taxonomic coverage of the understudied cryptic coral reef community in public databases. However, a ≥ 85% sequence identity cut-off provided more accurate results (0.7% errors) and enabled phylum level identifications of 86.3% of the sequence reads. With over 1600 ARMS deployed, standardizing methods and improving databases are imperative to provide unprecedented global baseline assessments of understudied cryptic marine species in a rapidly changing world. PMID:28430780

Metagenomics Reveals Pervasive Bacterial Populations and Reduced Community Diversity across the Alaska Tundra Ecosystem

DOE PAGES

Johnston, Eric R.; Rodriguez-R, Luis M.; Luo, Chengwei; ...

2016-04-25

How soil microbial communities contrast with respect to taxonomic and functional composition within and between ecosystems remains an unresolved question that is central to predicting how global anthropogenic change will affect soil functioning and services. In particular, it remains unclear how small-scale observations of soil communities based on the typical volume sampled (1-2 g) are generalizable to ecosystem-scale responses and processes. This is especially relevant for remote, northern latitude soils, which are challenging to sample and are also thought to be more vulnerable to climate change compared to temperate soils. Here, we employed well-replicated shotgun metagenome and 16S rRNA genemore » amplicon sequencing to characterize community composition and metabolic potential in Alaskan tundra soils, combining our own datasets with those publically available from distant tundra and temperate grassland and agriculture habitats. We found that the abundance of many taxa and metabolic functions differed substantially between tundra soil metagenomes relative to those from temperate soils, and that a high degree of OTU-sharing exists between tundra locations. Tundra soils were an order of magnitude less complex than their temperate counterparts, allowing for near-complete coverage of microbial community richness (~92% breadth) by sequencing, and the recovery of 27 high-quality, almost complete ( > 80% completeness) population bins. These population bins, collectively, made up to ~10% of the metagenomic datasets, and represented diverse taxonomic groups and metabolic lifestyles tuned toward sulfur cycling, hydrogen metabolism, methanotrophy, and organic matter oxidation. Several population bins, including members of Acidobacteria, Actinobacteria, and Proteobacteria, were also present in geographically distant (~100-530 km apart) tundra habitats (full genome representation and up to 99.6% genome-derived average nucleotide identity). Collectively, our results revealed that Alaska tundra microbial communities are less diverse and more homogenous across spatial scales than previously anticipated, and provided DNA sequences of abundant populations and genes that would be relevant for future studies of the effects of environmental change on tundra ecosystems.« less

Metagenomics Reveals Pervasive Bacterial Populations and Reduced Community Diversity across the Alaska Tundra Ecosystem.

PubMed

Johnston, Eric R; Rodriguez-R, Luis M; Luo, Chengwei; Yuan, Mengting M; Wu, Liyou; He, Zhili; Schuur, Edward A G; Luo, Yiqi; Tiedje, James M; Zhou, Jizhong; Konstantinidis, Konstantinos T

2016-01-01

How soil microbial communities contrast with respect to taxonomic and functional composition within and between ecosystems remains an unresolved question that is central to predicting how global anthropogenic change will affect soil functioning and services. In particular, it remains unclear how small-scale observations of soil communities based on the typical volume sampled (1-2 g) are generalizable to ecosystem-scale responses and processes. This is especially relevant for remote, northern latitude soils, which are challenging to sample and are also thought to be more vulnerable to climate change compared to temperate soils. Here, we employed well-replicated shotgun metagenome and 16S rRNA gene amplicon sequencing to characterize community composition and metabolic potential in Alaskan tundra soils, combining our own datasets with those publically available from distant tundra and temperate grassland and agriculture habitats. We found that the abundance of many taxa and metabolic functions differed substantially between tundra soil metagenomes relative to those from temperate soils, and that a high degree of OTU-sharing exists between tundra locations. Tundra soils were an order of magnitude less complex than their temperate counterparts, allowing for near-complete coverage of microbial community richness (~92% breadth) by sequencing, and the recovery of 27 high-quality, almost complete (>80% completeness) population bins. These population bins, collectively, made up to ~10% of the metagenomic datasets, and represented diverse taxonomic groups and metabolic lifestyles tuned toward sulfur cycling, hydrogen metabolism, methanotrophy, and organic matter oxidation. Several population bins, including members of Acidobacteria, Actinobacteria, and Proteobacteria, were also present in geographically distant (~100-530 km apart) tundra habitats (full genome representation and up to 99.6% genome-derived average nucleotide identity). Collectively, our results revealed that Alaska tundra microbial communities are less diverse and more homogenous across spatial scales than previously anticipated, and provided DNA sequences of abundant populations and genes that would be relevant for future studies of the effects of environmental change on tundra ecosystems.

Metagenomics Reveals Pervasive Bacterial Populations and Reduced Community Diversity across the Alaska Tundra Ecosystem

PubMed Central

Johnston, Eric R.; Rodriguez-R, Luis M.; Luo, Chengwei; Yuan, Mengting M.; Wu, Liyou; He, Zhili; Schuur, Edward A. G.; Luo, Yiqi; Tiedje, James M.; Zhou, Jizhong; Konstantinidis, Konstantinos T.

2016-01-01

How soil microbial communities contrast with respect to taxonomic and functional composition within and between ecosystems remains an unresolved question that is central to predicting how global anthropogenic change will affect soil functioning and services. In particular, it remains unclear how small-scale observations of soil communities based on the typical volume sampled (1–2 g) are generalizable to ecosystem-scale responses and processes. This is especially relevant for remote, northern latitude soils, which are challenging to sample and are also thought to be more vulnerable to climate change compared to temperate soils. Here, we employed well-replicated shotgun metagenome and 16S rRNA gene amplicon sequencing to characterize community composition and metabolic potential in Alaskan tundra soils, combining our own datasets with those publically available from distant tundra and temperate grassland and agriculture habitats. We found that the abundance of many taxa and metabolic functions differed substantially between tundra soil metagenomes relative to those from temperate soils, and that a high degree of OTU-sharing exists between tundra locations. Tundra soils were an order of magnitude less complex than their temperate counterparts, allowing for near-complete coverage of microbial community richness (~92% breadth) by sequencing, and the recovery of 27 high-quality, almost complete (>80% completeness) population bins. These population bins, collectively, made up to ~10% of the metagenomic datasets, and represented diverse taxonomic groups and metabolic lifestyles tuned toward sulfur cycling, hydrogen metabolism, methanotrophy, and organic matter oxidation. Several population bins, including members of Acidobacteria, Actinobacteria, and Proteobacteria, were also present in geographically distant (~100–530 km apart) tundra habitats (full genome representation and up to 99.6% genome-derived average nucleotide identity). Collectively, our results revealed that Alaska tundra microbial communities are less diverse and more homogenous across spatial scales than previously anticipated, and provided DNA sequences of abundant populations and genes that would be relevant for future studies of the effects of environmental change on tundra ecosystems. PMID:27199914

Genome Wide Characterization of Simple Sequence Repeats in Cucumber

USDA-ARS?s Scientific Manuscript database

The whole genome sequence of the cucumber cultivar Gy14 was recently sequenced at 15× coverage with the Roche 454 Titanium technology. The microsatellite DNA sequences (simple sequence repeats, SSRs) in the assembled scaffolds were computationally explored and characterized. A total of 112,073 SSRs ...

CNNdel: Calling Structural Variations on Low Coverage Data Based on Convolutional Neural Networks

PubMed Central

2017-01-01

Many structural variations (SVs) detection methods have been proposed due to the popularization of next-generation sequencing (NGS). These SV calling methods use different SV-property-dependent features; however, they all suffer from poor accuracy when running on low coverage sequences. The union of results from these tools achieves fairly high sensitivity but still produces low accuracy on low coverage sequence data. That is, these methods contain many false positives. In this paper, we present CNNdel, an approach for calling deletions from paired-end reads. CNNdel gathers SV candidates reported by multiple tools and then extracts features from aligned BAM files at the positions of candidates. With labeled feature-expressed candidates as a training set, CNNdel trains convolutional neural networks (CNNs) to distinguish true unlabeled candidates from false ones. Results show that CNNdel works well with NGS reads from 26 low coverage genomes of the 1000 Genomes Project. The paper demonstrates that convolutional neural networks can automatically assign the priority of SV features and reduce the false positives efficaciously. PMID:28630866

Reproducibility and Consistency of In Vitro Nucleosome Reconstitutions Demonstrated by Invitrosome Isolation and Sequencing

PubMed Central

Kempton, Colton E.; Heninger, Justin R.; Johnson, Steven M.

2014-01-01

Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reasons for performing in vitro nucleosome reconstitution experiments is to identify how the underlying DNA sequence will influence a nucleosome's position in the absence of other compounding cellular factors. However, concerns have been raised about the reproducibility of data generated from these kinds of experiments. Here we present data for in vitro nucleosome reconstitution experiments performed on linear plasmid DNA that demonstrate that, when coverage is deep enough, these reconstitution experiments are exquisitely reproducible and highly consistent. Our data also suggests that a coverage depth of 35X be maintained for maximal confidence when assaying nucleosome positions, but lower coverage levels may be generally sufficient. These coverage depth recommendations are sufficient in the experimental system and conditions used in this study, but may vary depending on the exact parameters used in other systems. PMID:25093869

Accounting for Errors in Low Coverage High-Throughput Sequencing Data When Constructing Genetic Maps Using Biparental Outcrossed Populations

PubMed Central

Bilton, Timothy P.; Schofield, Matthew R.; Black, Michael A.; Chagné, David; Wilcox, Phillip L.; Dodds, Ken G.

2018-01-01

Next-generation sequencing is an efficient method that allows for substantially more markers than previous technologies, providing opportunities for building high-density genetic linkage maps, which facilitate the development of nonmodel species’ genomic assemblies and the investigation of their genes. However, constructing genetic maps using data generated via high-throughput sequencing technology (e.g., genotyping-by-sequencing) is complicated by the presence of sequencing errors and genotyping errors resulting from missing parental alleles due to low sequencing depth. If unaccounted for, these errors lead to inflated genetic maps. In addition, map construction in many species is performed using full-sibling family populations derived from the outcrossing of two individuals, where unknown parental phase and varying segregation types further complicate construction. We present a new methodology for modeling low coverage sequencing data in the construction of genetic linkage maps using full-sibling populations of diploid species, implemented in a package called GUSMap. Our model is based on the Lander–Green hidden Markov model but extended to account for errors present in sequencing data. We were able to obtain accurate estimates of the recombination fractions and overall map distance using GUSMap, while most existing mapping packages produced inflated genetic maps in the presence of errors. Our results demonstrate the feasibility of using low coverage sequencing data to produce genetic maps without requiring extensive filtering of potentially erroneous genotypes, provided that the associated errors are correctly accounted for in the model. PMID:29487138

Accounting for Errors in Low Coverage High-Throughput Sequencing Data When Constructing Genetic Maps Using Biparental Outcrossed Populations.

PubMed

Bilton, Timothy P; Schofield, Matthew R; Black, Michael A; Chagné, David; Wilcox, Phillip L; Dodds, Ken G

2018-05-01

Next-generation sequencing is an efficient method that allows for substantially more markers than previous technologies, providing opportunities for building high-density genetic linkage maps, which facilitate the development of nonmodel species' genomic assemblies and the investigation of their genes. However, constructing genetic maps using data generated via high-throughput sequencing technology ( e.g. , genotyping-by-sequencing) is complicated by the presence of sequencing errors and genotyping errors resulting from missing parental alleles due to low sequencing depth. If unaccounted for, these errors lead to inflated genetic maps. In addition, map construction in many species is performed using full-sibling family populations derived from the outcrossing of two individuals, where unknown parental phase and varying segregation types further complicate construction. We present a new methodology for modeling low coverage sequencing data in the construction of genetic linkage maps using full-sibling populations of diploid species, implemented in a package called GUSMap. Our model is based on the Lander-Green hidden Markov model but extended to account for errors present in sequencing data. We were able to obtain accurate estimates of the recombination fractions and overall map distance using GUSMap, while most existing mapping packages produced inflated genetic maps in the presence of errors. Our results demonstrate the feasibility of using low coverage sequencing data to produce genetic maps without requiring extensive filtering of potentially erroneous genotypes, provided that the associated errors are correctly accounted for in the model. Copyright © 2018 Bilton et al.

Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.

PubMed

Shapland, Elaine B; Holmes, Victor; Reeves, Christopher D; Sorokin, Elena; Durot, Maxime; Platt, Darren; Allen, Christopher; Dean, Jed; Serber, Zach; Newman, Jack; Chandran, Sunil

2015-07-17

In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.

The diploid genome sequence of an Asian individual

PubMed Central

Wang, Jun; Wang, Wei; Li, Ruiqiang; Li, Yingrui; Tian, Geng; Goodman, Laurie; Fan, Wei; Zhang, Junqing; Li, Jun; Zhang, Juanbin; Guo, Yiran; Feng, Binxiao; Li, Heng; Lu, Yao; Fang, Xiaodong; Liang, Huiqing; Du, Zhenglin; Li, Dong; Zhao, Yiqing; Hu, Yujie; Yang, Zhenzhen; Zheng, Hancheng; Hellmann, Ines; Inouye, Michael; Pool, John; Yi, Xin; Zhao, Jing; Duan, Jinjie; Zhou, Yan; Qin, Junjie; Ma, Lijia; Li, Guoqing; Yang, Zhentao; Zhang, Guojie; Yang, Bin; Yu, Chang; Liang, Fang; Li, Wenjie; Li, Shaochuan; Li, Dawei; Ni, Peixiang; Ruan, Jue; Li, Qibin; Zhu, Hongmei; Liu, Dongyuan; Lu, Zhike; Li, Ning; Guo, Guangwu; Zhang, Jianguo; Ye, Jia; Fang, Lin; Hao, Qin; Chen, Quan; Liang, Yu; Su, Yeyang; san, A.; Ping, Cuo; Yang, Shuang; Chen, Fang; Li, Li; Zhou, Ke; Zheng, Hongkun; Ren, Yuanyuan; Yang, Ling; Gao, Yang; Yang, Guohua; Li, Zhuo; Feng, Xiaoli; Kristiansen, Karsten; Wong, Gane Ka-Shu; Nielsen, Rasmus; Durbin, Richard; Bolund, Lars; Zhang, Xiuqing; Li, Songgang; Yang, Huanming; Wang, Jian

2009-01-01

Here we present the first diploid genome sequence of an Asian individual. The genome was sequenced to 36-fold average coverage using massively parallel sequencing technology. We aligned the short reads onto the NCBI human reference genome to 99.97% coverage, and guided by the reference genome, we used uniquely mapped reads to assemble a high-quality consensus sequence for 92% of the Asian individual's genome. We identified approximately 3 million single-nucleotide polymorphisms (SNPs) inside this region, of which 13.6% were not in the dbSNP database. Genotyping analysis showed that SNP identification had high accuracy and consistency, indicating the high sequence quality of this assembly. We also carried out heterozygote phasing and haplotype prediction against HapMap CHB and JPT haplotypes (Chinese and Japanese, respectively), sequence comparison with the two available individual genomes (J. D. Watson and J. C. Venter), and structural variation identification. These variations were considered for their potential biological impact. Our sequence data and analyses demonstrate the potential usefulness of next-generation sequencing technologies for personal genomics. PMID:18987735

Expanding Kenya's protected areas under the Convention on Biological Diversity to maximize coverage of plant diversity.

PubMed

Scherer, Laura; Curran, Michael; Alvarez, Miguel

2017-04-01

Biodiversity is highly valuable and critically threatened by anthropogenic degradation of the natural environment. In response, governments have pledged enhanced protected-area coverage, which requires scarce biological data to identify conservation priorities. To assist this effort, we mapped conservation priorities in Kenya based on maximizing alpha (species richness) and beta diversity (species turnover) of plant communities while minimizing economic costs. We used plant-cover percentages from vegetation surveys of over 2000 plots to build separate models for each type of diversity. Opportunity and management costs were based on literature data and interviews with conservation organizations. Species richness was predicted to be highest in a belt from Lake Turkana through Mount Kenya and in a belt parallel to the coast, and species turnover was predicted to be highest in western Kenya and along the coast. Our results suggest the expanding reserve network should focus on the coast and northeastern provinces of Kenya, where new biological surveys would also fill biological data gaps. Meeting the Convention on Biological Diversity target of 17% terrestrial coverage by 2020 would increase representation of Kenya's plant communities by 75%. However, this would require about 50 times more funds than Kenya has received thus far from the Global Environment Facility. © 2016 Society for Conservation Biology.

GeneImp: Fast Imputation to Large Reference Panels Using Genotype Likelihoods from Ultralow Coverage Sequencing

PubMed Central

Spiliopoulou, Athina; Colombo, Marco; Orchard, Peter; Agakov, Felix; McKeigue, Paul

2017-01-01

We address the task of genotype imputation to a dense reference panel given genotype likelihoods computed from ultralow coverage sequencing as inputs. In this setting, the data have a high-level of missingness or uncertainty, and are thus more amenable to a probabilistic representation. Most existing imputation algorithms are not well suited for this situation, as they rely on prephasing for computational efficiency, and, without definite genotype calls, the prephasing task becomes computationally expensive. We describe GeneImp, a program for genotype imputation that does not require prephasing and is computationally tractable for whole-genome imputation. GeneImp does not explicitly model recombination, instead it capitalizes on the existence of large reference panels—comprising thousands of reference haplotypes—and assumes that the reference haplotypes can adequately represent the target haplotypes over short regions unaltered. We validate GeneImp based on data from ultralow coverage sequencing (0.5×), and compare its performance to the most recent version of BEAGLE that can perform this task. We show that GeneImp achieves imputation quality very close to that of BEAGLE, using one to two orders of magnitude less time, without an increase in memory complexity. Therefore, GeneImp is the first practical choice for whole-genome imputation to a dense reference panel when prephasing cannot be applied, for instance, in datasets produced via ultralow coverage sequencing. A related future application for GeneImp is whole-genome imputation based on the off-target reads from deep whole-exome sequencing. PMID:28348060

Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak

PubMed Central

2010-01-01

Background The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity. Results We generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html. Conclusions This genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations. PMID:21092232

A computational method for detecting copy number variations using scale-space filtering

PubMed Central

2013-01-01

Background As next-generation sequencing technology made rapid and cost-effective sequencing available, the importance of computational approaches in finding and analyzing copy number variations (CNVs) has been amplified. Furthermore, most genome projects need to accurately analyze sequences with fairly low-coverage read data. It is urgently needed to develop a method to detect the exact types and locations of CNVs from low coverage read data. Results Here, we propose a new CNV detection method, CNV_SS, which uses scale-space filtering. The scale-space filtering is evaluated by applying to the read coverage data the Gaussian convolution for various scales according to a given scaling parameter. Next, by differentiating twice and finding zero-crossing points, inflection points of scale-space filtered read coverage data are calculated per scale. Then, the types and the exact locations of CNVs are obtained by analyzing the finger print map, the contours of zero-crossing points for various scales. Conclusions The performance of CNV_SS showed that FNR and FPR stay in the range of 1.27% to 2.43% and 1.14% to 2.44%, respectively, even at a relatively low coverage (0.5x ≤C ≤2x). CNV_SS gave also much more effective results than the conventional methods in the evaluation of FNR, at 3.82% at least and 76.97% at most even when the coverage level of read data is low. CNV_SS source code is freely available from http://dblab.hallym.ac.kr/CNV SS/. PMID:23418726

Error and Error Mitigation in Low-Coverage Genome Assemblies

PubMed Central

Hubisz, Melissa J.; Lin, Michael F.; Kellis, Manolis; Siepel, Adam

2011-01-01

The recent release of twenty-two new genome sequences has dramatically increased the data available for mammalian comparative genomics, but twenty of these new sequences are currently limited to ∼2× coverage. Here we examine the extent of sequencing error in these 2× assemblies, and its potential impact in downstream analyses. By comparing 2× assemblies with high-quality sequences from the ENCODE regions, we estimate the rate of sequencing error to be 1–4 errors per kilobase. While this error rate is fairly modest, sequencing error can still have surprising effects. For example, an apparent lineage-specific insertion in a coding region is more likely to reflect sequencing error than a true biological event, and the length distribution of coding indels is strongly distorted by error. We find that most errors are contributed by a small fraction of bases with low quality scores, in particular, by the ends of reads in regions of single-read coverage in the assembly. We explore several approaches for automatic sequencing error mitigation (SEM), making use of the localized nature of sequencing error, the fact that it is well predicted by quality scores, and information about errors that comes from comparisons across species. Our automatic methods for error mitigation cannot replace the need for additional sequencing, but they do allow substantial fractions of errors to be masked or eliminated at the cost of modest amounts of over-correction, and they can reduce the impact of error in downstream phylogenomic analyses. Our error-mitigated alignments are available for download. PMID:21340033

Single nucleotide polymorphism analysis of Korean native chickens using next generation sequencing data.

PubMed

Seo, Dong-Won; Oh, Jae-Don; Jin, Shil; Song, Ki-Duk; Park, Hee-Bok; Heo, Kang-Nyeong; Shin, Younhee; Jung, Myunghee; Park, Junhyung; Jo, Cheorun; Lee, Hak-Kyo; Lee, Jun-Heon

2015-02-01

There are five native chicken lines in Korea, which are mainly classified by plumage colors (black, white, red, yellow, gray). These five lines are very important genetic resources in the Korean poultry industry. Based on a next generation sequencing technology, whole genome sequence and reference assemblies were performed using Gallus_gallus_4.0 (NCBI) with whole genome sequences from these lines to identify common and novel single nucleotide polymorphisms (SNPs). We obtained 36,660,731,136 ± 1,257,159,120 bp of raw sequence and average 26.6-fold of 25-29 billion reference assembly sequences representing 97.288 % coverage. Also, 4,006,068 ± 97,534 SNPs were observed from 29 autosomes and the Z chromosome and, of these, 752,309 SNPs are the common SNPs across lines. Among the identified SNPs, the number of novel- and known-location assigned SNPs was 1,047,951 ± 14,956 and 2,948,648 ± 81,414, respectively. The number of unassigned known SNPs was 1,181 ± 150 and unassigned novel SNPs was 8,238 ± 1,019. Synonymous SNPs, non-synonymous SNPs, and SNPs having character changes were 26,266 ± 1,456, 11,467 ± 604, 8,180 ± 458, respectively. Overall, 443,048 ± 26,389 SNPs in each bird were identified by comparing with dbSNP in NCBI. The presently obtained genome sequence and SNP information in Korean native chickens have wide applications for further genome studies such as genetic diversity studies to detect causative mutations for economic and disease related traits.

A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies.

PubMed

Utturkar, Sagar M; Klingeman, Dawn M; Hurt, Richard A; Brown, Steven D

2017-01-01

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.

Misconceptions on Missing Data in RAD-seq Phylogenetics with a Deep-scale Example from Flowering Plants.

PubMed

Eaton, Deren A R; Spriggs, Elizabeth L; Park, Brian; Donoghue, Michael J

2017-05-01

Restriction-site associated DNA (RAD) sequencing and related methods rely on the conservation of enzyme recognition sites to isolate homologous DNA fragments for sequencing, with the consequence that mutations disrupting these sites lead to missing information. There is thus a clear expectation for how missing data should be distributed, with fewer loci recovered between more distantly related samples. This observation has led to a related expectation: that RAD-seq data are insufficiently informative for resolving deeper scale phylogenetic relationships. Here we investigate the relationship between missing information among samples at the tips of a tree and information at edges within it. We re-analyze and review the distribution of missing data across ten RAD-seq data sets and carry out simulations to determine expected patterns of missing information. We also present new empirical results for the angiosperm clade Viburnum (Adoxaceae, with a crown age >50 Ma) for which we examine phylogenetic information at different depths in the tree and with varied sequencing effort. The total number of loci, the proportion that are shared, and phylogenetic informativeness varied dramatically across the examined RAD-seq data sets. Insufficient or uneven sequencing coverage accounted for similar proportions of missing data as dropout from mutation-disruption. Simulations reveal that mutation-disruption, which results in phylogenetically distributed missing data, can be distinguished from the more stochastic patterns of missing data caused by low sequencing coverage. In Viburnum, doubling sequencing coverage nearly doubled the number of parsimony informative sites, and increased by >10X the number of loci with data shared across >40 taxa. Our analysis leads to a set of practical recommendations for maximizing phylogenetic information in RAD-seq studies. [hierarchical redundancy; phylogenetic informativeness; quartet informativeness; Restriction-site associated DNA (RAD) sequencing; sequencing coverage; Viburnum.]. © The authors 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Spatial scaling of bacterial community diversity at shallow hydrothermal vents: a global comparison

NASA Astrophysics Data System (ADS)

Pop Ristova, P.; Hassenrueck, C.; Molari, M.; Fink, A.; Bühring, S. I.

2016-02-01

Marine shallow hydrothermal vents are extreme environments, often characterized by discharge of fluids with e.g. high temperatures, low pH, and laden with elements toxic to higher organisms. They occur at continental margins around the world's oceans, but represent fragmented, isolated habitats of locally small areal coverage. Microorganisms contribute the main biomass at shallow hydrothermal vent ecosystems and build the basis of the food chain by autotrophic fixation of carbon both via chemosynthesis and photosynthesis, occurring simultaneously. Despite their importance and unique capacity to adapt to these extreme environments, little is known about the spatial scales on which the alpha- and beta-diversity of microbial communities vary at shallow vents, and how the geochemical habitat heterogeneity influences shallow vent biodiversity. Here for the first time we investigated the spatial scaling of microbial biodiversity patterns and their interconnectivity at geochemically diverse shallow vents on a global scale. This study presents data on the comparison of bacterial community structures on large (> 1000 km) and small (0.1 - 100 m) spatial scales as derived from ARISA and Illumina sequencing. Despite the fragmented global distribution of shallow hydrothermal vents, similarity of vent bacterial communities decreased with geographic distance, confirming the ubiquity of distance-decay relationship. Moreover, at all investigated vents, pH was the main factor locally structuring these communities, while temperature influenced both the alpha- and beta-diversity.

Challenges of coverage policy development for next-generation tumor sequencing panels: experts and payers weigh in.

PubMed

Trosman, Julia R; Weldon, Christine B; Kelley, R Kate; Phillips, Kathryn A

2015-03-01

Next-generation tumor sequencing (NGTS) panels, which include multiple established and novel targets across cancers, are emerging in oncology practice, but lack formal positive coverage by US payers. Lack of coverage may impact access and adoption. This study identified challenges of NGTS coverage by private payers. We conducted semi-structured interviews with 14 NGTS experts on potential NGTS benefits, and with 10 major payers, representing more than 125,000,000 enrollees, on NGTS coverage considerations. We used the framework approach of qualitative research for study design and thematic analyses and simple frequencies to further describe findings. All interviewed payers see potential NGTS benefits, but all noted challenges to formal coverage: 80% state that inherent features of NGTS do not fit the medical necessity definition required for coverage, 70% view NGTS as a bundle of targets versus comprehensive tumor characterization and may evaluate each target individually, and 70% express skepticism regarding new evidence methods proposed for NGTS. Fifty percent of payers expressed sufficient concerns about NGTS adoption and implementation that will preclude their ability to issue positive coverage policies. Payers perceive that NGTS holds significant promise but, in its current form, poses disruptive challenges to coverage policy frameworks. Proactive multidisciplinary efforts to define the direction for NGTS development, evidence generation, and incorporation into coverage policy are necessary to realize its promise and provide patient access. This study contributes to current literature, as possibly the first study to directly interview US payers on NGTS coverage and reimbursement. Copyright © 2015 by the National Comprehensive Cancer Network.

Challenges of Coverage Policy Development for Next-Generation Tumor Sequencing Panels: Experts and Payers Weigh In

PubMed Central

Trosman, Julia R.; Weldon, Christine B.; Kate Kelley, R.; Phillips, Kathryn A.

2015-01-01

Background Next-generation tumor sequencing (NGTS) panels, which include multiple established and novel targets across cancers, are emerging in oncology practice, but lack formal positive coverage by US payers. Lack of coverage may impact access and adoption. This study identified challenges of NGTS coverage by private payers. Methods We conducted semi-structured interviews with 14 NGTS experts on potential NGTS benefits, and with 10 major payers, representing more than 125,000,000 enrollees, on NGTS coverage considerations. We used the framework approach of qualitative research for study design and thematic analyses and simple frequencies to further describe findings. Results All interviewed payers see potential NGTS benefits, but all noted challenges to formal coverage: 80% state that inherent features of NGTS do not fit the medical necessity definition required for coverage, 70% view NGTS as a bundle of targets versus comprehensive tumor characterization and may evaluate each target individually, and 70% express skepticism regarding new evidence methods proposed for NGTS. Fifty percent of payers expressed sufficient concerns about NGTS adoption and implementation that will preclude their ability to issue positive coverage policies. Conclusions Payers perceive that NGTS holds significant promise but, in its current form, poses disruptive challenges to coverage policy frameworks. Proactive multidisciplinary efforts to define the direction for NGTS development, evidence generation, and incorporation into coverage policy are necessary to realize its promise and provide patient access. This study contributes to current literature, as possibly the first study to directly interview US payers on NGTS coverage and reimbursement. PMID:25736008

Deep whole-genome sequencing of 90 Han Chinese genomes.

PubMed

Lan, Tianming; Lin, Haoxiang; Zhu, Wenjuan; Laurent, Tellier Christian Asker Melchior; Yang, Mengcheng; Liu, Xin; Wang, Jun; Wang, Jian; Yang, Huanming; Xu, Xun; Guo, Xiaosen

2017-09-01

Next-generation sequencing provides a high-resolution insight into human genetic information. However, the focus of previous studies has primarily been on low-coverage data due to the high cost of sequencing. Although the 1000 Genomes Project and the Haplotype Reference Consortium have both provided powerful reference panels for imputation, low-frequency and novel variants remain difficult to discover and call with accuracy on the basis of low-coverage data. Deep sequencing provides an optimal solution for the problem of these low-frequency and novel variants. Although whole-exome sequencing is also a viable choice for exome regions, it cannot account for noncoding regions, sometimes resulting in the absence of important, causal variants. For Han Chinese populations, the majority of variants have been discovered based upon low-coverage data from the 1000 Genomes Project. However, high-coverage, whole-genome sequencing data are limited for any population, and a large amount of low-frequency, population-specific variants remain uncharacterized. We have performed whole-genome sequencing at a high depth (∼×80) of 90 unrelated individuals of Chinese ancestry, collected from the 1000 Genomes Project samples, including 45 Northern Han Chinese and 45 Southern Han Chinese samples. Eighty-three of these 90 have been sequenced by the 1000 Genomes Project. We have identified 12 568 804 single nucleotide polymorphisms, 2 074 210 short InDels, and 26 142 structural variations from these 90 samples. Compared to the Han Chinese data from the 1000 Genomes Project, we have found 7 000 629 novel variants with low frequency (defined as minor allele frequency < 5%), including 5 813 503 single nucleotide polymorphisms, 1 169 199 InDels, and 17 927 structural variants. Using deep sequencing data, we have built a greatly expanded spectrum of genetic variation for the Han Chinese genome. Compared to the 1000 Genomes Project, these Han Chinese deep sequencing data enhance the characterization of a large number of low-frequency, novel variants. This will be a valuable resource for promoting Chinese genetics research and medical development. Additionally, it will provide a valuable supplement to the 1000 Genomes Project, as well as to other human genome projects. © The Authors 2017. Published by Oxford University Press.

Exploiting long read sequencing technologies to establish high quality highly contiguous pig reference genome assemblies

USDA-ARS?s Scientific Manuscript database

The current pig reference genome sequence (Sscrofa10.2) was established using Sanger sequencing and following the clone-by-clone hierarchical shotgun sequencing approach used in the public human genome project. However, as sequence coverage was low (4-6x) the resulting assembly was only of draft qua...

Accurate and exact CNV identification from targeted high-throughput sequence data.

PubMed

Nord, Alex S; Lee, Ming; King, Mary-Claire; Walsh, Tom

2011-04-12

Massively parallel sequencing of barcoded DNA samples significantly increases screening efficiency for clinically important genes. Short read aligners are well suited to single nucleotide and indel detection. However, methods for CNV detection from targeted enrichment are lacking. We present a method combining coverage with map information for the identification of deletions and duplications in targeted sequence data. Sequencing data is first scanned for gains and losses using a comparison of normalized coverage data between samples. CNV calls are confirmed by testing for a signature of sequences that span the CNV breakpoint. With our method, CNVs can be identified regardless of whether breakpoints are within regions targeted for sequencing. For CNVs where at least one breakpoint is within targeted sequence, exact CNV breakpoints can be identified. In a test data set of 96 subjects sequenced across ~1 Mb genomic sequence using multiplexing technology, our method detected mutations as small as 31 bp, predicted quantitative copy count, and had a low false-positive rate. Application of this method allows for identification of gains and losses in targeted sequence data, providing comprehensive mutation screening when combined with a short read aligner.

Rare Cell Detection by Single-Cell RNA Sequencing as Guided by Single-Molecule RNA FISH.

PubMed

Torre, Eduardo; Dueck, Hannah; Shaffer, Sydney; Gospocic, Janko; Gupte, Rohit; Bonasio, Roberto; Kim, Junhyong; Murray, John; Raj, Arjun

2018-02-28

Although single-cell RNA sequencing can reliably detect large-scale transcriptional programs, it is unclear whether it accurately captures the behavior of individual genes, especially those that express only in rare cells. Here, we use single-molecule RNA fluorescence in situ hybridization as a gold standard to assess trade-offs in single-cell RNA-sequencing data for detecting rare cell expression variability. We quantified the gene expression distribution for 26 genes that range from ubiquitous to rarely expressed and found that the correspondence between estimates across platforms improved with both transcriptome coverage and increased number of cells analyzed. Further, by characterizing the trade-off between transcriptome coverage and number of cells analyzed, we show that when the number of genes required to answer a given biological question is small, then greater transcriptome coverage is more important than analyzing large numbers of cells. More generally, our report provides guidelines for selecting quality thresholds for single-cell RNA-sequencing experiments aimed at rare cell analyses. Copyright © 2018 Elsevier Inc. All rights reserved.

Real-world clinical effectiveness, regulatory transparency and payer coverage: three ingredients for translating pharmacogenomics into clinical practice.

PubMed

Frueh, Felix W

2010-05-01

The past decade of pharmacogenomics was driven by the sequencing of the human genome to create ever denser maps of genetic variations for studying the diversity across individuals. Today, genotyping technology is available at a fraction of the cost of what it was 10 years ago and many pharmacogenomic variations have been studied in detail. Still, we are only starting to gain an understanding of how pharmacogenomic-guided drug therapy affects clinical outcomes: real-world studies that demonstrate the clinical effectiveness and address the economic implications of pharmacogenomics are needed to help decide when and how to implement pharmacogenomics in clinical practice, how to regulate pharmacogenomic testing and how the healthcare system will integrate this new science into an environment of rapidly increasing cost.

Optimization of conditions to sequence long cDNAs from viruses

USDA-ARS?s Scientific Manuscript database

Fourth generation sequencing with the Minion nanopore sequencer provides opportunity to obtain deep coverage and long read for single molecules. This will benefit studies on RNA viruses. In the past, Sanger, Illumina, and Ion Torrent sequencing have been utilized to study RNA viruses. Both technique...

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes.

PubMed

Haiminen, Niina; Feltus, F Alex; Parida, Laxmi

2011-04-15

We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies.

Identification of mutated driver pathways in cancer using a multi-objective optimization model.

PubMed

Zheng, Chun-Hou; Yang, Wu; Chong, Yan-Wen; Xia, Jun-Feng

2016-05-01

New-generation high-throughput technologies, including next-generation sequencing technology, have been extensively applied to solve biological problems. As a result, large cancer genomics projects such as the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium are producing large amount of rich and diverse data in multiple cancer types. The identification of mutated driver genes and driver pathways from these data is a significant challenge. Genome aberrations in cancer cells can be divided into two types: random 'passenger mutation' and functional 'driver mutation'. In this paper, we introduced a Multi-objective Optimization model based on a Genetic Algorithm (MOGA) to solve the maximum weight submatrix problem, which can be employed to identify driver genes and driver pathways promoting cancer proliferation. The maximum weight submatrix problem defined to find mutated driver pathways is based on two specific properties, i.e., high coverage and high exclusivity. The multi-objective optimization model can adjust the trade-off between high coverage and high exclusivity. We proposed an integrative model by combining gene expression data and mutation data to improve the performance of the MOGA algorithm in a biological context. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modeling the SHG activities of diverse protein crystals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J., E-mail: gsimpson@purdue.edu

2012-11-01

The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed. A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much ofmore » the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.« less

Archaeal β diversity patterns under the seafloor along geochemical gradients

NASA Astrophysics Data System (ADS)

Koyano, Hitoshi; Tsubouchi, Taishi; Kishino, Hirohisa; Akutsu, Tatsuya

2014-09-01

Recently, deep drilling into the seafloor has revealed that there are vast sedimentary ecosystems of diverse microorganisms, particularly archaea, in subsurface areas. We investigated the β diversity patterns of archaeal communities in sediment layers under the seafloor and their determinants. This study was accomplished by analyzing large environmental samples of 16S ribosomal RNA gene sequences and various geochemical data collected from a sediment core of 365.3 m, obtained by drilling into the seafloor off the east coast of the Shimokita Peninsula. To extract the maximum amount of information from these environmental samples, we first developed a method for measuring β diversity using sequence data by applying probability theory on a set of strings developed by two of the authors in a previous publication. We introduced an index of β diversity between sequence populations from which the sequence data were sampled. We then constructed an estimator of the β diversity index based on the sequence data and demonstrated that it converges to the β diversity index between sequence populations with probability of 1 as the number of sampled sequences increases. Next, we applied this new method to quantify β diversities between archaeal sequence populations under the seafloor and constructed a quantitative model of the estimated β diversity patterns. Nearly 90% of the variation in the archaeal β diversity was explained by a model that included as variables the differences in the abundances of chlorine, iodine, and carbon between the sediment layers.

78 FR 54553 - Taking of Threatened or Endangered Marine Mammals Incidental to Commercial Fishing Operations...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-04

... serious injuries and mortalities. The Center for Biological Diversity, Turtle Island Restoration Network... percent coverage level. Similarly, Center for Biological Diversity, Turtle Island Restoration Network, and.... Comment 7: The Center for Biological Diversity, Turtle Island Restoration Network, and Oceana opposed the...

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

NASA Astrophysics Data System (ADS)

He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

2017-05-01

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative

PubMed Central

Khafizov, Kamil; Madrid-Aliste, Carlos; Almo, Steven C.; Fiser, Andras

2014-01-01

The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins—including proteins for which reliable homology models can be generated—on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long. PMID:24567391

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative.

PubMed

Khafizov, Kamil; Madrid-Aliste, Carlos; Almo, Steven C; Fiser, Andras

2014-03-11

The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins--including proteins for which reliable homology models can be generated--on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long.

Developing market class specific InDel markers from next generation sequence data in Phaseolus vulgaris L.

PubMed

Moghaddam, Samira Mafi; Song, Qijian; Mamidi, Sujan; Schmutz, Jeremy; Lee, Rian; Cregan, Perry; Osorno, Juan M; McClean, Phillip E

2014-01-01

Next generation sequence data provides valuable information and tools for genetic and genomic research and offers new insights useful for marker development. This data is useful for the design of accurate and user-friendly molecular tools. Common bean (Phaseolus vulgaris L.) is a diverse crop in which separate domestication events happened in each gene pool followed by race and market class diversification that has resulted in different morphological characteristics in each commercial market class. This has led to essentially independent breeding programs within each market class which in turn has resulted in limited within market class sequence variation. Sequence data from selected genotypes of five bean market classes (pinto, black, navy, and light and dark red kidney) were used to develop InDel-based markers specific to each market class. Design of the InDel markers was conducted through a combination of assembly, alignment and primer design software using 1.6× to 5.1× coverage of Illumina GAII sequence data for each of the selected genotypes. The procedure we developed for primer design is fast, accurate, less error prone, and higher throughput than when they are designed manually. All InDel markers are easy to run and score with no need for PCR optimization. A total of 2687 InDel markers distributed across the genome were developed. To highlight their usefulness, they were employed to construct a phylogenetic tree and a genetic map, showing that InDel markers are reliable, simple, and accurate.

Developing market class specific InDel markers from next generation sequence data in Phaseolus vulgaris L.

PubMed Central

Moghaddam, Samira Mafi; Song, Qijian; Mamidi, Sujan; Schmutz, Jeremy; Lee, Rian; Cregan, Perry; Osorno, Juan M.; McClean, Phillip E.

2013-01-01

Next generation sequence data provides valuable information and tools for genetic and genomic research and offers new insights useful for marker development. This data is useful for the design of accurate and user-friendly molecular tools. Common bean (Phaseolus vulgaris L.) is a diverse crop in which separate domestication events happened in each gene pool followed by race and market class diversification that has resulted in different morphological characteristics in each commercial market class. This has led to essentially independent breeding programs within each market class which in turn has resulted in limited within market class sequence variation. Sequence data from selected genotypes of five bean market classes (pinto, black, navy, and light and dark red kidney) were used to develop InDel-based markers specific to each market class. Design of the InDel markers was conducted through a combination of assembly, alignment and primer design software using 1.6× to 5.1× coverage of Illumina GAII sequence data for each of the selected genotypes. The procedure we developed for primer design is fast, accurate, less error prone, and higher throughput than when they are designed manually. All InDel markers are easy to run and score with no need for PCR optimization. A total of 2687 InDel markers distributed across the genome were developed. To highlight their usefulness, they were employed to construct a phylogenetic tree and a genetic map, showing that InDel markers are reliable, simple, and accurate. PMID:24860578

Genome Survey Sequencing of Luffa Cylindrica L. and Microsatellite High Resolution Melting (SSR-HRM) Analysis for Genetic Relationship of Luffa Genotypes.

PubMed

An, Jianyu; Yin, Mengqi; Zhang, Qin; Gong, Dongting; Jia, Xiaowen; Guan, Yajing; Hu, Jin

2017-09-11

Luffa cylindrica (L.) Roem. is an economically important vegetable crop in China. However, the genomic information on this species is currently unknown. In this study, for the first time, a genome survey of L. cylindrica was carried out using next-generation sequencing (NGS) technology. In total, 43.40 Gb sequence data of L. cylindrica , about 54.94× coverage of the estimated genome size of 789.97 Mb, were obtained from HiSeq 2500 sequencing, in which the guanine plus cytosine (GC) content was calculated to be 37.90%. The heterozygosity of genome sequences was only 0.24%. In total, 1,913,731 contigs (>200 bp) with 525 bp N 50 length and 1,410,117 scaffolds (>200 bp) with 885.01 Mb total length were obtained. From the initial assembled L. cylindrica genome, 431,234 microsatellites (SSRs) (≥5 repeats) were identified. The motif types of SSR repeats included 62.88% di-nucleotide, 31.03% tri-nucleotide, 4.59% tetra-nucleotide, 0.96% penta-nucleotide and 0.54% hexa-nucleotide. Eighty genomic SSR markers were developed, and 51/80 primers could be used in both "Zheda 23" and "Zheda 83". Nineteen SSRs were used to investigate the genetic diversity among 32 accessions through SSR-HRM analysis. The unweighted pair group method analysis (UPGMA) dendrogram tree was built by calculating the SSR-HRM raw data. SSR-HRM could be effectively used for genotype relationship analysis of Luffa species.

Estimates of abundance and diversity of Shewanella genus in natural and engineered aqueous environments with newly designed primers.

PubMed

Li, Bing-Bing; Cheng, Yuan-Yuan; Fan, Yang-Yang; Liu, Dong-Feng; Fang, Cai-Yun; Wu, Chao; Li, Wen-Wei; Yang, Zong-Chuang; Yu, Han-Qing

2018-05-12

Shewanella species have a diverse respiratory ability and wide distribution in environments and play an important role in bioremediation and the biogeochemical cycles of elements. Primers with more accuracy and broader coverage are required with consideration of the increasing number of Shewanella species and evaluation of their roles in various environments. In this work, a new primer set of 640F/815R was developed to quantify the abundance of Shewanella species in natural and engineered environments. In silico tools for primer evaluation, quantitative polymerase chain reaction (qPCR) and clone library results showed that 640F/815R had a higher specificity and coverage than the previous primers in quantitative analysis of Shewanella. Another newly developed primer pair of 211F/815cR was also adopted to analyze the Shewanella diversity and demonstrated to be the best candidate in terms of specificity and coverage. We detected more Shewanella-related species in freshwater environments and found them to be substantially different from those in marine environments. Abundance and diversity of Shewanella species in wastewater treatment plants were largely affected by the process and operating conditions. Overall, this study suggests that investigations of abundance and diversity of Shewanella in various environments are of great importance to evaluate their ecophysiology and potential ecological roles. Copyright © 2018 Elsevier B.V. All rights reserved.

ViDiT-CACTUS: an inexpensive and versatile library preparation and sequence analysis method for virus discovery and other microbiology applications.

PubMed

Verhoeven, Joost Theo Petra; Canuti, Marta; Munro, Hannah J; Dufour, Suzanne C; Lang, Andrew S

2018-04-19

High-throughput sequencing (HTS) technologies are becoming increasingly important within microbiology research, but aspects of library preparation, such as high cost per sample or strict input requirements, make HTS difficult to implement in some niche applications and for research groups on a budget. To answer these necessities, we developed ViDiT, a customizable, PCR-based, extremely low-cost (<5 US dollars per sample) and versatile library preparation method, and CACTUS, an analysis pipeline designed to rely on cloud computing power to generate high-quality data from ViDiT-based experiments without the need of expensive servers. We demonstrate here the versatility and utility of these methods within three fields of microbiology: virus discovery, amplicon-based viral genome sequencing and microbiome profiling. ViDiT-CACTUS allowed the identification of viral fragments from 25 different viral families from 36 oropharyngeal-cloacal swabs collected from wild birds, the sequencing of three almost complete genomes of avian influenza A viruses (>90% coverage), and the characterization and functional profiling of the complete microbial diversity (bacteria, archaea, viruses) within a deep-sea carnivorous sponge. ViDiT-CACTUS demonstrated its validity in a wide range of microbiology applications and its simplicity and modularity make it easily implementable in any molecular biology laboratory, towards various research goals.

Transcriptome sequencing for high throughput SNP development and genetic mapping in Pea

PubMed Central

2014-01-01

Background Pea has a complex genome of 4.3 Gb for which only limited genomic resources are available to date. Although SNP markers are now highly valuable for research and modern breeding, only a few are described and used in pea for genetic diversity and linkage analysis. Results We developed a large resource by cDNA sequencing of 8 genotypes representative of modern breeding material using the Roche 454 technology, combining both long reads (400 bp) and high coverage (3.8 million reads, reaching a total of 1,369 megabases). Sequencing data were assembled and generated a 68 K unigene set, from which 41 K were annotated from their best blast hit against the model species Medicago truncatula. Annotated contigs showed an even distribution along M. truncatula pseudochromosomes, suggesting a good representation of the pea genome. 10 K pea contigs were found to be polymorphic among the genetic material surveyed, corresponding to 35 K SNPs. We validated a subset of 1538 SNPs through the GoldenGate assay, proving their ability to structure a diversity panel of breeding germplasm. Among them, 1340 were genetically mapped and used to build a new consensus map comprising a total of 2070 markers. Based on blast analysis, we could establish 1252 bridges between our pea consensus map and the pseudochromosomes of M. truncatula, which provides new insight on synteny between the two species. Conclusions Our approach created significant new resources in pea, i.e. the most comprehensive genetic map to date tightly linked to the model species M. truncatula and a large SNP resource for both academic research and breeding. PMID:24521263

Analysis of expressed sequence tags from Actinidia: applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

PubMed Central

Crowhurst, Ross N; Gleave, Andrew P; MacRae, Elspeth A; Ampomah-Dwamena, Charles; Atkinson, Ross G; Beuning, Lesley L; Bulley, Sean M; Chagne, David; Marsh, Ken B; Matich, Adam J; Montefiori, Mirco; Newcomb, Richard D; Schaffer, Robert J; Usadel, Björn; Allan, Andrew C; Boldingh, Helen L; Bowen, Judith H; Davy, Marcus W; Eckloff, Rheinhart; Ferguson, A Ross; Fraser, Lena G; Gera, Emma; Hellens, Roger P; Janssen, Bart J; Klages, Karin; Lo, Kim R; MacDiarmid, Robin M; Nain, Bhawana; McNeilage, Mark A; Rassam, Maysoon; Richardson, Annette C; Rikkerink, Erik HA; Ross, Gavin S; Schröder, Roswitha; Snowden, Kimberley C; Souleyre, Edwige JF; Templeton, Matt D; Walton, Eric F; Wang, Daisy; Wang, Mindy Y; Wang, Yanming Y; Wood, Marion; Wu, Rongmei; Yauk, Yar-Khing; Laing, William A

2008-01-01

Background Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia. PMID:18655731

Deep whole-genome sequencing of 100 southeast Asian Malays.

PubMed

Wong, Lai-Ping; Ong, Rick Twee-Hee; Poh, Wan-Ting; Liu, Xuanyao; Chen, Peng; Li, Ruoying; Lam, Kevin Koi-Yau; Pillai, Nisha Esakimuthu; Sim, Kar-Seng; Xu, Haiyan; Sim, Ngak-Leng; Teo, Shu-Mei; Foo, Jia-Nee; Tan, Linda Wei-Lin; Lim, Yenly; Koo, Seok-Hwee; Gan, Linda Seo-Hwee; Cheng, Ching-Yu; Wee, Sharon; Yap, Eric Peng-Huat; Ng, Pauline Crystal; Lim, Wei-Yen; Soong, Richie; Wenk, Markus Rene; Aung, Tin; Wong, Tien-Yin; Khor, Chiea-Chuen; Little, Peter; Chia, Kee-Seng; Teo, Yik-Ying

2013-01-10

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Deep Whole-Genome Sequencing of 100 Southeast Asian Malays

PubMed Central

Wong, Lai-Ping; Ong, Rick Twee-Hee; Poh, Wan-Ting; Liu, Xuanyao; Chen, Peng; Li, Ruoying; Lam, Kevin Koi-Yau; Pillai, Nisha Esakimuthu; Sim, Kar-Seng; Xu, Haiyan; Sim, Ngak-Leng; Teo, Shu-Mei; Foo, Jia-Nee; Tan, Linda Wei-Lin; Lim, Yenly; Koo, Seok-Hwee; Gan, Linda Seo-Hwee; Cheng, Ching-Yu; Wee, Sharon; Yap, Eric Peng-Huat; Ng, Pauline Crystal; Lim, Wei-Yen; Soong, Richie; Wenk, Markus Rene; Aung, Tin; Wong, Tien-Yin; Khor, Chiea-Chuen; Little, Peter; Chia, Kee-Seng; Teo, Yik-Ying

2013-01-01

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies. PMID:23290073

Genetic Diversity and Population Structure of Rice Varieties Cultivated in Temperate Regions.

PubMed

Reig-Valiente, Juan L; Viruel, Juan; Sales, Ester; Marqués, Luis; Terol, Javier; Gut, Marta; Derdak, Sophia; Talón, Manuel; Domingo, Concha

2016-12-01

After its domestication, rice cultivation expanded from tropical regions towards northern latitudes with temperate climate in a progressive process to overcome limiting photoperiod and temperature conditions. This process has originated a wide range of diversity that can be regarded as a valuable resource for crop improvement. In general, current rice breeding programs have to deal with a lack of both germplasm accessions specifically adapted to local agro-environmental conditions and adapted donors carrying desired agronomical traits. Comprehensive maps of genome variability and population structure would facilitate genome-wide association studies of complex traits, functional gene investigations and the selection of appropriate donors for breeding purposes. A collection of 217 rice varieties mainly cultivated in temperate regions was generated. The collection encompasses modern elite and old cultivars, as well as traditional landraces covering a wide genetic diversity available for rice breeders. Whole Genome Sequencing was performed on 14 cultivars representative of the collection and the genomic profiles of all cultivars were constructed using a panel of 2697 SNPs with wide coverage throughout the rice genome, obtained from the sequencing data. The population structure and genetic relationship analyses showed a strong substructure in the temperate rice population, predominantly based on grain type and the origin of the cultivars. Dendrogram also agrees population structure results. Based on SNP markers, we have elucidated the genetic relationship and the degree of genetic diversity among a collection of 217 temperate rice varieties possessing an enormous variety of agromorphological and physiological characters. Taken together, the data indicated the occurrence of relatively high gene flow and elevated rates of admixture between cultivars grown in remote regions, probably favoured by local breeding activities. The results of this study significantly expand the current genetic resources available for temperate varieties of rice, providing a valuable tool for future association mapping studies.

Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing

PubMed Central

Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

2015-01-01

The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions. PMID:26067561

Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing

NASA Astrophysics Data System (ADS)

Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

2015-06-01

The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.

Diversity and Structure of Fungal Communities in Neotropical Rainforest Soils: The Effect of Host Recurrence.

PubMed

Schimann, Heidy; Bach, Cyrille; Lengelle, Juliette; Louisanna, Eliane; Barantal, Sandra; Murat, Claude; Buée, Marc

2017-02-01

The patterns of the distribution of fungal species and their potential interactions with trees remain understudied in Neotropical rainforests, which harbor more than 16,000 tree species, mostly dominated by endomycorrhizal trees. Our hypothesis was that tree species shape the non-mycorrhizal fungal assemblages in soil and litter and that the diversity of fungal communities in these two compartments is partly dependent on the coverage of trees in the Neotropical rainforest. In French Guiana, a long-term plantation and a natural forest were selected to test this hypothesis. Fungal ITS1 regions were sequenced from soil and litter samples from within the vicinity of tree species. A broad range of fungal taxa was found, with 42 orders and 14 classes. Significant spatial heterogeneity in the fungal communities was found without strong variation in the species richness and evenness among the tree plots. However, tree species shaped the fungal assemblages in the soil and litter, explaining up to 18 % of the variation among the communities in the natural forest. These results demonstrate that vegetation cover has an important effect on the structure of fungal assemblages inhabiting the soil and litter in Amazonian forests, illustrating the relative impact of deterministic processes on fungal community structures in these highly diverse ecosystems.

Diversity and functions of bacterial community in drinking water biofilms revealed by high-throughput sequencing.

PubMed

Chao, Yuanqing; Mao, Yanping; Wang, Zhiping; Zhang, Tong

2015-06-12

The development of biofilms in drinking water (DW) systems may cause various problems to water quality. To investigate the community structure of biofilms on different pipe materials and the global/specific metabolic functions of DW biofilms, PCR-based 454 pyrosequencing data for 16S rRNA genes and Illumina metagenomic data were generated and analysed. Considerable differences in bacterial diversity and taxonomic structure were identified between biofilms formed on stainless steel and biofilms formed on plastics, indicating that the metallic materials facilitate the formation of higher diversity biofilms. Moreover, variations in several dominant genera were observed during biofilm formation. Based on PCA analysis, the global functions in the DW biofilms were similar to other DW metagenomes. Beyond the global functions, the occurrences and abundances of specific protective genes involved in the glutathione metabolism, the SoxRS system, the OxyR system, RpoS regulated genes, and the production/degradation of extracellular polymeric substances were also evaluated. A near-complete and low-contamination draft genome was constructed from the metagenome of the DW biofilm, based on the coverage and tetranucleotide frequencies, and identified as a Bradyrhizobiaceae-like bacterium according to a phylogenetic analysis. Our findings provide new insight into DW biofilms, especially in terms of their metabolic functions.

Strategies for Achieving High Sequencing Accuracy for Low Diversity Samples and Avoiding Sample Bleeding Using Illumina Platform

PubMed Central

Mitra, Abhishek; Skrzypczak, Magdalena; Ginalski, Krzysztof; Rowicka, Maga

2015-01-01

Sequencing microRNA, reduced representation sequencing, Hi-C technology and any method requiring the use of in-house barcodes result in sequencing libraries with low initial sequence diversity. Sequencing such data on the Illumina platform typically produces low quality data due to the limitations of the Illumina cluster calling algorithm. Moreover, even in the case of diverse samples, these limitations are causing substantial inaccuracies in multiplexed sample assignment (sample bleeding). Such inaccuracies are unacceptable in clinical applications, and in some other fields (e.g. detection of rare variants). Here, we discuss how both problems with quality of low-diversity samples and sample bleeding are caused by incorrect detection of clusters on the flowcell during initial sequencing cycles. We propose simple software modifications (Long Template Protocol) that overcome this problem. We present experimental results showing that our Long Template Protocol remarkably increases data quality for low diversity samples, as compared with the standard analysis protocol; it also substantially reduces sample bleeding for all samples. For comprehensiveness, we also discuss and compare experimental results from alternative approaches to sequencing low diversity samples. First, we discuss how the low diversity problem, if caused by barcodes, can be avoided altogether at the barcode design stage. Second and third, we present modified guidelines, which are more stringent than the manufacturer’s, for mixing low diversity samples with diverse samples and lowering cluster density, which in our experience consistently produces high quality data from low diversity samples. Fourth and fifth, we present rescue strategies that can be applied when sequencing results in low quality data and when there is no more biological material available. In such cases, we propose that the flowcell be re-hybridized and sequenced again using our Long Template Protocol. Alternatively, we discuss how analysis can be repeated from saved sequencing images using the Long Template Protocol to increase accuracy. PMID:25860802

Implementation of Whole Genome Sequencing (WGS) for Identification and Characterization of Shiga Toxin-Producing Escherichia coli (STEC) in the United States

PubMed Central

Lindsey, Rebecca L.; Pouseele, Hannes; Chen, Jessica C.; Strockbine, Nancy A.; Carleton, Heather A.

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS) benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org) and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x coverage, the different databases accurately predicted serotype, virulence, and resistance from WGS data, providing a fast and cheaper alternative to conventional typing techniques. PMID:27242777

Improved Analysis of Nanopore Sequence Data and Scanning Nanopore Techniques

NASA Astrophysics Data System (ADS)

Szalay, Tamas

The field of nanopore research has been driven by the need to inexpensively and rapidly sequence DNA. In order to help realize this goal, this thesis describes the PoreSeq algorithm that identifies and corrects errors in real-world nanopore sequencing data and improves the accuracy of de novo genome assembly with increasing coverage depth. The approach relies on modeling the possible sources of uncertainty that occur as DNA advances through the nanopore and then using this model to find the sequence that best explains multiple reads of the same region of DNA. PoreSeq increases nanopore sequencing read accuracy of M13 bacteriophage DNA from 85% to 99% at 100X coverage. We also use the algorithm to assemble E. coli with 30X coverage and the lambda genome at a range of coverages from 3X to 50X. Additionally, we classify sequence variants at an order of magnitude lower coverage than is possible with existing methods. This thesis also reports preliminary progress towards controlling the motion of DNA using two nanopores instead of one. The speed at which the DNA travels through the nanopore needs to be carefully controlled to facilitate the detection of individual bases. A second nanopore in close proximity to the first could be used to slow or stop the motion of the DNA in order to enable a more accurate readout. The fabrication process for a new pyramidal nanopore geometry was developed in order to facilitate the positioning of the nanopores. This thesis demonstrates that two of them can be placed close enough to interact with a single molecule of DNA, which is a prerequisite for being able to use the driving force of the pores to exert fine control over the motion of the DNA. Another strategy for reading the DNA is to trap it completely with one pore and to move the second nanopore instead. To that end, this thesis also shows that a single strand of immobilized DNA can be captured in a scanning nanopore and examined for a full hour, with data from many scans at many different voltages obtained in order to detect a bound protein placed partway along the molecule.

Implementation of Whole Genome Sequencing (WGS) for Identification and Characterization of Shiga Toxin-Producing Escherichia coli (STEC) in the United States.

PubMed

Lindsey, Rebecca L; Pouseele, Hannes; Chen, Jessica C; Strockbine, Nancy A; Carleton, Heather A

2016-01-01

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS) benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org) and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x coverage, the different databases accurately predicted serotype, virulence, and resistance from WGS data, providing a fast and cheaper alternative to conventional typing techniques.

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

PubMed

Bombarely, Aureliano; Moser, Michel; Amrad, Avichai; Bao, Manzhu; Bapaume, Laure; Barry, Cornelius S; Bliek, Mattijs; Boersma, Maaike R; Borghi, Lorenzo; Bruggmann, Rémy; Bucher, Marcel; D'Agostino, Nunzio; Davies, Kevin; Druege, Uwe; Dudareva, Natalia; Egea-Cortines, Marcos; Delledonne, Massimo; Fernandez-Pozo, Noe; Franken, Philipp; Grandont, Laurie; Heslop-Harrison, J S; Hintzsche, Jennifer; Johns, Mitrick; Koes, Ronald; Lv, Xiaodan; Lyons, Eric; Malla, Diwa; Martinoia, Enrico; Mattson, Neil S; Morel, Patrice; Mueller, Lukas A; Muhlemann, Joëlle; Nouri, Eva; Passeri, Valentina; Pezzotti, Mario; Qi, Qinzhou; Reinhardt, Didier; Rich, Melanie; Richert-Pöggeler, Katja R; Robbins, Tim P; Schatz, Michael C; Schranz, M Eric; Schuurink, Robert C; Schwarzacher, Trude; Spelt, Kees; Tang, Haibao; Urbanus, Susan L; Vandenbussche, Michiel; Vijverberg, Kitty; Villarino, Gonzalo H; Warner, Ryan M; Weiss, Julia; Yue, Zhen; Zethof, Jan; Quattrocchio, Francesca; Sims, Thomas L; Kuhlemeier, Cris

2016-05-27

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

EXAMINING EVIDENCE IN U.S. PAYER COVERAGE POLICIES FOR MULTI-GENE PANELS AND SEQUENCING TESTS

PubMed Central

Chambers, James D.; Saret, Cayla J.; Anderson, Jordan E.; Deverka, Patricia A.; Douglas, Michael P.; Phillips, Kathryn A.

2017-01-01

Objectives The aim of this study was to examine the evidence payers cited in their coverage policies for multi-gene panels and sequencing tests (panels), and to compare these findings with the evidence payers cited in their coverage policies for other types of medical interventions. Methods We used the University of California at San Francisco TRANSPERS Payer Coverage Registry to identify coverage policies for panels issued by five of the largest US private payers. We reviewed each policy and categorized the evidence cited within as: clinical studies, systematic reviews, technology assessments, cost-effectiveness analyses (CEAs), budget impact studies, and clinical guidelines. We compared the evidence cited in these coverage policies for panels with the evidence cited in policies for other intervention types (pharmaceuticals, medical devices, diagnostic tests and imaging, and surgical interventions) as reported in a previous study. Results Fifty-five coverage policies for panels were included. On average, payers cited clinical guidelines in 84 percent of their coverage policies (range, 73–100 percent), clinical studies in 69 percent (50–87 percent), technology assessments 47 percent (33–86 percent), systematic reviews or meta-analyses 31 percent (7–71 percent), and CEAs 5 percent (0–7 percent). No payers cited budget impact studies in their policies. Payers less often cited clinical studies, systematic reviews, technology assessments, and CEAs in their coverage policies for panels than in their policies for other intervention types. Payers cited clinical guidelines in a comparable proportion of policies for panels and other technology types. Conclusions Payers in our sample less often cited clinical studies and other evidence types in their coverage policies for panels than they did in their coverage policies for other types of medical interventions. PMID:29065945

EXAMINING EVIDENCE IN U.S. PAYER COVERAGE POLICIES FOR MULTI-GENE PANELS AND SEQUENCING TESTS.

PubMed

Chambers, James D; Saret, Cayla J; Anderson, Jordan E; Deverka, Patricia A; Douglas, Michael P; Phillips, Kathryn A

2017-01-01

The aim of this study was to examine the evidence payers cited in their coverage policies for multi-gene panels and sequencing tests (panels), and to compare these findings with the evidence payers cited in their coverage policies for other types of medical interventions. We used the University of California at San Francisco TRANSPERS Payer Coverage Registry to identify coverage policies for panels issued by five of the largest US private payers. We reviewed each policy and categorized the evidence cited within as: clinical studies, systematic reviews, technology assessments, cost-effectiveness analyses (CEAs), budget impact studies, and clinical guidelines. We compared the evidence cited in these coverage policies for panels with the evidence cited in policies for other intervention types (pharmaceuticals, medical devices, diagnostic tests and imaging, and surgical interventions) as reported in a previous study. Fifty-five coverage policies for panels were included. On average, payers cited clinical guidelines in 84 percent of their coverage policies (range, 73-100 percent), clinical studies in 69 percent (50-87 percent), technology assessments 47 percent (33-86 percent), systematic reviews or meta-analyses 31 percent (7-71 percent), and CEAs 5 percent (0-7 percent). No payers cited budget impact studies in their policies. Payers less often cited clinical studies, systematic reviews, technology assessments, and CEAs in their coverage policies for panels than in their policies for other intervention types. Payers cited clinical guidelines in a comparable proportion of policies for panels and other technology types. Payers in our sample less often cited clinical studies and other evidence types in their coverage policies for panels than they did in their coverage policies for other types of medical interventions.

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer.

PubMed

Court, Colin M; Ankeny, Jacob S; Sho, Shonan; Hou, Shuang; Li, Qingyu; Hsieh, Carolyn; Song, Min; Liao, Xinfang; Rochefort, Matthew M; Wainberg, Zev A; Graeber, Thomas G; Tseng, Hsian-Rong; Tomlinson, James S

2016-09-01

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Tertiary alphabet for the observable protein structural universe.

PubMed

Mackenzie, Craig O; Zhou, Jianfu; Grigoryan, Gevorg

2016-11-22

Here, we systematically decompose the known protein structural universe into its basic elements, which we dub tertiary structural motifs (TERMs). A TERM is a compact backbone fragment that captures the secondary, tertiary, and quaternary environments around a given residue, comprising one or more disjoint segments (three on average). We seek the set of universal TERMs that capture all structure in the Protein Data Bank (PDB), finding remarkable degeneracy. Only ∼600 TERMs are sufficient to describe 50% of the PDB at sub-Angstrom resolution. However, more rare geometries also exist, and the overall structural coverage grows logarithmically with the number of TERMs. We go on to show that universal TERMs provide an effective mapping between sequence and structure. We demonstrate that TERM-based statistics alone are sufficient to recapitulate close-to-native sequences given either NMR or X-ray backbones. Furthermore, sequence variability predicted from TERM data agrees closely with evolutionary variation. Finally, locations of TERMs in protein chains can be predicted from sequence alone based on sequence signatures emergent from TERM instances in the PDB. For multisegment motifs, this method identifies spatially adjacent fragments that are not contiguous in sequence-a major bottleneck in structure prediction. Although all TERMs recur in diverse proteins, some appear specialized for certain functions, such as interface formation, metal coordination, or even water binding. Structural biology has benefited greatly from previously observed degeneracies in structure. The decomposition of the known structural universe into a finite set of compact TERMs offers exciting opportunities toward better understanding, design, and prediction of protein structure.

An expanded mammal mitogenome dataset from Southeast Asia

PubMed Central

Ramos-Madrigal, Jazmín; Peñaloza, Fernando; Liu, Shanlin; Mikkel-Holger, S. Sinding; Riddhi, P. Patel; Martins, Renata; Lenz, Dorina; Fickel, Jörns; Roos, Christian; Shamsir, Mohd Shahir; Azman, Mohammad Shahfiz; Burton, K. Lim; Stephen, J. Rossiter; Wilting, Andreas

2017-01-01

Abstract Southeast (SE) Asia is 1 of the most biodiverse regions in the world, and it holds approximately 20% of all mammal species. Despite this, the majority of SE Asia's genetic diversity is still poorly characterized. The growing interest in using environmental DNA to assess and monitor SE Asian species, in particular threatened mammals—has created the urgent need to expand the available reference database of mitochondrial barcode and complete mitogenome sequences. We have partially addressed this need by generating 72 new mitogenome sequences reconstructed from DNA isolated from a range of historical and modern tissue samples. Approximately 55 gigabases of raw sequence were generated. From this data, we assembled 72 complete mitogenome sequences, with an average depth of coverage of ×102.9 and ×55.2 for modern samples and historical samples, respectively. This dataset represents 52 species, of which 30 species had no previous mitogenome data available. The mitogenomes were geotagged to their sampling location, where known, to display a detailed geographical distribution of the species. Our new database of 52 taxa will strongly enhance the utility of environmental DNA approaches for monitoring mammals in SE Asia as it greatly increases the likelihoods that identification of metabarcoding sequencing reads can be assigned to reference sequences. This magnifies the confidence in species detections and thus allows more robust surveys and monitoring programmes of SE Asia's threatened mammal biodiversity. The extensive collections of historical samples from SE Asia in western and SE Asian museums should serve as additional valuable material to further enrich this reference database. PMID:28873965

An expanded mammal mitogenome dataset from Southeast Asia.

PubMed

Mohd Salleh, Faezah; Ramos-Madrigal, Jazmín; Peñaloza, Fernando; Liu, Shanlin; Mikkel-Holger, S Sinding; Riddhi, P Patel; Martins, Renata; Lenz, Dorina; Fickel, Jörns; Roos, Christian; Shamsir, Mohd Shahir; Azman, Mohammad Shahfiz; Burton, K Lim; Stephen, J Rossiter; Wilting, Andreas; Gilbert, M Thomas P

2017-08-01

Southeast (SE) Asia is 1 of the most biodiverse regions in the world, and it holds approximately 20% of all mammal species. Despite this, the majority of SE Asia's genetic diversity is still poorly characterized. The growing interest in using environmental DNA to assess and monitor SE Asian species, in particular threatened mammals-has created the urgent need to expand the available reference database of mitochondrial barcode and complete mitogenome sequences. We have partially addressed this need by generating 72 new mitogenome sequences reconstructed from DNA isolated from a range of historical and modern tissue samples. Approximately 55 gigabases of raw sequence were generated. From this data, we assembled 72 complete mitogenome sequences, with an average depth of coverage of ×102.9 and ×55.2 for modern samples and historical samples, respectively. This dataset represents 52 species, of which 30 species had no previous mitogenome data available. The mitogenomes were geotagged to their sampling location, where known, to display a detailed geographical distribution of the species. Our new database of 52 taxa will strongly enhance the utility of environmental DNA approaches for monitoring mammals in SE Asia as it greatly increases the likelihoods that identification of metabarcoding sequencing reads can be assigned to reference sequences. This magnifies the confidence in species detections and thus allows more robust surveys and monitoring programmes of SE Asia's threatened mammal biodiversity. The extensive collections of historical samples from SE Asia in western and SE Asian museums should serve as additional valuable material to further enrich this reference database. © The Author 2017. Published by Oxford University Press.

A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Jr., Richard A.

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted.more » PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Furthermore, our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.« less

A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies

DOE PAGES

Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Jr., Richard A.; ...

2017-07-18

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted.more » PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Furthermore, our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.« less

A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies

PubMed Central

Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Richard A.; Brown, Steven D.

2017-01-01

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences. PMID:28769883

Evidence-Based Assessment of Depression in Adults

ERIC Educational Resources Information Center

Joiner, Thomas E.; Walker, Rheeda L.; Pettit, Jeremy W.; Perez, Marisol; Cukrowicz, Kelly C.

2005-01-01

From diverse perspectives, there is little doubt that depressive symptoms cohere to form a valid and distinct syndrome. Research indicates that an evidence-based assessment of depression would include (a) measures with adequate psychometric properties; (b) adequate coverage of symptoms; (c) adequate coverage of depressed mood, anhedonia, and…

Predictors of elevational biodiversity gradients change from single taxa to the multi-taxa community level

PubMed Central

Peters, Marcell K.; Hemp, Andreas; Appelhans, Tim; Behler, Christina; Classen, Alice; Detsch, Florian; Ensslin, Andreas; Ferger, Stefan W.; Frederiksen, Sara B.; Gebert, Friederike; Haas, Michael; Helbig-Bonitz, Maria; Hemp, Claudia; Kindeketa, William J.; Mwangomo, Ephraim; Ngereza, Christine; Otte, Insa; Röder, Juliane; Rutten, Gemma; Schellenberger Costa, David; Tardanico, Joseph; Zancolli, Giulia; Deckert, Jürgen; Eardley, Connal D.; Peters, Ralph S.; Rödel, Mark-Oliver; Schleuning, Matthias; Ssymank, Axel; Kakengi, Victor; Zhang, Jie; Böhning-Gaese, Katrin; Brandl, Roland; Kalko, Elisabeth K.V.; Kleyer, Michael; Nauss, Thomas; Tschapka, Marco; Fischer, Markus; Steffan-Dewenter, Ingolf

2016-01-01

The factors determining gradients of biodiversity are a fundamental yet unresolved topic in ecology. While diversity gradients have been analysed for numerous single taxa, progress towards general explanatory models has been hampered by limitations in the phylogenetic coverage of past studies. By parallel sampling of 25 major plant and animal taxa along a 3.7 km elevational gradient on Mt. Kilimanjaro, we quantify cross-taxon consensus in diversity gradients and evaluate predictors of diversity from single taxa to a multi-taxa community level. While single taxa show complex distribution patterns and respond to different environmental factors, scaling up diversity to the community level leads to an unambiguous support for temperature as the main predictor of species richness in both plants and animals. Our findings illuminate the influence of taxonomic coverage for models of diversity gradients and point to the importance of temperature for diversification and species coexistence in plant and animal communities. PMID:28004657

Identification, characterization and distribution of transposable elements in the flax (Linum usitatissimum L.) genome.

PubMed

González, Leonardo Galindo; Deyholos, Michael K

2012-11-21

Flax (Linum usitatissimum L.) is an important crop for the production of bioproducts derived from its seed and stem fiber. Transposable elements (TEs) are widespread in plant genomes and are a key component of their evolution. The availability of a genome assembly of flax (Linum usitatissimum) affords new opportunities to explore the diversity of TEs and their relationship to genes and gene expression. Four de novo repeat identification algorithms (PILER, RepeatScout, LTR_finder and LTR_STRUC) were applied to the flax genome assembly. The resulting library of flax repeats was combined with the RepBase Viridiplantae division and used with RepeatMasker to identify TEs coverage in the genome. LTR retrotransposons were the most abundant TEs (17.2% genome coverage), followed by Long Interspersed Nuclear Element (LINE) retrotransposons (2.10%) and Mutator DNA transposons (1.99%). Comparison of putative flax TEs to flax transcript databases indicated that TEs are not highly expressed in flax. However, the presence of recent insertions, defined by 100% intra-element LTR similarity, provided evidence for recent TE activity. Spatial analysis showed TE-rich regions, gene-rich regions as well as regions with similar genes and TE density. Monte Carlo simulations for the 71 largest scaffolds (≥ 1 Mb each) did not show any regional differences in the frequency of TE overlap with gene coding sequences. However, differences between TE superfamilies were found in their proximity to genes. Genes within TE-rich regions also appeared to have lower transcript expression, based on EST abundance. When LTR elements were compared, Copia showed more diversity, recent insertions and conserved domains than the Gypsy, demonstrating their importance in genome evolution. The calculated 23.06% TE coverage of the flax WGS assembly is at the low end of the range of TE coverages reported in other eudicots, although this estimate does not include TEs likely found in unassembled repetitive regions of the genome. Since enrichment for TEs in genomic regions was associated with reduced expression of neighbouring genes, and many members of the Copia LTR superfamily are inserted close to coding regions, we suggest Copia elements have a greater influence on recent flax genome evolution while Gypsy elements have become residual and highly mutated.

Identification, characterization and distribution of transposable elements in the flax (Linum usitatissimum L.) genome

PubMed Central

2012-01-01

Background Flax (Linum usitatissimum L.) is an important crop for the production of bioproducts derived from its seed and stem fiber. Transposable elements (TEs) are widespread in plant genomes and are a key component of their evolution. The availability of a genome assembly of flax (Linum usitatissimum) affords new opportunities to explore the diversity of TEs and their relationship to genes and gene expression. Results Four de novo repeat identification algorithms (PILER, RepeatScout, LTR_finder and LTR_STRUC) were applied to the flax genome assembly. The resulting library of flax repeats was combined with the RepBase Viridiplantae division and used with RepeatMasker to identify TEs coverage in the genome. LTR retrotransposons were the most abundant TEs (17.2% genome coverage), followed by Long Interspersed Nuclear Element (LINE) retrotransposons (2.10%) and Mutator DNA transposons (1.99%). Comparison of putative flax TEs to flax transcript databases indicated that TEs are not highly expressed in flax. However, the presence of recent insertions, defined by 100% intra-element LTR similarity, provided evidence for recent TE activity. Spatial analysis showed TE-rich regions, gene-rich regions as well as regions with similar genes and TE density. Monte Carlo simulations for the 71 largest scaffolds (≥ 1 Mb each) did not show any regional differences in the frequency of TE overlap with gene coding sequences. However, differences between TE superfamilies were found in their proximity to genes. Genes within TE-rich regions also appeared to have lower transcript expression, based on EST abundance. When LTR elements were compared, Copia showed more diversity, recent insertions and conserved domains than the Gypsy, demonstrating their importance in genome evolution. Conclusions The calculated 23.06% TE coverage of the flax WGS assembly is at the low end of the range of TE coverages reported in other eudicots, although this estimate does not include TEs likely found in unassembled repetitive regions of the genome. Since enrichment for TEs in genomic regions was associated with reduced expression of neighbouring genes, and many members of the Copia LTR superfamily are inserted close to coding regions, we suggest Copia elements have a greater influence on recent flax genome evolution while Gypsy elements have become residual and highly mutated. PMID:23171245

High-resolution characterization of a hepatocellular carcinoma genome.

PubMed

Totoki, Yasushi; Tatsuno, Kenji; Yamamoto, Shogo; Arai, Yasuhito; Hosoda, Fumie; Ishikawa, Shumpei; Tsutsumi, Shuichi; Sonoda, Kohtaro; Totsuka, Hirohiko; Shirakihara, Takuya; Sakamoto, Hiromi; Wang, Linghua; Ojima, Hidenori; Shimada, Kazuaki; Kosuge, Tomoo; Okusaka, Takuji; Kato, Kazuto; Kusuda, Jun; Yoshida, Teruhiko; Aburatani, Hiroyuki; Shibata, Tatsuhiro

2011-05-01

Hepatocellular carcinoma, one of the most common virus-associated cancers, is the third most frequent cause of cancer-related death worldwide. By massively parallel sequencing of a primary hepatitis C virus-positive hepatocellular carcinoma (36× coverage) and matched lymphocytes (>28× coverage) from the same individual, we identified more than 11,000 somatic substitutions of the tumor genome that showed predominance of T>C/A>G transition and a decrease of the T>C substitution on the transcribed strand, suggesting preferential DNA repair. Gene annotation enrichment analysis of 63 validated non-synonymous substitutions revealed enrichment of phosphoproteins. We further validated 22 chromosomal rearrangements, generating four fusion transcripts that had altered transcriptional regulation (BCORL1-ELF4) or promoter activity. Whole-exome sequencing at a higher sequence depth (>76× coverage) revealed a TSC1 nonsense substitution in a subpopulation of the tumor cells. This first high-resolution characterization of a virus-associated cancer genome identified previously uncharacterized mutation patterns, intra-chromosomal rearrangements and fusion genes, as well as genetic heterogeneity within the tumor.

Multiple hybrid de novo genome assembly of finger millet, an orphan allotetraploid crop

PubMed Central

Hatakeyama, Masaomi; Aluri, Sirisha; Balachadran, Mathi Thumilan; Sivarajan, Sajeevan Radha; Patrignani, Andrea; Grüter, Simon; Poveda, Lucy; Shimizu-Inatsugi, Rie; Baeten, John; Francoijs, Kees-Jan; Nataraja, Karaba N; Reddy, Yellodu A Nanja; Phadnis, Shamprasad; Ravikumar, Ramapura L; Schlapbach, Ralph; Sreeman, Sheshshayee M; Shimizu, Kentaro K

2018-01-01

Abstract Finger millet (Eleusine coracana (L.) Gaertn) is an important crop for food security because of its tolerance to drought, which is expected to be exacerbated by global climate changes. Nevertheless, it is often classified as an orphan/underutilized crop because of the paucity of scientific attention. Among several small millets, finger millet is considered as an excellent source of essential nutrient elements, such as iron and zinc; hence, it has potential as an alternate coarse cereal. However, high-quality genome sequence data of finger millet are currently not available. One of the major problems encountered in the genome assembly of this species was its polyploidy, which hampers genome assembly compared with a diploid genome. To overcome this problem, we sequenced its genome using diverse technologies with sufficient coverage and assembled it via a novel multiple hybrid assembly workflow that combines next-generation with single-molecule sequencing, followed by whole-genome optical mapping using the Bionano Irys® system. The total number of scaffolds was 1,897 with an N50 length >2.6 Mb and detection of 96% of the universal single-copy orthologs. The majority of the homeologs were assembled separately. This indicates that the proposed workflow is applicable to the assembly of other allotetraploid genomes. PMID:28985356

The international Genome sample resource (IGSR): A worldwide collection of genome variation incorporating the 1000 Genomes Project data.

PubMed

Clarke, Laura; Fairley, Susan; Zheng-Bradley, Xiangqun; Streeter, Ian; Perry, Emily; Lowy, Ernesto; Tassé, Anne-Marie; Flicek, Paul

2017-01-04

The International Genome Sample Resource (IGSR; http://www.internationalgenome.org) expands in data type and population diversity the resources from the 1000 Genomes Project. IGSR represents the largest open collection of human variation data and provides easy access to these resources. IGSR was established in 2015 to maintain and extend the 1000 Genomes Project data, which has been widely used as a reference set of human variation and by researchers developing analysis methods. IGSR has mapped all of the 1000 Genomes sequence to the newest human reference (GRCh38), and will release updated variant calls to ensure maximal usefulness of the existing data. IGSR is collecting new structural variation data on the 1000 Genomes samples from long read sequencing and other technologies, and will collect relevant functional data into a single comprehensive resource. IGSR is extending coverage with new populations sequenced by collaborating groups. Here, we present the new data and analysis that IGSR has made available. We have also introduced a new data portal that increases discoverability of our data-previously only browseable through our FTP site-by focusing on particular samples, populations or data sets of interest. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes

PubMed Central

2011-01-01

Background We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. Results The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. Conclusions BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies. PMID:21496274

Molecular Diagnosis of Usher Syndrome: Application of Two Different Next Generation Sequencing-Based Procedures

PubMed Central

Licastro, Danilo; Mutarelli, Margherita; Peluso, Ivana; Neveling, Kornelia; Wieskamp, Nienke; Rispoli, Rossella; Vozzi, Diego; Athanasakis, Emmanouil; D'Eustacchio, Angela; Pizzo, Mariateresa; D'Amico, Francesca; Ziviello, Carmela; Simonelli, Francesca; Fabretto, Antonella; Scheffer, Hans; Gasparini, Paolo; Banfi, Sandro; Nigro, Vincenzo

2012-01-01

Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified. PMID:22952768

A RESTful application programming interface for the PubMLST molecular typing and genome databases

PubMed Central

Bray, James E.; Maiden, Martin C. J.

2017-01-01

Abstract Molecular typing is used to differentiate microorganisms at the subspecies or strain level for epidemiological investigations, infection control, public health and environmental sampling. DNA sequence-based typing methods require authoritative databases that link sequence variants to nomenclature in order to facilitate communication and comparison of identified types in national or global settings. The PubMLST website (https://pubmlst.org/) fulfils this role for over a hundred microorganisms for which it hosts curated molecular sequence typing data, providing sequence and allelic profile definitions for multi-locus sequence typing (MLST) and single-gene typing approaches. In recent years, these have expanded to cover the whole genome with schemes such as core genome MLST (cgMLST) and whole genome MLST (wgMLST) which catalogue the allelic diversity found in hundreds to thousands of genes. These approaches provide a common nomenclature for high-resolution strain characterization and comparison. Molecular typing information is linked to isolate provenance, phenotype, and increasingly genome assemblies, providing a resource for outbreak investigation and research in to population structure, gene association, global epidemiology and vaccine coverage. A Representational State Transfer (REST) Application Programming Interface (API) has been developed for the PubMLST website to make these large quantities of structured molecular typing and whole genome sequence data available for programmatic access by any third party application. The API is an integral component of the Bacterial Isolate Genome Sequence Database (BIGSdb) platform that is used to host PubMLST resources, and exposes all public data within the site. In addition to data browsing, searching and download, the API supports authentication and submission of new data to curator queues. Database URL: http://rest.pubmlst.org/ PMID:29220452

Evaluating imputation algorithms for low-depth genotyping-by-sequencing (GBS) data

USDA-ARS?s Scientific Manuscript database

Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordabl...

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

PubMed Central

Lucero, Mary E.; Unc, Adrian; Cooke, Peter; Dowd, Scot; Sun, Shulei

2011-01-01

Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities. PMID:21437280

EEG microstates during resting represent personality differences.

PubMed

Schlegel, Felix; Lehmann, Dietrich; Faber, Pascal L; Milz, Patricia; Gianotti, Lorena R R

2012-01-01

We investigated the spontaneous brain electric activity of 13 skeptics and 16 believers in paranormal phenomena; they were university students assessed with a self-report scale about paranormal beliefs. 33-channel EEG recordings during no-task resting were processed as sequences of momentary potential distribution maps. Based on the maps at peak times of Global Field Power, the sequences were parsed into segments of quasi-stable potential distribution, the 'microstates'. The microstates were clustered into four classes of map topographies (A-D). Analysis of the microstate parameters time coverage, occurrence frequency and duration as well as the temporal sequence (syntax) of the microstate classes revealed significant differences: Believers had a higher coverage and occurrence of class B, tended to decreased coverage and occurrence of class C, and showed a predominant sequence of microstate concatenations from A to C to B to A that was reversed in skeptics (A to B to C to A). Microstates of different topographies, putative "atoms of thought", are hypothesized to represent different types of information processing.The study demonstrates that personality differences can be detected in resting EEG microstate parameters and microstate syntax. Microstate analysis yielded no conclusive evidence for the hypothesized relation between paranormal belief and schizophrenia.

A case of canine borreliosis in Iran caused by Borrelia persica.

PubMed

Shirani, Darush; Rakhshanpoor, Alaleh; Cutler, Sally Jane; Ghazinezhad, Behnaz; Naddaf, Saied Reza

2016-04-01

Tick-borne relapsing fever is an endemic disease in Iran, with most cases attributed to infection by Borrelia persica, which is transmitted by Ornithodoros tholozani soft ticks. Here, we report spirochetemia in blood of a puppy residing in Tehran, Iran. The causative species was identified by use of highly discriminative IGS sequencing; the 489 bp IGS sequence obtained in our study showed 99% identity (100% coverage) when compared with B. persica sequences derived from clinical cases or from O. tholozani ticks. Our IGS sequence also showed 99% similarity over 414 bp (85% coverage) with a strain from a domestic dog, and 96% over 328 bp (69% coverage) with a strain from a domestic cat. Pet-keeping in cosmopolitan cities like Tehran has become increasingly popular in recent years. Animals are often transported into the city in cages or cardboard boxes that might also harbor minute tick larvae and/or early stages of the nymphs bringing them into the urban environment. This may pose a threat to household members who buy and keep these puppies and as a result may come into close contact with infected ticks. Copyright © 2016 Elsevier GmbH. All rights reserved.

ReadXplorer—visualization and analysis of mapped sequences

PubMed Central

Hilker, Rolf; Stadermann, Kai Bernd; Doppmeier, Daniel; Kalinowski, Jörn; Stoye, Jens; Straube, Jasmin; Winnebald, Jörn; Goesmann, Alexander

2014-01-01

Motivation: Fast algorithms and well-arranged visualizations are required for the comprehensive analysis of the ever-growing size of genomic and transcriptomic next-generation sequencing data. Results: ReadXplorer is a software offering straightforward visualization and extensive analysis functions for genomic and transcriptomic DNA sequences mapped on a reference. A unique specialty of ReadXplorer is the quality classification of the read mappings. It is incorporated in all analysis functions and displayed in ReadXplorer's various synchronized data viewers for (i) the reference sequence, its base coverage as (ii) normalizable plot and (iii) histogram, (iv) read alignments and (v) read pairs. ReadXplorer's analysis capability covers RNA secondary structure prediction, single nucleotide polymorphism and deletion–insertion polymorphism detection, genomic feature and general coverage analysis. Especially for RNA-Seq data, it offers differential gene expression analysis, transcription start site and operon detection as well as RPKM value and read count calculations. Furthermore, ReadXplorer can combine or superimpose coverage of different datasets. Availability and implementation: ReadXplorer is available as open-source software at http://www.readxplorer.org along with a detailed manual. Contact: rhilker@mikrobio.med.uni-giessen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24790157

Phylogenomics from Whole Genome Sequences Using aTRAM.

PubMed

Allen, Julie M; Boyd, Bret; Nguyen, Nam-Phuong; Vachaspati, Pranjal; Warnow, Tandy; Huang, Daisie I; Grady, Patrick G S; Bell, Kayce C; Cronk, Quentin C B; Mugisha, Lawrence; Pittendrigh, Barry R; Leonardi, M Soledad; Reed, David L; Johnson, Kevin P

2017-09-01

Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes ($<$1000 Mbp) it is feasible to sequence the entire genome at modest coverage ($10-30\\times$). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single-copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out-groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close- to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above $5-10\\times$, aTRAM was successful at assembling 80-90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.]. © The Author(s) 2017. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Low diversity, activity, and density of transposable elements in five avian genomes.

PubMed

Gao, Bo; Wang, Saisai; Wang, Yali; Shen, Dan; Xue, Songlei; Chen, Cai; Cui, Hengmi; Song, Chengyi

2017-07-01

In this study, we conducted the activity, diversity, and density analysis of transposable elements (TEs) across five avian genomes (budgerigar, chicken, turkey, medium ground finch, and zebra finch) to explore the potential reason of small genome sizes of birds. We found that these avian genomes exhibited low density of TEs by about 10% of genome coverages and low diversity of TEs with the TE landscapes dominated by CR1 and ERV elements, and contrasting proliferation dynamics both between TE types and between species were observed across the five avian genomes. Phylogenetic analysis revealed that CR1 clade was more diverse in the family structure compared with R2 clade in birds; avian ERVs were classified into four clades (alpha, beta, gamma, and ERV-L) and belonged to three classes of ERV with an uneven distributed in these lineages. The activities of DNA and SINE TEs were very low in the evolution history of avian genomes; most LINEs and LTRs were ancient copies with a substantial decrease of activity in recent, with only LTRs and LINEs in chicken and zebra finch exhibiting weak activity in very recent, and very few TEs were intact; however, the recent activity may be underestimated due to the sequencing/assembly technologies in some species. Overall, this study demonstrates low diversity, activity, and density of TEs in the five avian species; highlights the differences of TEs in these lineages; and suggests that the current and recent activity of TEs in avian genomes is very limited, which may be one of the reasons of small genome sizes in birds.

HetMappsS: Heterozygous mapping strategy for high resolution Genotyping-by-Sequencing Markers

USDA-ARS?s Scientific Manuscript database

Reduced representation genotyping approaches, such as genotyping-by-sequencing (GBS), provide opportunities to generate high-resolution genetic maps at a low per-sample cost. However, missing data and non-uniform sequence coverage can complicate map creation in highly heterozygous species. To facili...

Method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation in conjunction with mass spectrometric analysis

DOEpatents

Laskin, Julia [Richland, WA; Futrell, Jean H [Richland, WA

2008-04-29

The invention relates to a method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation (SID) in conjunction with mass spectrometric analysis. Results demonstrate formation of a wide distribution of structure-specific fragments having wide sequence coverage useful for sequencing and identifying the complex molecules.

Whole genome sequence analysis of BT-474 using complete Genomics' standard and long fragment read technologies.

PubMed

Ciotlos, Serban; Mao, Qing; Zhang, Rebecca Yu; Li, Zhenyu; Chin, Robert; Gulbahce, Natali; Liu, Sophie Jia; Drmanac, Radoje; Peters, Brock A

2016-01-01

The cell line BT-474 is a popular cell line for studying the biology of cancer and developing novel drugs. However, there is no complete, published genome sequence for this highly utilized scientific resource. In this study we sought to provide a comprehensive and useful data set for the scientific community by generating a whole genome sequence for BT-474. Five μg of genomic DNA, isolated from an early passage of the BT-474 cell line, was used to generate a whole genome sequence (114X coverage) using Complete Genomics' standard sequencing process. To provide additional variant phasing and structural variation data we also processed and analyzed two separate libraries of 5 and 6 individual cells to depths of 99X and 87X, respectively, using Complete Genomics' Long Fragment Read (LFR) technology. BT-474 is a highly aneuploid cell line with an extremely complex genome sequence. This ~300X total coverage genome sequence provides a more complete understanding of this highly utilized cell line at the genomic level.

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.

PubMed

Wang, Zhiwen; Hobson, Neil; Galindo, Leonardo; Zhu, Shilin; Shi, Daihu; McDill, Joshua; Yang, Linfeng; Hawkins, Simon; Neutelings, Godfrey; Datla, Raju; Lambert, Georgina; Galbraith, David W; Grassa, Christopher J; Geraldes, Armando; Cronk, Quentin C; Cullis, Christopher; Dash, Prasanta K; Kumar, Polumetla A; Cloutier, Sylvie; Sharpe, Andrew G; Wong, Gane K-S; Wang, Jun; Deyholos, Michael K

2012-11-01

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity

PubMed Central

Cousins, Matthew M.; Ou, San-San; Wawer, Maria J.; Munshaw, Supriya; Swan, David; Magaret, Craig A.; Mullis, Caroline E.; Serwadda, David; Porcella, Stephen F.; Gray, Ronald H.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

2012-01-01

Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution. PMID:22785188

Caught in the middle with multiple displacement amplification: the myth of pooling for avoiding multiple displacement amplification bias in a metagenome.

PubMed

Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric

2014-01-30

Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.

Sequenced sorghum mutant library- an efficient platform for discovery of causal gene mutations

USDA-ARS?s Scientific Manuscript database

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. We applied whole-genome sequencing to 256 phenotyped mutant lines of sorghum (Sorghum bicolor L. Moench) to 16x coverage. Comparisons with the reference sequence revealed >1.8 million canonical EMS-induced G/C to A...

Launch mission summary and terminal countdown, Delta 153 Satellite Business Systems satellite (SBS-A)

NASA Technical Reports Server (NTRS)

1980-01-01

A brief summary of the launch vehicle, spacecraft, and mission is contained. Information relative to launch windows, vehicle telemetry coverage, realtime data flow, telemetry coverage by station, selected trajectory information, and a brief sequence of flight events is also included.

Tertiary alphabet for the observable protein structural universe

PubMed Central

Mackenzie, Craig O.; Zhou, Jianfu; Grigoryan, Gevorg

2016-01-01

Here, we systematically decompose the known protein structural universe into its basic elements, which we dub tertiary structural motifs (TERMs). A TERM is a compact backbone fragment that captures the secondary, tertiary, and quaternary environments around a given residue, comprising one or more disjoint segments (three on average). We seek the set of universal TERMs that capture all structure in the Protein Data Bank (PDB), finding remarkable degeneracy. Only ∼600 TERMs are sufficient to describe 50% of the PDB at sub-Angstrom resolution. However, more rare geometries also exist, and the overall structural coverage grows logarithmically with the number of TERMs. We go on to show that universal TERMs provide an effective mapping between sequence and structure. We demonstrate that TERM-based statistics alone are sufficient to recapitulate close-to-native sequences given either NMR or X-ray backbones. Furthermore, sequence variability predicted from TERM data agrees closely with evolutionary variation. Finally, locations of TERMs in protein chains can be predicted from sequence alone based on sequence signatures emergent from TERM instances in the PDB. For multisegment motifs, this method identifies spatially adjacent fragments that are not contiguous in sequence—a major bottleneck in structure prediction. Although all TERMs recur in diverse proteins, some appear specialized for certain functions, such as interface formation, metal coordination, or even water binding. Structural biology has benefited greatly from previously observed degeneracies in structure. The decomposition of the known structural universe into a finite set of compact TERMs offers exciting opportunities toward better understanding, design, and prediction of protein structure. PMID:27810958

Whole-genome sequencing approaches for conservation biology: Advantages, limitations and practical recommendations.

PubMed

Fuentes-Pardo, Angela P; Ruzzante, Daniel E

2017-10-01

Whole-genome resequencing (WGR) is a powerful method for addressing fundamental evolutionary biology questions that have not been fully resolved using traditional methods. WGR includes four approaches: the sequencing of individuals to a high depth of coverage with either unresolved or resolved haplotypes, the sequencing of population genomes to a high depth by mixing equimolar amounts of unlabelled-individual DNA (Pool-seq) and the sequencing of multiple individuals from a population to a low depth (lcWGR). These techniques require the availability of a reference genome. This, along with the still high cost of shotgun sequencing and the large demand for computing resources and storage, has limited their implementation in nonmodel species with scarce genomic resources and in fields such as conservation biology. Our goal here is to describe the various WGR methods, their pros and cons and potential applications in conservation biology. WGR offers an unprecedented marker density and surveys a wide diversity of genetic variations not limited to single nucleotide polymorphisms (e.g., structural variants and mutations in regulatory elements), increasing their power for the detection of signatures of selection and local adaptation as well as for the identification of the genetic basis of phenotypic traits and diseases. Currently, though, no single WGR approach fulfils all requirements of conservation genetics, and each method has its own limitations and sources of potential bias. We discuss proposed ways to minimize such biases. We envision a not distant future where the analysis of whole genomes becomes a routine task in many nonmodel species and fields including conservation biology. © 2017 John Wiley & Sons Ltd.

Genome Survey Sequencing of Luffa Cylindrica L. and Microsatellite High Resolution Melting (SSR-HRM) Analysis for Genetic Relationship of Luffa Genotypes

PubMed Central

An, Jianyu; Yin, Mengqi; Zhang, Qin; Gong, Dongting; Jia, Xiaowen; Guan, Yajing; Hu, Jin

2017-01-01

Luffa cylindrica (L.) Roem. is an economically important vegetable crop in China. However, the genomic information on this species is currently unknown. In this study, for the first time, a genome survey of L. cylindrica was carried out using next-generation sequencing (NGS) technology. In total, 43.40 Gb sequence data of L. cylindrica, about 54.94× coverage of the estimated genome size of 789.97 Mb, were obtained from HiSeq 2500 sequencing, in which the guanine plus cytosine (GC) content was calculated to be 37.90%. The heterozygosity of genome sequences was only 0.24%. In total, 1,913,731 contigs (>200 bp) with 525 bp N50 length and 1,410,117 scaffolds (>200 bp) with 885.01 Mb total length were obtained. From the initial assembled L. cylindrica genome, 431,234 microsatellites (SSRs) (≥5 repeats) were identified. The motif types of SSR repeats included 62.88% di-nucleotide, 31.03% tri-nucleotide, 4.59% tetra-nucleotide, 0.96% penta-nucleotide and 0.54% hexa-nucleotide. Eighty genomic SSR markers were developed, and 51/80 primers could be used in both “Zheda 23” and “Zheda 83”. Nineteen SSRs were used to investigate the genetic diversity among 32 accessions through SSR-HRM analysis. The unweighted pair group method analysis (UPGMA) dendrogram tree was built by calculating the SSR-HRM raw data. SSR-HRM could be effectively used for genotype relationship analysis of Luffa species. PMID:28891982

Developmental validation of a custom panel including 273 SNPs for forensic application using Ion Torrent PGM.

PubMed

Zhang, Suhua; Bian, Yingnan; Chen, Anqi; Zheng, Hancheng; Gao, Yuzhen; Hou, Yiping; Li, Chengtao

2017-03-01

Utilizing massively parallel sequencing (MPS) technology for SNP testing in forensic genetics is becoming attractive because of the shortcomings of STR markers, such as their high mutation rates and disadvantages associated with the current PCR-CE method as well as its limitations regarding multiplex capabilities. MPS offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. In this study, we designed a customized SNP panel that includes 273 forensically relevant identity SNPs chosen from SNPforID, IISNP, and the HapMap database as well as previously related studies and evaluated the levels of genotyping precision, sequence coverage, sensitivity and SNP performance using the Ion Torrent PGM. In a concordant study of the custom MPS-SNP panel, only four MPS callings were missing due to coverage reads that were too low (<20), whereas the others were fully concordant with Sanger's sequencing results across the two control samples, that is, 9947A and 9948. The analyses indicated a balanced coverage among the included loci, with the exception of the 16 SNPs that were used to detect an inconsistent allele balance and/or lower coverage reads among 50 tested individuals from the Chinese HAN population and the above controls. With the exception of the 16 poorly performing SNPs, the sequence coverage obtained was extensive for the bulk of the SNPs, and only three Y-SNPs (rs16980601, rs11096432, rs3900) showed a mean coverage below 1000. Analyses of the dilution series of control DNA 9948 yielded reproducible results down to 1ng of DNA input. In addition, we provide an analysis tool for automated data quality control and genotyping checks, and we conclude that the SNP targets are polymorphic and independent in the Chinese HAN population. In summary, the evaluation of the sensitivity, accuracy and genotyping performance provides strong support for the application of MPS technology in forensic SNP analysis, and the assay offers a straightforward sample-to-genotype workflow that could be beneficial in forensic casework with respect to both individual identification and complex kinship issues. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Caititu: a tool to graphically represent peptide sequence coverage and domain distribution.

PubMed

Carvalho, Paulo C; Junqueira, Magno; Valente, Richard H; Domont, Gilberto B

2008-10-07

Here we present Caititu, an easy-to-use proteomics software to graphically represent peptide sequence coverage and domain distribution for different correlated samples (e.g. originated from 2D gel spots) relatively to the full-sequence of the known protein they are related to. Although Caititu has a broad applicability, we exemplify its usefulness in Toxinology using snake venom as a model. For example, proteolytic processing may lead to inactivation or loss of domains. Therefore, our proposed graphic representation for peptides identified by two dimensional electrophoresis followed by mass spectrometric identification of excised spots can aid in inferring what kind of processing happened to the toxins, if any. Caititu is freely available to download at: http://pcarvalho.com/things/caititu.

High-quality mtDNA control region sequences from 680 individuals sampled across the Netherlands to establish a national forensic mtDNA reference database.

PubMed

Chaitanya, Lakshmi; van Oven, Mannis; Brauer, Silke; Zimmermann, Bettina; Huber, Gabriela; Xavier, Catarina; Parson, Walther; de Knijff, Peter; Kayser, Manfred

2016-03-01

The use of mitochondrial DNA (mtDNA) for maternal lineage identification often marks the last resort when investigating forensic and missing-person cases involving highly degraded biological materials. As with all comparative DNA testing, a match between evidence and reference sample requires a statistical interpretation, for which high-quality mtDNA population frequency data are crucial. Here, we determined, under high quality standards, the complete mtDNA control-region sequences of 680 individuals from across the Netherlands sampled at 54 sites, covering the entire country with 10 geographic sub-regions. The complete mtDNA control region (nucleotide positions 16,024-16,569 and 1-576) was amplified with two PCR primers and sequenced with ten different sequencing primers using the EMPOP protocol. Haplotype diversity of the entire sample set was very high at 99.63% and, accordingly, the random-match probability was 0.37%. No population substructure within the Netherlands was detected with our dataset. Phylogenetic analyses were performed to determine mtDNA haplogroups. Inclusion of these high-quality data in the EMPOP database (accession number: EMP00666) will improve its overall data content and geographic coverage in the interest of all EMPOP users worldwide. Moreover, this dataset will serve as (the start of) a national reference database for mtDNA applications in forensic and missing person casework in the Netherlands. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A genome sequence resource for the aye-aye (Daubentonia madagascariensis), a nocturnal lemur from Madagascar.

PubMed

Perry, George H; Reeves, Darryl; Melsted, Páll; Ratan, Aakrosh; Miller, Webb; Michelini, Katelyn; Louis, Edward E; Pritchard, Jonathan K; Mason, Christopher E; Gilad, Yoav

2012-01-01

We present a high-coverage draft genome assembly of the aye-aye (Daubentonia madagascariensis), a highly unusual nocturnal primate from Madagascar. Our assembly totals ~3.0 billion bp (3.0 Gb), roughly the size of the human genome, comprised of ~2.6 million scaffolds (N50 scaffold size = 13,597 bp) based on short paired-end sequencing reads. We compared the aye-aye genome sequence data with four other published primate genomes (human, chimpanzee, orangutan, and rhesus macaque) as well as with the mouse and dog genomes as nonprimate outgroups. Unexpectedly, we observed strong evidence for a relatively slow substitution rate in the aye-aye lineage compared with these and other primates. In fact, the aye-aye branch length is estimated to be ~10% shorter than that of the human lineage, which is known for its low substitution rate. This finding may be explained, in part, by the protracted aye-aye life-history pattern, including late weaning and age of first reproduction relative to other lemurs. Additionally, the availability of this draft lemur genome sequence allowed us to polarize nucleotide and protein sequence changes to the ancestral primate lineage-a critical period in primate evolution, for which the relevant fossil record is sparse. Finally, we identified 293,800 high-confidence single nucleotide polymorphisms in the donor individual for our aye-aye genome sequence, a captive-born individual from two wild-born parents. The resulting heterozygosity estimate of 0.051% is the lowest of any primate studied to date, which is understandable considering the aye-aye's extensive home-range size and relatively low population densities. Yet this level of genetic diversity also suggests that conservation efforts benefiting this unusual species should be prioritized, especially in the face of the accelerating degradation and fragmentation of Madagascar's forests.

Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits.

PubMed

Imprialou, Martha; Kahles, André; Steffen, Joshua G; Osborne, Edward J; Gan, Xiangchao; Lempe, Janne; Bhomra, Amarjit; Belfield, Eric; Visscher, Anne; Greenhalgh, Robert; Harberd, Nicholas P; Goram, Richard; Hein, Jotun; Robert-Seilaniantz, Alexandre; Jones, Jonathan; Stegle, Oliver; Kover, Paula; Tsiantis, Miltos; Nordborg, Magnus; Rätsch, Gunnar; Clark, Richard M; Mott, Richard

2017-04-01

To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii , isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions. Copyright © 2017 by the Genetics Society of America.

Draft versus finished sequence data for DNA and protein diagnostic signature development

PubMed Central

Gardner, Shea N.; Lam, Marisa W.; Smith, Jason R.; Torres, Clinton L.; Slezak, Tom R.

2005-01-01

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10−3–10−5 (∼8× coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of ∼1% (3× to 6× coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures. PMID:16243783

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing.

PubMed

Hribová, Eva; Neumann, Pavel; Matsumoto, Takashi; Roux, Nicolas; Macas, Jirí; Dolezel, Jaroslav

2010-09-16

Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection.

Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing

PubMed Central

2010-01-01

Background Bananas and plantains (Musa spp.) are grown in more than a hundred tropical and subtropical countries and provide staple food for hundreds of millions of people. They are seed-sterile crops propagated clonally and this makes them vulnerable to a rapid spread of devastating diseases and at the same time hampers breeding improved cultivars. Although the socio-economic importance of bananas and plantains cannot be overestimated, they remain outside the focus of major research programs. This slows down the study of nuclear genome and the development of molecular tools to facilitate banana improvement. Results In this work, we report on the first thorough characterization of the repeat component of the banana (M. acuminata cv. 'Calcutta 4') genome. Analysis of almost 100 Mb of sequence data (0.15× genome coverage) permitted partial sequence reconstruction and characterization of repetitive DNA, making up about 30% of the genome. The results showed that the banana repeats are predominantly made of various types of Ty1/copia and Ty3/gypsy retroelements representing 16 and 7% of the genome respectively. On the other hand, DNA transposons were found to be rare. In addition to new families of transposable elements, two new satellite repeats were discovered and found useful as cytogenetic markers. To help in banana sequence annotation, a specific Musa repeat database was created, and its utility was demonstrated by analyzing the repeat composition of 62 genomic BAC clones. Conclusion A low-depth 454 sequencing of banana nuclear genome provided the largest amount of DNA sequence data available until now for Musa and permitted reconstruction of most of the major types of DNA repeats. The information obtained in this study improves the knowledge of the long-range organization of banana chromosomes, and provides sequence resources needed for repeat masking and annotation during the Musa genome sequencing project. It also provides sequence data for isolation of DNA markers to be used in genetic diversity studies and in marker-assisted selection. PMID:20846365

Genomic Sequence around Butterfly Wing Development Genes: Annotation and Comparative Analysis

PubMed Central

Conceição, Inês C.; Long, Anthony D.; Gruber, Jonathan D.; Beldade, Patrícia

2011-01-01

Background Analysis of genomic sequence allows characterization of genome content and organization, and access beyond gene-coding regions for identification of functional elements. BAC libraries, where relatively large genomic regions are made readily available, are especially useful for species without a fully sequenced genome and can increase genomic coverage of phylogenetic and biological diversity. For example, no butterfly genome is yet available despite the unique genetic and biological properties of this group, such as diversified wing color patterns. The evolution and development of these patterns is being studied in a few target species, including Bicyclus anynana, where a whole-genome BAC library allows targeted access to large genomic regions. Methodology/Principal Findings We characterize ∼1.3 Mb of genomic sequence around 11 selected genes expressed in B. anynana developing wings. Extensive manual curation of in silico predictions, also making use of a large dataset of expressed genes for this species, identified repetitive elements and protein coding sequence, and highlighted an expansion of Alcohol dehydrogenase genes. Comparative analysis with orthologous regions of the lepidopteran reference genome allowed assessment of conservation of fine-scale synteny (with detection of new inversions and translocations) and of DNA sequence (with detection of high levels of conservation of non-coding regions around some, but not all, developmental genes). Conclusions The general properties and organization of the available B. anynana genomic sequence are similar to the lepidopteran reference, despite the more than 140 MY divergence. Our results lay the groundwork for further studies of new interesting findings in relation to both coding and non-coding sequence: 1) the Alcohol dehydrogenase expansion with higher similarity between the five tandemly-repeated B. anynana paralogs than with the corresponding B. mori orthologs, and 2) the high conservation of non-coding sequence around the genes wingless and Ecdysone receptor, both involved in multiple developmental processes including wing pattern formation. PMID:21909358

Ancestry estimation and control of population stratification for sequence-based association studies.

PubMed

Wang, Chaolong; Zhan, Xiaowei; Bragg-Gresham, Jennifer; Kang, Hyun Min; Stambolian, Dwight; Chew, Emily Y; Branham, Kari E; Heckenlively, John; Fulton, Robert; Wilson, Richard K; Mardis, Elaine R; Lin, Xihong; Swaroop, Anand; Zöllner, Sebastian; Abecasis, Gonçalo R

2014-04-01

Estimating individual ancestry is important in genetic association studies where population structure leads to false positive signals, although assigning ancestry remains challenging with targeted sequence data. We propose a new method for the accurate estimation of individual genetic ancestry, based on direct analysis of off-target sequence reads, and implement our method in the publicly available LASER software. We validate the method using simulated and empirical data and show that the method can accurately infer worldwide continental ancestry when used with sequencing data sets with whole-genome shotgun coverage as low as 0.001×. For estimates of fine-scale ancestry within Europe, the method performs well with coverage of 0.1×. On an even finer scale, the method improves discrimination between exome-sequenced study participants originating from different provinces within Finland. Finally, we show that our method can be used to improve case-control matching in genetic association studies and to reduce the risk of spurious findings due to population structure.

Language Development: Understanding Language Diversity in the Classroom

ERIC Educational Resources Information Center

Levey, Sandra; Polirstok, Susan

2010-01-01

Language Development: Understanding Language Diversity in the Classroom offers comprehensive coverage of the language development process for pre- and in-service teachers while emphasizing the factors that further academic success in the classroom, including literacy skills, phonological awareness, and narrative. With chapters written by respected…

On-Line Electrochemical Reduction of Disulfide Bonds: Improved FTICR-CID and -ETD Coverage of Oxytocin and Hepcidin

NASA Astrophysics Data System (ADS)

Nicolardi, Simone; Giera, Martin; Kooijman, Pieter; Kraj, Agnieszka; Chervet, Jean-Pierre; Deelder, André M.; van der Burgt, Yuri E. M.

2013-12-01

Particularly in the field of middle- and top-down peptide and protein analysis, disulfide bridges can severely hinder fragmentation and thus impede sequence analysis (coverage). Here we present an on-line/electrochemistry/ESI-FTICR-MS approach, which was applied to the analysis of the primary structure of oxytocin, containing one disulfide bridge, and of hepcidin, containing four disulfide bridges. The presented workflow provided up to 80 % (on-line) conversion of disulfide bonds in both peptides. With minimal sample preparation, such reduction resulted in a higher number of peptide backbone cleavages upon CID or ETD fragmentation, and thus yielded improved sequence coverage. The cycle times, including electrode recovery, were rapid and, therefore, might very well be coupled with liquid chromatography for protein or peptide separation, which has great potential for high-throughput analysis.

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

PubMed Central

Hurt, Richard A.; Robeson, Michael S.; Shakya, Migun; Moberly, James G.; Vishnivetskaya, Tatiana A.; Gu, Baohua; Elias, Dwayne A.

2014-01-01

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered. PMID:25033199

Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation.

PubMed

Artieri, Carlo G; Fraser, Hunter B

2014-12-01

The recent advent of ribosome profiling-sequencing of short ribosome-bound fragments of mRNA-has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling data set have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a method that implicated positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that it produces false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling. © 2014 Artieri and Fraser; Published by Cold Spring Harbor Laboratory Press.

New in-depth rainbow trout transcriptome reference and digital atlas of gene expression

USDA-ARS?s Scientific Manuscript database

Sequencing the rainbow trout genome is underway and a transcriptome reference sequence is required to help in genome assembly and gene discovery. Previously, we reported a transcriptome reference sequence using a 19X coverage of 454-pyrosequencing data. Although this work added a great wealth of ann...

Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.

PubMed

Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew

2017-11-06

Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

Hypersaline sapropels act as hotspots for microbial dark matter

DOE PAGES

Andrei, Adrian -Stefan; Baricz, Andreea; Robeson, Michael Scott; ...

2017-07-21

Present-day terrestrial analogue sites are crucial ground truth proxies for studying life in geochemical conditions close to those assumed to be present on early Earth or inferred to exist on other celestial bodies (e.g. Mars, Europa). Although hypersaline sapropels are border-of-life habitats with moderate occurrence, their microbiological and physicochemical characterization lags behind. Here, we study the diversity of life under low water activity by describing the prokaryotic communities from two disparate hypersaline sapropels (Transylvanian Basin, Romania) in relation to geochemical milieu and pore water chemistry, while inferring their role in carbon cycling by matching taxa to known taxon-specific biogeochemical functions.more » Furthermore, the polyphasic approach combined deep coverage SSU rRNA gene amplicon sequencing and bioinformatics with RT-qPCR and physicochemical investigations. We found that sapropels developed an analogous elemental milieu and harbored prokaryotes affiliated with fifty-nine phyla, among which the most abundant were Proteobacteria, Bacteroidetes and Chloroflexi. Containing thirty-two candidate divisions and possibly undocumented prokaryotic lineages, the hypersaline sapropels were found to accommodate one of the most diverse and novel ecosystems reported to date and may contribute to completing the phylogenetic branching of the tree of life.« less

An integrated map of genetic variation from 1,092 human genomes

PubMed Central

2012-01-01

Summary Through characterising the geographic and functional spectrum of human genetic variation, the 1000 Genomes Project aims to build a resource to help understand the genetic contribution to disease. We describe the genomes of 1,092 individuals from 14 populations, constructed using a combination of low-coverage whole-genome and exome sequencing. By developing methodologies to integrate information across multiple algorithms and diverse data sources we provide a validated haplotype map of 38 million SNPs, 1.4 million indels and over 14 thousand larger deletions. We show that individuals from different populations carry different profiles of rare and common variants and that low-frequency variants show substantial geographic differentiation, which is further increased by the action of purifying selection. We show that evolutionary conservation and coding consequence are key determinants of the strength of purifying selection, that rare-variant load varies substantially across biological pathways and that each individual harbours hundreds of rare non-coding variants at conserved sites, such as transcription-factor-motif disrupting changes. This resource, which captures up to 98% of accessible SNPs at a frequency of 1% in populations of medical genetics focus, enables analysis of common and low-frequency variants in individuals from diverse, including admixed, populations. PMID:23128226

Hypersaline sapropels act as hotspots for microbial dark matter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andrei, Adrian -Stefan; Baricz, Andreea; Robeson, Michael Scott

Present-day terrestrial analogue sites are crucial ground truth proxies for studying life in geochemical conditions close to those assumed to be present on early Earth or inferred to exist on other celestial bodies (e.g. Mars, Europa). Although hypersaline sapropels are border-of-life habitats with moderate occurrence, their microbiological and physicochemical characterization lags behind. Here, we study the diversity of life under low water activity by describing the prokaryotic communities from two disparate hypersaline sapropels (Transylvanian Basin, Romania) in relation to geochemical milieu and pore water chemistry, while inferring their role in carbon cycling by matching taxa to known taxon-specific biogeochemical functions.more » Furthermore, the polyphasic approach combined deep coverage SSU rRNA gene amplicon sequencing and bioinformatics with RT-qPCR and physicochemical investigations. We found that sapropels developed an analogous elemental milieu and harbored prokaryotes affiliated with fifty-nine phyla, among which the most abundant were Proteobacteria, Bacteroidetes and Chloroflexi. Containing thirty-two candidate divisions and possibly undocumented prokaryotic lineages, the hypersaline sapropels were found to accommodate one of the most diverse and novel ecosystems reported to date and may contribute to completing the phylogenetic branching of the tree of life.« less

Influence of Peanut Cultivars and Environmental Conditions on the Diversity and Community Composition of Pod Rot Soil Fungi in China.

PubMed

Wang, Mian; Chen, Mingna; Yang, Zhen; Chen, Na; Chi, Xiaoyuan; Pan, Lijuan; Wang, Tong; Yu, Shanlin; Guo, Xingqi

2017-12-01

Peanut yield and quality are seriously affected by pod rot pathogens worldwide, especially in China in recent years. The goals of this study are to analyze the structure of fungal communities of peanut pod rot in soil in three peanut cultivars and the correlation of pod rot with environmental variables using 454 pyrosequencing. A total of 46,723 internal transcribed spacer high-quality sequences were obtained and grouped into 1,706 operational taxonomic units at the 97% similarity cut-off level. The coverage, rank abundance, and the Chao 1 and Shannon diversity indices of the operational taxonomic units were analyzed. Members of the phylum Ascomycota were dominant, such as Fusarium , Chaetomium , Alternaria , and Sordariomycetes , followed by Basidiomycota. The results of the heatmap and redundancy analysis revealed significant variation in the composition of the fungal community among the three cultivar samples. The environmental conditions in different peanut cultivars may also influence on the structure of the fungal community. The results of this study suggest that the causal agent of peanut pod rot may be more complex, and cultivars and environmental conditions are both important contributors to the community structure of peanut pod rot fungi.

Switch from oral to inactivated poliovirus vaccine in Yogyakarta Province, Indonesia: summary of coverage, immunity, and environmental surveillance.

PubMed

Wahjuhono, Gendro; Revolusiana; Widhiastuti, Dyah; Sundoro, Julitasari; Mardani, Tri; Ratih, Woro Umi; Sutomo, Retno; Safitri, Ida; Sampurno, Ondri Dwi; Rana, Bardan; Roivainen, Merja; Kahn, Anna-Lea; Mach, Ondrej; Pallansch, Mark A; Sutter, Roland W

2014-11-01

Inactivated poliovirus vaccine (IPV) is rarely used in tropical developing countries. To generate additional scientific information, especially on the possible emergence of vaccine-derived polioviruses (VDPVs) in an IPV-only environment, we initiated an IPV introduction project in Yogyakarta, an Indonesian province. In this report, we present the coverage, immunity, and VDPV surveillance results. In Yogyakarta, we established environmental surveillance starting in 2004; and conducted routine immunization coverage and seroprevalence surveys before and after a September 2007 switch from oral poliovirus vaccine (OPV) to IPV, using standard coverage and serosurvey methods. Rates and types of polioviruses found in sewage samples were analyzed, and all poliovirus isolates after the switch were sequenced. Vaccination coverage (>95%) and immunity (approximately 100%) did not change substantially before and after the IPV switch. No VDPVs were detected. Before the switch, 58% of environmental samples contained Sabin poliovirus; starting 6 weeks after the switch, Sabin polioviruses were rarely isolated, and if they were, genetic sequencing suggested recent introductions. This project demonstrated that under almost ideal conditions (good hygiene, maintenance of universally high IPV coverage, and corresponding high immunity against polioviruses), no emergence and circulation of VDPV could be detected in a tropical developing country setting. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Exome sequencing for prenatal diagnosis of fetuses with sonographic abnormalities.

PubMed

Drury, Suzanne; Williams, Hywel; Trump, Natalie; Boustred, Christopher; Lench, Nicholas; Scott, Richard H; Chitty, Lyn S

2015-10-01

In the absence of aneuploidy or other pathogenic cytogenetic abnormality, fetuses with increased nuchal translucency (NT ≥ 3.5 mm) and/or other sonographic abnormalities have a greater incidence of genetic syndromes, but defining the underlying pathology can be challenging. Here, we investigate the value of whole exome sequencing in fetuses with sonographic abnormalities but normal microarray analysis. Whole exome sequencing was performed on DNA extracted from chorionic villi or amniocytes in 24 fetuses with unexplained ultrasound findings. In the first 14 cases sequencing was initially performed on fetal DNA only. For the remaining 10, the trio of fetus, mother and father was sequenced simultaneously. In 21% (5/24) cases, exome sequencing provided definitive diagnoses (Milroy disease, hypophosphatasia, achondrogenesis type 2, Freeman-Sheldon syndrome and Baraitser-Winter Syndrome). In a further case, a plausible diagnosis of orofaciodigital syndrome type 6 was made. In two others, a single mutation in an autosomal recessive gene was identified, but incomplete sequencing coverage precluded exclusion of the presence of a second mutation. Whole exome sequencing improves prenatal diagnosis in euploid fetuses with abnormal ultrasound scans. In order to expedite interpretation of results, trio sequencing should be employed, but interpretation can still be compromised by incomplete coverage of relevant genes. © 2015 John Wiley & Sons, Ltd.

Exploring Pandora's Box: Potential and Pitfalls of Low Coverage Genome Surveys for Evolutionary Biology

PubMed Central

Leese, Florian; Mayer, Christoph; Agrawal, Shobhit; Dambach, Johannes; Dietz, Lars; Doemel, Jana S.; Goodall-Copstake, William P.; Held, Christoph; Jackson, Jennifer A.; Lampert, Kathrin P.; Linse, Katrin; Macher, Jan N.; Nolzen, Jennifer; Raupach, Michael J.; Rivera, Nicole T.; Schubart, Christoph D.; Striewski, Sebastian; Tollrian, Ralph; Sands, Chester J.

2012-01-01

High throughput sequencing technologies are revolutionizing genetic research. With this “rise of the machines”, genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02–25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers. PMID:23185309

Verifying Digital Components of Physical Systems: Experimental Evaluation of Test Quality

NASA Astrophysics Data System (ADS)

Laputenko, A. V.; López, J. E.; Yevtushenko, N. V.

2018-03-01

This paper continues the study of high quality test derivation for verifying digital components which are used in various physical systems; those are sensors, data transfer components, etc. We have used logic circuits b01-b010 of the package of ITC'99 benchmarks (Second Release) for experimental evaluation which as stated before, describe digital components of physical systems designed for various applications. Test sequences are derived for detecting the most known faults of the reference logic circuit using three different approaches to test derivation. Three widely used fault types such as stuck-at-faults, bridges, and faults which slightly modify the behavior of one gate are considered as possible faults of the reference behavior. The most interesting test sequences are short test sequences that can provide appropriate guarantees after testing, and thus, we experimentally study various approaches to the derivation of the so-called complete test suites which detect all fault types. In the first series of experiments, we compare two approaches for deriving complete test suites. In the first approach, a shortest test sequence is derived for testing each fault. In the second approach, a test sequence is pseudo-randomly generated by the use of an appropriate software for logic synthesis and verification (ABC system in our study) and thus, can be longer. However, after deleting sequences detecting the same set of faults, a test suite returned by the second approach is shorter. The latter underlines the fact that in many cases it is useless to spend `time and efforts' for deriving a shortest distinguishing sequence; it is better to use the test minimization afterwards. The performed experiments also show that the use of only randomly generated test sequences is not very efficient since such sequences do not detect all the faults of any type. After reaching the fault coverage around 70%, saturation is observed, and the fault coverage cannot be increased anymore. For deriving high quality short test suites, the approach that is the combination of randomly generated sequences together with sequences which are aimed to detect faults not detected by random tests, allows to reach the good fault coverage using shortest test sequences.

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

PubMed

Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

Prescription Drug Diversion: Predictors of Illicit Acquisition and Redistribution in Three U.S. Metropolitan Areas.

PubMed

Harris, Shana; Nikulina, Valentina; Gelpí-Acosta, Camila; Morton, Cory; Newsome, Valerie; Gunn, Alana; Hoefinger, Heidi; Aikins, Ross; Smith, Vivian; Barry, Victoria; Downing, Martin J

2015-12-02

Prescription drug diversion, the transfer of prescription drugs from lawful to unlawful channels for distribution or use, is a problem in the United States. Despite the pervasiveness of diversion, there are gaps in the literature regarding characteristics of individuals who participate in the illicit trade of prescription drugs. This study examines a range of predictors (e.g., demographics, prescription insurance coverage, perceived risk associated with prescription drug diversion) of membership in three distinct diverter groups: individuals who illicitly acquire prescription drugs, those who redistribute them, and those who engage in both behaviors. Data were drawn from a cross-sectional Internet study ( N = 846) of prescription drug use and diversion patterns in New York City, South Florida, and Washington, D.C.. Participants were classified into diversion categories based on their self-reported involvement in the trade of prescription drugs. Group differences in background characteristics of diverter groups were assessed by Chi-Square tests and followed up with multivariate logistic regressions. While individuals in all diversion groups were more likely to be younger and have a licit prescription for any of the assessed drugs in the past year than those who did not divert, individuals who both acquire and redistribute are more likely to live in New York City, not have prescription insurance coverage, and perceive fewer legal risks of prescription drug diversion. Findings suggest that predictive characteristics vary according to diverter group.

Genomes of Salmonella with diverse patterns of antibiotic resistance (AR) revealed the dynamics of AR gene organization and detected resistance gene families found in Salmonella

USDA-ARS?s Scientific Manuscript database

We produced and assembled high quality draft genomes (~100X coverage) for 305 Salmonella from a diverse a group of over 100 serovars and diverse sources. Of these isolates, 119 were selected to capture a wide variety of different AR patterns. In our subsequent analyses we included 285 additional pub...

[Spatio-temporal distribution of carabids and spiders between semi-natural field margin and the adjacent crop fields in agricultural landscape].

PubMed

Zhang, Xu Zhu; Han, Yin; Yu, Zhen Rong; Liu, Yun Hui

2017-06-18

This study was conducted before and after harvesting of wheat and maize in a typical agricultural landscape of the North China Plain. We investigated the diversity of two important natural enemy groups, carabids and spiders, using pitfall traps at crop field margin with different vegetation structures and their neighboring crop field. Throughout the comparison of the spatial and temporal distribution of the diversity of carabids and spiders in field margin and neighboring field, and the investigation of the relationship between arthropod communities and vegetation structure, this study aimed to understand the role of semi-natural field margin in biodiversity conservation of different natural enemy taxa. Results showed that the abundance of spiders was significantly higher in field margin than in neighboring fields over the entire period. No significant difference of the diversity of carabids in field margin and crop field was observed, but the community composition was different. Number of spider families increased in field margin but deceased in crop field after harvesting, indicating a migration activity between field and field margin. Vegetation structure in the field margin had different association with carabids than with spiders, with diversity of dominant carabid species positively associated with herb coverage and negatively with wood coverage, while the diversity of spider family Linyphiidae was positively associated with herb coverage only. Semi-natural habitat benefited the conservation of the diversity of arthropod natural enemies in crop field via promoting their dispersal to crop field, while such impacts differed from different vegetation structures and varied from target beneficial natural enemy communities. Future studies should focus on in-depth understanding of the food and habitat source requirement of different natural enemy taxa, and hence to design suitable semi-natural habitats to maintain a high diversity of natural enemy communities.

Multifaceted diversity-area relationships reveal global hotspots of mammalian species, trait and lineage diversity.

PubMed

Mazel, Florent; Guilhaumon, François; Mouquet, Nicolas; Devictor, Vincent; Gravel, Dominique; Renaud, Julien; Cianciaruso, Marcus Vinicius; Loyola, Rafael Dias; Diniz-Filho, José Alexandre Felizola; Mouillot, David; Thuiller, Wilfried

2014-08-01

To define biome-scale hotspots of phylogenetic and functional mammalian biodiversity (PD and FD, respectively) and compare them to 'classical' hotspots based on species richness (SR) only. Global. SR, PD & FD were computed for 782 terrestrial ecoregions using distribution ranges of 4616 mammalian species. We used a set of comprehensive diversity indices unified by a recent framework that incorporates the species relative coverage in each ecoregion. We build large-scale multifaceted diversity-area relationships to rank ecoregions according to their levels of biodiversity while accounting for the effect of area on each diversity facet. Finally we defined hotspots as the top-ranked ecoregions. While ignoring species relative coverage led to a relative good congruence between biome top ranked SR, PD and FD hotspots, ecoregions harboring a rich and abundantly represented evolutionary history and functional diversity did not match with top ranked ecoregions defined by species richness. More importantly PD and FD hotspots showed important spatial mismatches. We also found that FD and PD generally reached their maximum values faster than species richness as a function of area. The fact that PD/FD reach faster their maximal value than SR may suggest that the two former facets might be less vulnerable to habitat loss than the latter. While this point is expected, it is the first time that it is quantified at global scale and should have important consequences in conservation. Incorporating species relative coverage into the delineation of multifaceted hotspots of diversity lead to weak congruence between SR, PD and FD hotspots. This means that maximizing species number may fail at preserving those nodes (in the phylogenetic or functional tree) that are relatively abundant in the ecoregion. As a consequence it may be of prime importance to adopt a multifaceted biodiversity perspective to inform conservation strategies at global scale.

Multifaceted diversity-area relationships reveal global hotspots of mammalian species, trait and lineage diversity

PubMed Central

Mazel, Florent; Guilhaumon, François; Mouquet, Nicolas; Devictor, Vincent; Gravel, Dominique; Renaud, Julien; Cianciaruso, Marcus Vinicius; Loyola, Rafael Dias; Diniz-Filho, José Alexandre Felizola; Mouillot, David; Thuiller, Wilfried

2014-01-01

Aim To define biome-scale hotspots of phylogenetic and functional mammalian biodiversity (PD and FD, respectively) and compare them to ‘classical’ hotspots based on species richness (SR) only. Location Global Methods SR, PD & FD were computed for 782 terrestrial ecoregions using distribution ranges of 4616 mammalian species. We used a set of comprehensive diversity indices unified by a recent framework that incorporates the species relative coverage in each ecoregion. We build large-scale multifaceted diversity-area relationships to rank ecoregions according to their levels of biodiversity while accounting for the effect of area on each diversity facet. Finally we defined hotspots as the top-ranked ecoregions. Results While ignoring species relative coverage led to a relative good congruence between biome top ranked SR, PD and FD hotspots, ecoregions harboring a rich and abundantly represented evolutionary history and functional diversity did not match with top ranked ecoregions defined by species richness. More importantly PD and FD hotspots showed important spatial mismatches. We also found that FD and PD generally reached their maximum values faster than species richness as a function of area. Main conclusions The fact that PD/FD reach faster their maximal value than SR may suggest that the two former facets might be less vulnerable to habitat loss than the latter. While this point is expected, it is the first time that it is quantified at global scale and should have important consequences in conservation. Incorporating species relative coverage into the delineation of multifaceted hotspots of diversity lead to weak congruence between SR, PD and FD hotspots. This means that maximizing species number may fail at preserving those nodes (in the phylogenetic or functional tree) that are relatively abundant in the ecoregion. As a consequence it may be of prime importance to adopt a multifaceted biodiversity perspective to inform conservation strategies at global scale. PMID:25071413

Deep sequencing with intronic capture enables identification of an APC exon 10 inversion in a patient with polyposis.

PubMed

Shirts, Brian H; Salipante, Stephen J; Casadei, Silvia; Ryan, Shawnia; Martin, Judith; Jacobson, Angela; Vlaskin, Tatyana; Koehler, Karen; Livingston, Robert J; King, Mary-Claire; Walsh, Tom; Pritchard, Colin C

2014-10-01

Single-exon inversions have rarely been described in clinical syndromes and are challenging to detect using Sanger sequencing. We report the case of a 40-year-old woman with adenomatous colon polyps too numerous to count and who had a complex inversion spanning the entire exon 10 in APC (the gene encoding for adenomatous polyposis coli), causing exon skipping and resulting in a frameshift and premature protein truncation. In this study, we employed complete APC gene sequencing using high-coverage next-generation sequencing by ColoSeq, analysis with BreakDancer and SLOPE software, and confirmatory transcript analysis. ColoSeq identified a complex small genomic rearrangement consisting of an inversion that results in translational skipping of exon 10 in the APC gene. This mutation would not have been detected by traditional sequencing or gene-dosage methods. We report a case of adenomatous polyposis resulting from a complex single-exon inversion. Our report highlights the benefits of large-scale sequencing methods that capture intronic sequences with high enough depth of coverage-as well as the use of informatics tools-to enable detection of small pathogenic structural rearrangements.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

PubMed

Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D; Nystul, Todd G; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E; Murphy, Terence D; Levis, Robert W; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A; Spradling, Allan C

2007-03-01

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

Gene family innovation, conservation and loss on the animal stem lineage.

PubMed

Richter, Daniel J; Fozouni, Parinaz; Eisen, Michael; King, Nicole

2018-05-31

Choanoflagellates, the closest living relatives of animals, can provide unique insights into the changes in gene content that preceded the origin of animals. However, only two choanoflagellate genomes are currently available, providing poor coverage of their diversity. We sequenced transcriptomes of 19 additional choanoflagellate species to produce a comprehensive reconstruction of the gains and losses that shaped the ancestral animal gene repertoire. We identified ~1,944 gene families that originated on the animal stem lineage, of which only 39 are conserved across all animals in our study. In addition, ~372 gene families previously thought to be animal-specific, including Notch, Delta, and homologs of the animal Toll-like receptor genes, instead evolved prior to the animal-choanoflagellate divergence. Our findings contribute to an increasingly detailed portrait of the gene families that defined the biology of the Urmetazoan and that may underpin core features of extant animals. © 2018, Richter et al.

XX/XY Sex Chromosomes in the South American Dwarf Gecko (Gonatodes humeralis).

PubMed

Gamble, Tony; McKenna, Erin; Meyer, Wyatt; Nielsen, Stuart V; Pinto, Brendan J; Scantlebury, Daniel P; Higham, Timothy E

2018-05-11

Sex-specific genetic markers identified using restriction site-associated DNA sequencing, or RADseq, permits the recognition of a species' sex chromosome system in cases where standard cytogenetic methods fail. Thus, species with male-specific RAD markers have an XX/XY sex chromosome system (male heterogamety) while species with female-specific RAD markers have a ZZ/ZW sex chromosome (female heterogamety). Here, we use RADseq data from 5 male and 5 female South American dwarf geckos (Gonatodes humeralis) to identify an XX/XY sex chromosome system. This is the first confidently known sex chromosome system in a Gonatodes species. We used a low-coverage de novo G. humeralis genome assembly to design PCR primers to validate the male-specificity of a subset of the sex-specific RADseq markers and describe how even modest genome assemblies can facilitate the design of sex-specific PCR primers in species with diverse sex chromosome systems.

Genome sequence and genetic diversity of the common carp, Cyprinus carpio.

PubMed

Xu, Peng; Zhang, Xiaofeng; Wang, Xumin; Li, Jiongtang; Liu, Guiming; Kuang, Youyi; Xu, Jian; Zheng, Xianhu; Ren, Lufeng; Wang, Guoliang; Zhang, Yan; Huo, Linhe; Zhao, Zixia; Cao, Dingchen; Lu, Cuiyun; Li, Chao; Zhou, Yi; Liu, Zhanjiang; Fan, Zhonghua; Shan, Guangle; Li, Xingang; Wu, Shuangxiu; Song, Lipu; Hou, Guangyuan; Jiang, Yanliang; Jeney, Zsigmond; Yu, Dan; Wang, Li; Shao, Changjun; Song, Lai; Sun, Jing; Ji, Peifeng; Wang, Jian; Li, Qiang; Xu, Liming; Sun, Fanyue; Feng, Jianxin; Wang, Chenghui; Wang, Shaolin; Wang, Baosen; Li, Yan; Zhu, Yaping; Xue, Wei; Zhao, Lan; Wang, Jintu; Gu, Ying; Lv, Weihua; Wu, Kejing; Xiao, Jingfa; Wu, Jiayan; Zhang, Zhang; Yu, Jun; Sun, Xiaowen

2014-11-01

The common carp, Cyprinus carpio, is one of the most important cyprinid species and globally accounts for 10% of freshwater aquaculture production. Here we present a draft genome of domesticated C. carpio (strain Songpu), whose current assembly contains 52,610 protein-coding genes and approximately 92.3% coverage of its paleotetraploidized genome (2n = 100). The latest round of whole-genome duplication has been estimated to have occurred approximately 8.2 million years ago. Genome resequencing of 33 representative individuals from worldwide populations demonstrates a single origin for C. carpio in 2 subspecies (C. carpio Haematopterus and C. carpio carpio). Integrative genomic and transcriptomic analyses were used to identify loci potentially associated with traits including scaling patterns and skin color. In combination with the high-resolution genetic map, the draft genome paves the way for better molecular studies and improved genome-assisted breeding of C. carpio and other closely related species.

Necessary Sequencing Depth and Clustering Method to Obtain Relatively Stable Diversity Patterns in Studying Fish Gut Microbiota.

PubMed

Xiao, Fanshu; Yu, Yuhe; Li, Jinjin; Juneau, Philippe; Yan, Qingyun

2018-05-25

The 16S rRNA gene is one of the most commonly used molecular markers for estimating bacterial diversity during the past decades. However, there is no consistency about the sequencing depth (from thousand to millions of sequences per sample), and the clustering methods used to generate OTUs may also be different among studies. These inconsistent premises make effective comparisons among studies difficult or unreliable. This study aims to examine the necessary sequencing depth and clustering method that would be needed to ensure a stable diversity patterns for studying fish gut microbiota. A total number of 42 samples dataset of Siniperca chuatsi (carnivorous fish) gut microbiota were used to test how the sequencing depth and clustering may affect the alpha and beta diversity patterns of fish intestinal microbiota. Interestingly, we found that the sequencing depth (resampling 1000-11,000 per sample) and the clustering methods (UPARSE and UCLUST) did not bias the estimates of the diversity patterns during the fish development from larva to adult. Although we should acknowledge that a suitable sequencing depth may differ case by case, our finding indicates that a shallow sequencing such as 1000 sequences per sample may be also enough to reflect the general diversity patterns of fish gut microbiota. However, we have shown in the present study that strict pre-processing of the original sequences is required to ensure reliable results. This study provides evidences to help making a strong scientific choice of the sequencing depth and clustering method for future studies on fish gut microbiota patterns, but at the same time reducing as much as possible the costs related to the analysis.

[Effects of legume-oat intercropping on abundance and community structure of soil N2-fixing bacteria].

PubMed

Yang, Ya Dong; Feng, Xiao Min; Hu, Yue Gao; Ren, Chang Zhong; Zeng, Zhao Hai

2017-03-18

In this study, real-time PCR and high-throughput sequencing approaches were employed to investigate the abundance and community structure of N 2 -fixing bacteria in a field experiment with three planting patterns (Oat monoculture, O; Soybean-oat intercropping, OSO; Mung bean-oat intercropping, OMO). The results showed that soil chemical properties varied significantly in different soil samples (P＜0.05). The abundance of nifH gene varied from 1.75×10 10 to 7.37×10 10 copies·g -1 dry soil in all soil samples. The copy numbers of nifH gene in OSO and OMO were 2.18, 2.64, and 1.92, 2.57 times as much as that in O at jointing and mature stages, with a significant decline from jointing to mature stage for all treatments (P＜0.05). Rarefaction curve and cove-rage results proved the nifH gene sequencing results were reliable, and the diversity index showed that the N 2 -fixing bacteria diversity of OSO was much higher than that of O. Azohydromonas, Azotobacter, Bradyrhizobium, Skermanella and other groups that could not be classified are the dominant genera, with significant differences in proportion of these dominant groups observed among all soil samples (P＜0.05). Venn and PCA analysis indicated that there were greater differences of nifH gene communities between jointing and mature stages; however, the OSO and OMO had similar communities in both stages. All these results confirmed that legume-oat intercropping significantly increased the abundance and changed the community composition of N 2 -fixing bacteria in oat soils.

Defining the diverse spectrum of inversions, complex structural variation, and chromothripsis in the morbid human genome.

PubMed

Collins, Ryan L; Brand, Harrison; Redin, Claire E; Hanscom, Carrie; Antolik, Caroline; Stone, Matthew R; Glessner, Joseph T; Mason, Tamara; Pregno, Giulia; Dorrani, Naghmeh; Mandrile, Giorgia; Giachino, Daniela; Perrin, Danielle; Walsh, Cole; Cipicchio, Michelle; Costello, Maura; Stortchevoi, Alexei; An, Joon-Yong; Currall, Benjamin B; Seabra, Catarina M; Ragavendran, Ashok; Margolin, Lauren; Martinez-Agosto, Julian A; Lucente, Diane; Levy, Brynn; Sanders, Stephan J; Wapner, Ronald J; Quintero-Rivera, Fabiola; Kloosterman, Wigard; Talkowski, Michael E

2017-03-06

Structural variation (SV) influences genome organization and contributes to human disease. However, the complete mutational spectrum of SV has not been routinely captured in disease association studies. We sequenced 689 participants with autism spectrum disorder (ASD) and other developmental abnormalities to construct a genome-wide map of large SV. Using long-insert jumping libraries at 105X mean physical coverage and linked-read whole-genome sequencing from 10X Genomics, we document seven major SV classes at ~5 kb SV resolution. Our results encompass 11,735 distinct large SV sites, 38.1% of which are novel and 16.8% of which are balanced or complex. We characterize 16 recurrent subclasses of complex SV (cxSV), revealing that: (1) cxSV are larger and rarer than canonical SV; (2) each genome harbors 14 large cxSV on average; (3) 84.4% of large cxSVs involve inversion; and (4) most large cxSV (93.8%) have not been delineated in previous studies. Rare SVs are more likely to disrupt coding and regulatory non-coding loci, particularly when truncating constrained and disease-associated genes. We also identify multiple cases of catastrophic chromosomal rearrangements known as chromoanagenesis, including somatic chromoanasynthesis, and extreme balanced germline chromothripsis events involving up to 65 breakpoints and 60.6 Mb across four chromosomes, further defining rare categories of extreme cxSV. These data provide a foundational map of large SV in the morbid human genome and demonstrate a previously underappreciated abundance and diversity of cxSV that should be considered in genomic studies of human disease.

Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: a multidisciplinary development unifying molecular biology, chemical and electronics engineering.

PubMed

Kumar Khanna, Vinod

2007-01-01

The current status and research trends of detection techniques for DNA-based analysis such as DNA finger printing, sequencing, biochips and allied fields are examined. An overview of main detectors is presented vis-à-vis these DNA operations. The biochip method is explained, the role of micro- and nanoelectronic technologies in biochip realization is highlighted, various optical and electrical detection principles employed in biochips are indicated, and the operational mechanisms of these detection devices are described. Although a diversity of biochips for diagnostic and therapeutic applications has been demonstrated in research laboratories worldwide, only some of these chips have entered the clinical market, and more chips are awaiting commercialization. The necessity of tagging is eliminated in refractive-index change based devices, but the basic flaw of indirect nature of most detection methodologies can only be overcome by generic and/or reagentless DNA sensors such as the conductance-based approach and the DNA-single electron transistor (DNA-SET) structure. Devices of the electrical detection-based category are expected to pave the pathway for the next-generation DNA chips. The review provides a comprehensive coverage of the detection technologies for DNA finger printing, sequencing and related techniques, encompassing a variety of methods from the primitive art to the state-of-the-art scenario as well as promising methods for the future.

The role of the RAS pathway in iAMP21-ALL

PubMed Central

Ryan, S L; Matheson, E; Grossmann, V; Sinclair, P; Bashton, M; Schwab, C; Towers, W; Partington, M; Elliott, A; Minto, L; Richardson, S; Rahman, T; Keavney, B; Skinner, R; Bown, N; Haferlach, T; Vandenberghe, P; Haferlach, C; Santibanez-Koref, M; Moorman, A V; Kohlmann, A; Irving, J A E; Harrison, C J

2016-01-01

Intrachromosomal amplification of chromosome 21 (iAMP21) identifies a high-risk subtype of acute lymphoblastic leukaemia (ALL), requiring intensive treatment to reduce their relapse risk. Improved understanding of the genomic landscape of iAMP21-ALL will ascertain whether these patients may benefit from targeted therapy. We performed whole-exome sequencing of eight iAMP21-ALL samples. The mutation rate was dramatically disparate between cases (average 24.9, range 5–51) and a large number of novel variants were identified, including frequent mutation of the RAS/MEK/ERK pathway. Targeted sequencing of a larger cohort revealed that 60% (25/42) of diagnostic iAMP21-ALL samples harboured 42 distinct RAS pathway mutations. High sequencing coverage demonstrated heterogeneity in the form of multiple RAS pathway mutations within the same sample and diverse variant allele frequencies (VAFs) (2–52%), similar to other subtypes of ALL. Constitutive RAS pathway activation was observed in iAMP21 samples that harboured mutations in the predominant clone (⩾35% VAF). Viable iAMP21 cells from primary xenografts showed reduced viability in response to the MEK1/2 inhibitor, selumetinib, in vitro. As clonal (⩾35% VAF) mutations were detected in 26% (11/42) of iAMP21-ALL, this evidence of response to RAS pathway inhibitors may offer the possibility to introduce targeted therapy to improve therapeutic efficacy in these high-risk patients. PMID:27168466

The Y-Chromosome Tree Bursts into Leaf: 13,000 High-Confidence SNPs Covering the Majority of Known Clades

PubMed Central

Hallast, Pille; Batini, Chiara; Zadik, Daniel; Maisano Delser, Pierpaolo; Wetton, Jon H.; Arroyo-Pardo, Eduardo; Cavalleri, Gianpiero L.; de Knijff, Peter; Destro Bisol, Giovanni; Dupuy, Berit Myhre; Eriksen, Heidi A.; Jorde, Lynn B.; King, Turi E.; Larmuseau, Maarten H.; López de Munain, Adolfo; López-Parra, Ana M.; Loutradis, Aphrodite; Milasin, Jelena; Novelletto, Andrea; Pamjav, Horolma; Sajantila, Antti; Schempp, Werner; Sears, Matt; Tolun, Aslıhan; Tyler-Smith, Chris; Van Geystelen, Anneleen; Watkins, Scott; Winney, Bruce; Jobling, Mark A.

2015-01-01

Many studies of human populations have used the male-specific region of the Y chromosome (MSY) as a marker, but MSY sequence variants have traditionally been subject to ascertainment bias. Also, dating of haplogroups has relied on Y-specific short tandem repeats (STRs), involving problems of mutation rate choice, and possible long-term mutation saturation. Next-generation sequencing can ascertain single nucleotide polymorphisms (SNPs) in an unbiased way, leading to phylogenies in which branch-lengths are proportional to time, and allowing the times-to-most-recent-common-ancestor (TMRCAs) of nodes to be estimated directly. Here we describe the sequencing of 3.7 Mb of MSY in each of 448 human males at a mean coverage of 51×, yielding 13,261 high-confidence SNPs, 65.9% of which are previously unreported. The resulting phylogeny covers the majority of the known clades, provides date estimates of nodes, and constitutes a robust evolutionary framework for analyzing the history of other classes of mutation. Different clades within the tree show subtle but significant differences in branch lengths to the root. We also apply a set of 23 Y-STRs to the same samples, allowing SNP- and STR-based diversity and TMRCA estimates to be systematically compared. Ongoing purifying selection is suggested by our analysis of the phylogenetic distribution of nonsynonymous variants in 15 MSY single-copy genes. PMID:25468874

Multiple hybrid de novo genome assembly of finger millet, an orphan allotetraploid crop.

PubMed

Hatakeyama, Masaomi; Aluri, Sirisha; Balachadran, Mathi Thumilan; Sivarajan, Sajeevan Radha; Patrignani, Andrea; Grüter, Simon; Poveda, Lucy; Shimizu-Inatsugi, Rie; Baeten, John; Francoijs, Kees-Jan; Nataraja, Karaba N; Reddy, Yellodu A Nanja; Phadnis, Shamprasad; Ravikumar, Ramapura L; Schlapbach, Ralph; Sreeman, Sheshshayee M; Shimizu, Kentaro K

2017-09-05

Finger millet (Eleusine coracana (L.) Gaertn) is an important crop for food security because of its tolerance to drought, which is expected to be exacerbated by global climate changes. Nevertheless, it is often classified as an orphan/underutilized crop because of the paucity of scientific attention. Among several small millets, finger millet is considered as an excellent source of essential nutrient elements, such as iron and zinc; hence, it has potential as an alternate coarse cereal. However, high-quality genome sequence data of finger millet are currently not available. One of the major problems encountered in the genome assembly of this species was its polyploidy, which hampers genome assembly compared with a diploid genome. To overcome this problem, we sequenced its genome using diverse technologies with sufficient coverage and assembled it via a novel multiple hybrid assembly workflow that combines next-generation with single-molecule sequencing, followed by whole-genome optical mapping using the Bionano Irys® system. The total number of scaffolds was 1,897 with an N50 length >2.6 Mb and detection of 96% of the universal single-copy orthologs. The majority of the homeologs were assembled separately. This indicates that the proposed workflow is applicable to the assembly of other allotetraploid genomes. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Evolution of plant virus movement proteins from the 30K superfamily and of their homologs integrated in plant genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mushegian, Arcady R., E-mail: mushegian2@gmail.com; Elena, Santiago F., E-mail: sfelena@ibmcp.upv.es; The Santa Fe Institute, Santa Fe, NM 87501

Homologs of Tobacco mosaic virus 30K cell-to-cell movement protein are encoded by diverse plant viruses. Mechanisms of action and evolutionary origins of these proteins remain obscure. We expand the picture of conservation and evolution of the 30K proteins, producing sequence alignment of the 30K superfamily with the broadest phylogenetic coverage thus far and illuminating structural features of the core all-beta fold of these proteins. Integrated copies of pararetrovirus 30K movement genes are prevalent in euphyllophytes, with at least one copy intact in nearly every examined species, and mRNAs detected for most of them. Sequence analysis suggests repeated integrations, pseudogenizations, andmore » positive selection in those provirus genes. An unannotated 30K-superfamily gene in Arabidopsis thaliana genome is likely expressed as a fusion with the At1g37113 transcript. This molecular background of endopararetrovirus gene products in plants may change our view of virus infection and pathogenesis, and perhaps of cellular homeostasis in the hosts. - Highlights: • Sequence region shared by plant virus “30K” movement proteins has an all-beta fold. • Most euphyllophyte genomes contain integrated copies of pararetroviruses. • These integrated virus genomes often include intact movement protein genes. • Molecular evidence suggests that these “30K” genes may be selected for function.« less

Diversity Issues and Business Education.

ERIC Educational Resources Information Center

Foxman, Ellen; And Others

1995-01-01

Thirty-two marketing textbooks were examined for content related to workforce diversity. Age was most frequently discussed at young/old extremes, although 53% of the population are in middle age groups. Racial/ethnic coverage was 60% about Hispanics, although African Americans have a larger population and Asians are the fastest growing group.…

Food nanotechnology in the news. Coverage patterns and thematic emphases during the last decade.

PubMed

Dudo, Anthony; Choi, Doo-Hun; Scheufele, Dietram A

2011-02-01

For novel issues like food nanotechnology, media can play an important role in shaping the awareness and mental associations that underlie public opinion. Seeking to complement recent research exploring public opinion formation about food nanotechnology, this study tracks the evolution of U.S. newspaper coverage of food nanotechnology, identifying the descriptive and thematic traits that have characterized this coverage over time. We use a rigorous methodology to examine the levels of coverage, authorship patterns, and thematic emphases exhibited in the American journalistic narrative about this burgeoning application of nanoscience. Our findings indicate that U.S. newspaper coverage of food nanotechnology is relatively modest in terms of how often it has been covered, its thematic diversity, and the level of journalistic expertise from which it was produced. To our knowledge, this is the first study to empirically assess journalistic coverage of food nanotechnology. Copyright © 2010 Elsevier Ltd. All rights reserved.

Microbial community analysis of the hypersaline water of the Dead Sea using high-throughput amplicon sequencing.

PubMed

Jacob, Jacob H; Hussein, Emad I; Shakhatreh, Muhamad Ali K; Cornelison, Christopher T

2017-10-01

Amplicon sequencing using next-generation technology (bTEFAP ® ) has been utilized in describing the diversity of Dead Sea microbiota. The investigated area is a well-known salt lake in the western part of Jordan found in the lowest geographical location in the world (more than 420 m below sea level) and characterized by extreme salinity (approximately, 34%) in addition to other extreme conditions (low pH, unique ionic composition different from sea water). DNA was extracted from Dead Sea water. A total of 314,310 small subunit RNA (SSU rRNA) sequences were parsed, and 288,452 sequences were then clustered. For alpha diversity analysis, sample was rarefied to 3,000 sequences. The Shannon-Wiener index curve plot reached a plateau at approximately 3,000 sequences indicating that sequencing depth was sufficient to capture the full scope of microbial diversity. Archaea was found to be dominating the sequences (52%), whereas Bacteria constitute 45% of the sequences. Altogether, prokaryotic sequences (which constitute 97% of all sequences) were found to predominate. The findings expand on previous studies by using high-throughput amplicon sequencing to describe the microbial community in an environment which in recent years has been shown to hide some interesting diversity. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Analysis of bacterial xylose isomerase gene diversity using gene-targeted metagenomics.

PubMed

Nurdiani, Dini; Ito, Michihiro; Maruyama, Toru; Terahara, Takeshi; Mori, Tetsushi; Ugawa, Shin; Takeyama, Haruko

2015-08-01

Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating <90% identity with known XIs in the database accounted for 89% of the total xylA phylotypes. The differences among xylA members and compositions within each soil sample were significantly smaller than they were between different soils based on a UniFrac distance analysis, suggesting soil-specific xylA genotypes and taxonomic compositions. The differences among xylA members and their compositions in the soil were strongly correlated with 16S rRNA variation between soil samples, also assessed by amplicon pyrosequencing. This is the first report of xylA diversity in environmental samples assessed by amplicon pyrosequencing. Our data provide information regarding xylA diversity in nature, and can be a basis for the screening of novel xylA genotypes for practical applications. Copyright © 2015. Published by Elsevier B.V.

The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing a new model for studying the evolution of cuticle function

PubMed Central

Yeats, Trevor H.; Buda, Gregory J.; Wang, Zhonghua; Chehanovsky, Noam; Moyle, Leonie C.; Jetter, Reinhard; Schaffer, Arthur A.; Rose, Jocelyn K.C.

2013-01-01

Summary The cuticle covers the aerial epidermis of land plants and plays a primary role in water regulation and protection from external stresses. Remarkable species diversity in the structure and composition of its components, cutin and wax, have been catalogued, but few functional or genetic correlations have emerged. Tomato (Solanum lycopersicum) is part of a complex of closely related wild species endemic to the northern Andes and the Galapagos Islands (Solanum Sect. Lycopersicon). Although sharing an ancestor less than seven million years ago, these species are found in diverse environments and are subject to unique selective pressures. Furthermore, they are genetically tractable, since they can be crossed with S. lycopersicum, which has a sequenced genome. With the aim of evaluating the relationships between evolution, structure and function of the cuticle, we characterized the morphological and chemical diversity of fruit cuticles of seven species from Solanum Sect. Lycopersicon. Striking differences in cuticular architecture and quantities of cutin and waxes were observed, with wild species wax coverage exceeding that of S. lycopersicum by up to seven fold. Wax composition varied in the occurrence of wax esters and triterpenoid isomers. Using a S. habrochaites introgression line population, we mapped triterpenoid differences to a genomic region that includes two S. lycopersicum triterpene synthases. Based on known metabolic pathways for acyl wax compounds, hypotheses are discussed to explain the appearance of wax esters with atypical chain lengths. These results establish a model system for understanding the ecological and evolutionary functional genomics of plant cuticles. PMID:22007785

Low diversity in the mitogenome of sperm whales revealed by next-generation sequencing

Treesearch

Alana Alexander; Debbie Steel; Beth Slikas; Kendra Hoekzema; Colm Carraher; Matthew Parks; Richard Cronn; C. Scott Baker

2012-01-01

Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20...

Comparison of the Diversity of Basidiomycetes from Dead Wood of the Manchurian fir (Abies holophylla) as Evaluated by Fruiting Body Collection, Mycelial Isolation, and 454 Sequencing.

PubMed

Jang, Yeongseon; Jang, Seokyoon; Min, Mihee; Hong, Joo-Hyun; Lee, Hanbyul; Lee, Hwanhwi; Lim, Young Woon; Kim, Jae-Jin

2015-10-01

In this study, three different methods (fruiting body collection, mycelial isolation, and 454 sequencing) were implemented to determine the diversity of wood-inhabiting basidiomycetes from dead Manchurian fir (Abies holophylla). The three methods recovered similar species richness (26 species from fruiting bodies, 32 species from mycelia, and 32 species from 454 sequencing), but Fisher's alpha, Shannon-Wiener, Simpson's diversity indices of fungal communities indicated fruiting body collection and mycelial isolation displayed higher diversity compared with 454 sequencing. In total, 75 wood-inhabiting basidiomycetes were detected. The most frequently observed species were Heterobasidion orientale (fruiting body collection), Bjerkandera adusta (mycelial isolation), and Trichaptum fusco-violaceum (454 sequencing). Only two species, Hymenochaete yasudae and Hypochnicium karstenii, were detected by all three methods. This result indicated that Manchurian fir harbors a diverse basidiomycetous fungal community and for complete estimation of fungal diversity, multiple methods should be used. Further studies are required to understand their ecology in the context of forest ecosystems.

Massively parallel pyrosequencing of the mitochondrial genome with the 454 methodology in forensic genetics.

PubMed

Mikkelsen, Martin; Frank-Hansen, Rune; Hansen, Anders J; Morling, Niels

2014-09-01

of sequencing of whole mitochondrial genome, HV1 and HV2 DNA with the second generation system (SGS) Roche 454 GS Junior were compared with results of Sanger sequencing and SNP typing with SNaPshot single base extension detected with MALDI-TOF and capillary electrophoresis. We investigated the performance of the software analysis of the data, reproducibility, ability to sequence homopolymeric regions, detection of mixtures and heteroplasmy as well as the implications of the depth of coverage. We found full reproducibility between samples sequenced twice with SGS. We found close to full concordance between the mtDNA sequences of 26 samples obtained with (1) the 454 SGS method using a depth of coverage above 100 and (2) Sanger sequencing and SNP typing. The discrepancies were primarily observed in homopolymeric regions. The 454 SGS method was able to sequence 95% of the reads correctly in homopolymers up to 4 bases, and up to 6 bases could be sequenced with similar success if the results were carefully, visually inspected. The 454 technology was able to detect mixtures or heteroplasmy of approximately 10%. We detected previously unreported heteroplasmy in the GM9947A component of the NIST human mitochondrial DNA SRM-2392 standard reference material. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Testing genotyping strategies for ultra-deep sequencing of a co-amplifying gene family: MHC class I in a passerine bird.

PubMed

Biedrzycka, Aleksandra; Sebastian, Alvaro; Migalska, Magdalena; Westerdahl, Helena; Radwan, Jacek

2017-07-01

Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage. © 2016 John Wiley & Sons Ltd.

AD-LIBS: inferring ancestry across hybrid genomes using low-coverage sequence data.

PubMed

Schaefer, Nathan K; Shapiro, Beth; Green, Richard E

2017-04-04

Inferring the ancestry of each region of admixed individuals' genomes is useful in studies ranging from disease gene mapping to speciation genetics. Current methods require high-coverage genotype data and phased reference panels, and are therefore inappropriate for many data sets. We present a software application, AD-LIBS, that uses a hidden Markov model to infer ancestry across hybrid genomes without requiring variant calling or phasing. This approach is useful for non-model organisms and in cases of low-coverage data, such as ancient DNA. We demonstrate the utility of AD-LIBS with synthetic data. We then use AD-LIBS to infer ancestry in two published data sets: European human genomes with Neanderthal ancestry and brown bear genomes with polar bear ancestry. AD-LIBS correctly infers 87-91% of ancestry in simulations and produces ancestry maps that agree with published results and global ancestry estimates in humans. In brown bears, we find more polar bear ancestry than has been published previously, using both AD-LIBS and an existing software application for local ancestry inference, HAPMIX. We validate AD-LIBS polar bear ancestry maps by recovering a geographic signal within bears that mirrors what is seen in SNP data. Finally, we demonstrate that AD-LIBS is more effective than HAPMIX at inferring ancestry when preexisting phased reference data are unavailable and genomes are sequenced to low coverage. AD-LIBS is an effective tool for ancestry inference that can be used even when few individuals are available for comparison or when genomes are sequenced to low coverage. AD-LIBS is therefore likely to be useful in studies of non-model or ancient organisms that lack large amounts of genomic DNA. AD-LIBS can therefore expand the range of studies in which admixture mapping is a viable tool.

Exploring fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing

NASA Astrophysics Data System (ADS)

Zhang, Xiao-Yong; Wang, Guang-Hua; Xu, Xin-Ya; Nong, Xu-Hua; Wang, Jie; Amin, Muhammad; Qi, Shu-Hua

2016-10-01

The present study investigated the fungal diversity in four different deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS1). A total of 40,297 fungal ITS1 sequences clustered into 420 operational taxonomic units (OTUs) with 97% sequence similarity and 170 taxa were recovered from these sediments. Most ITS1 sequences (78%) belonged to the phylum Ascomycota, followed by Basidiomycota (17.3%), Zygomycota (1.5%) and Chytridiomycota (0.8%), and a small proportion (2.4%) belonged to unassigned fungal phyla. Compared with previous studies on fungal diversity of sediments from deep-sea environments by culture-dependent approach and clone library analysis, the present result suggested that Illumina sequencing had been dramatically accelerating the discovery of fungal community of deep-sea sediments. Furthermore, our results revealed that Sordariomycetes was the most diverse and abundant fungal class in this study, challenging the traditional view that the diversity of Sordariomycetes phylotypes was low in the deep-sea environments. In addition, more than 12 taxa accounted for 21.5% sequences were found to be rarely reported as deep-sea fungi, suggesting the deep-sea sediments from Okinawa Trough harbored a plethora of different fungal communities compared with other deep-sea environments. To our knowledge, this study is the first exploration of the fungal diversity in deep-sea sediments from Okinawa Trough using high-throughput Illumina sequencing.

Broad and Cross-Clade CD4+ T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

PubMed Central

Almeida, Rafael Ribeiro; Rosa, Daniela Santoro; Ribeiro, Susan Pereira; Santana, Vinicius Canato; Kallás, Esper Georges; Sidney, John; Sette, Alessandro; Kalil, Jorge; Cunha-Neto, Edecio

2012-01-01

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4+ T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4+ T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4+ T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IAb and IAd. Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4+ and CD8+ T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4+ and CD8+ T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS. PMID:23028895

High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers.

PubMed

Hou, Weiguo; Wang, Shang; Briggs, Brandon R; Li, Gaoyuan; Xie, Wei; Dong, Hailiang

2018-01-01

Myocyanophages, a group of viruses infecting cyanobacteria, are abundant and play important roles in elemental cycling. Here we investigated the particle-associated viral communities retained on 0.2 μm filters and in sediment samples (representing ancient cyanophage communities) from four ocean and three lake locations, using high-throughput sequencing and a newly designed primer pair targeting a gene fragment (∼145-bp in length) encoding the cyanophage gp23 major capsid protein (MCP). Diverse viral communities were detected in all samples. The fragments of 142-, 145-, and 148-bp in length were most abundant in the amplicons, and most sequences (>92%) belonged to cyanophages. Additionally, different sequencing depths resulted in different diversity estimates of the viral community. Operational taxonomic units obtained from deep sequencing of the MCP gene covered the majority of those obtained from shallow sequencing, suggesting that deep sequencing exhibited a more complete picture of cyanophage community than shallow sequencing. Our results also revealed a wide geographic distribution of marine myocyanophages, i.e., higher dissimilarities of the myocyanophage communities corresponded with the larger distances between the sampling sites. Collectively, this study suggests that the newly designed primer pair can be effectively used to study the community and diversity of myocyanophage from different environments, and the high-throughput sequencing represents a good method to understand viral diversity.

High Diversity of Myocyanophage in Various Aquatic Environments Revealed by High-Throughput Sequencing of Major Capsid Protein Gene With a New Set of Primers

PubMed Central

Hou, Weiguo; Wang, Shang; Briggs, Brandon R.; Li, Gaoyuan; Xie, Wei; Dong, Hailiang

2018-01-01

Myocyanophages, a group of viruses infecting cyanobacteria, are abundant and play important roles in elemental cycling. Here we investigated the particle-associated viral communities retained on 0.2 μm filters and in sediment samples (representing ancient cyanophage communities) from four ocean and three lake locations, using high-throughput sequencing and a newly designed primer pair targeting a gene fragment (∼145-bp in length) encoding the cyanophage gp23 major capsid protein (MCP). Diverse viral communities were detected in all samples. The fragments of 142-, 145-, and 148-bp in length were most abundant in the amplicons, and most sequences (>92%) belonged to cyanophages. Additionally, different sequencing depths resulted in different diversity estimates of the viral community. Operational taxonomic units obtained from deep sequencing of the MCP gene covered the majority of those obtained from shallow sequencing, suggesting that deep sequencing exhibited a more complete picture of cyanophage community than shallow sequencing. Our results also revealed a wide geographic distribution of marine myocyanophages, i.e., higher dissimilarities of the myocyanophage communities corresponded with the larger distances between the sampling sites. Collectively, this study suggests that the newly designed primer pair can be effectively used to study the community and diversity of myocyanophage from different environments, and the high-throughput sequencing represents a good method to understand viral diversity.

Sequence Capture versus Restriction Site Associated DNA Sequencing for Shallow Systematics.

PubMed

Harvey, Michael G; Smith, Brian Tilston; Glenn, Travis C; Faircloth, Brant C; Brumfield, Robb T

2016-09-01

Sequence capture and restriction site associated DNA sequencing (RAD-Seq) are two genomic enrichment strategies for applying next-generation sequencing technologies to systematics studies. At shallow timescales, such as within species, RAD-Seq has been widely adopted among researchers, although there has been little discussion of the potential limitations and benefits of RAD-Seq and sequence capture. We discuss a series of issues that may impact the utility of sequence capture and RAD-Seq data for shallow systematics in non-model species. We review prior studies that used both methods, and investigate differences between the methods by re-analyzing existing RAD-Seq and sequence capture data sets from a Neotropical bird (Xenops minutus). We suggest that the strengths of RAD-Seq data sets for shallow systematics are the wide dispersion of markers across the genome, the relative ease and cost of laboratory work, the deep coverage and read overlap at recovered loci, and the high overall information that results. Sequence capture's benefits include flexibility and repeatability in the genomic regions targeted, success using low-quality samples, more straightforward read orthology assessment, and higher per-locus information content. The utility of a method in systematics, however, rests not only on its performance within a study, but on the comparability of data sets and inferences with those of prior work. In RAD-Seq data sets, comparability is compromised by low overlap of orthologous markers across species and the sensitivity of genetic diversity in a data set to an interaction between the level of natural heterozygosity in the samples examined and the parameters used for orthology assessment. In contrast, sequence capture of conserved genomic regions permits interrogation of the same loci across divergent species, which is preferable for maintaining comparability among data sets and studies for the purpose of drawing general conclusions about the impact of historical processes across biotas. We argue that sequence capture should be given greater attention as a method of obtaining data for studies in shallow systematics and comparative phylogeography. © The Author(s) 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Outbreak investigation for toxigenic Corynebacterium diphtheriae wound infections in refugees from Northeast Africa and Syria in Switzerland and Germany by whole genome sequencing.

PubMed

Meinel, D M; Kuehl, R; Zbinden, R; Boskova, V; Garzoni, C; Fadini, D; Dolina, M; Blümel, B; Weibel, T; Tschudin-Sutter, S; Widmer, A F; Bielicki, J A; Dierig, A; Heininger, U; Konrad, R; Berger, A; Hinic, V; Goldenberger, D; Blaich, A; Stadler, T; Battegay, M; Sing, A; Egli, A

2016-12-01

Toxigenic Corynebacterium diphtheriae is an important and potentially fatal threat to patients and public health. During the current dramatic influx of refugees into Europe, our objective was to use whole genome sequencing for the characterization of a suspected outbreak of C. diphtheriae wound infections among refugees. After conventional culture, we identified C. diphtheriae using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and investigated toxigenicity by PCR. Whole genome sequencing was performed on a MiSeq Illumina with >70×coverage, 2×250 bp read length, and mapping against a reference genome. Twenty cases of cutaneous C. diphtheriae in refugees from East African countries and Syria identified between April and August 2015 were included. Patients presented with wound infections shortly after arrival in Switzerland and Germany. Toxin production was detected in 9/20 (45%) isolates. Whole genome sequencing-based typing revealed relatedness between isolates using neighbour-joining algorithms. We detected three separate clusters among epidemiologically related refugees. Although the isolates within a cluster showed strong relatedness, isolates differed by >50 nucleotide polymorphisms. Toxigenic C. diphtheriae associated wound infections are currently observed more frequently in Europe, due to refugees travelling under poor hygienic conditions. Close genetic relatedness of C. diphtheriae isolates from 20 refugees with wound infections indicates likely transmission between patients. However, the diversity within each cluster and phylogenetic time-tree analysis suggest that transmissions happened several months ago, most likely outside Europe. Whole genome sequencing offers the potential to describe outbreaks at very high resolution and is a helpful tool in infection tracking and identification of transmission routes. Copyright © 2016. Published by Elsevier Ltd.

Community Structure and Function of High-Temperature Chlorophototrophic Microbial Mats Inhabiting Diverse Geothermal Environments

PubMed Central

Klatt, Christian G.; Inskeep, William P.; Herrgard, Markus J.; Jay, Zackary J.; Rusch, Douglas B.;  Tringe, Susannah G.; Niki Parenteau, M.; Ward, David M.; Boomer, Sarah M.; Bryant, Donald A.;  Miller, Scott R.

2013-01-01

Six phototrophic microbial mat communities from different geothermal springs (YNP) were studied using metagenome sequencing and geochemical analyses. The primary goals of this work were to determine differences in community composition of high-temperature phototrophic mats distributed across the Yellowstone geothermal ecosystem, and to identify metabolic attributes of predominant organisms present in these communities that may correlate with environmental attributes important in niche differentiation. Random shotgun metagenome sequences from six phototrophic communities (average ∼53 Mbp/site) were subjected to multiple taxonomic, phylogenetic, and functional analyses. All methods, including G + C content distribution, MEGAN analyses, and oligonucleotide frequency-based clustering, provided strong support for the dominant community members present in each site. Cyanobacteria were only observed in non-sulfidic sites; de novo assemblies were obtained for Synechococcus-like populations at Chocolate Pots (CP_7) and Fischerella-like populations at White Creek (WC_6). Chloroflexi-like sequences (esp. Roseiflexus and/or Chloroflexus spp.) were observed in all six samples and contained genes involved in bacteriochlorophyll biosynthesis and the 3-hydroxypropionate carbon fixation pathway. Other major sequence assemblies were obtained for a Chlorobiales population from CP_7 (proposed family Thermochlorobacteriaceae), and an anoxygenic, sulfur-oxidizing Thermochromatium-like (Gamma-proteobacteria) population from Bath Lake Vista Annex (BLVA_20). Additional sequence coverage is necessary to establish more complete assemblies of other novel bacteria in these sites (e.g., Bacteroidetes and Firmicutes); however, current assemblies suggested that several of these organisms play important roles in heterotrophic and fermentative metabolisms. Definitive linkages were established between several of the dominant phylotypes present in these habitats and important functional processes such as photosynthesis, carbon fixation, sulfur oxidation, and fermentation. PMID:23761787

Assessing DNA Barcodes for Species Identification in North American Reptiles and Amphibians in Natural History Collections.

PubMed

Chambers, E Anne; Hebert, Paul D N

2016-01-01

High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale.

Assessing DNA Barcodes for Species Identification in North American Reptiles and Amphibians in Natural History Collections

PubMed Central

Chambers, E. Anne; Hebert, Paul D. N.

2016-01-01

Background High rates of species discovery and loss have led to the urgent need for more rapid assessment of species diversity in the herpetofauna. DNA barcoding allows for the preliminary identification of species based on sequence divergence. Prior DNA barcoding work on reptiles and amphibians has revealed higher biodiversity counts than previously estimated due to cases of cryptic and undiscovered species. Past studies have provided DNA barcodes for just 14% of the North American herpetofauna, revealing the need for expanded coverage. Methodology/Principal Findings This study extends the DNA barcode reference library for North American herpetofauna, assesses the utility of this approach in aiding species delimitation, and examines the correspondence between current species boundaries and sequence clusters designated by the BIN system. Sequences were obtained from 730 specimens, representing 274 species (43%) from the North American herpetofauna. Mean intraspecific divergences were 1% and 3%, while average congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded with current species boundaries in 79% of amphibians, 100% of turtles, and 60% of squamates. Deep divergences (>2%) were noted in 35% of squamate and 16% of amphibian species, and low divergences (<2%) occurred in 12% of reptiles and 23% of amphibians, patterns reflected in BIN assignments. Sequence recovery declined with specimen age, and variation in recovery success was noted among collections. Within collections, barcodes effectively flagged seven mislabeled tissues, and barcode fragments were recovered from five formalin-fixed specimens. Conclusions/Significance This study demonstrates that DNA barcodes can effectively flag errors in museum collections, while BIN splits and merges reveal taxa belonging to deeply diverged or hybridizing lineages. This study is the first effort to compile a reference library of DNA barcodes for herpetofauna on a continental scale. PMID:27116180

Targeted gene panel sequencing in children with very early onset inflammatory bowel disease--evaluation and prospective analysis.

PubMed

Kammermeier, Jochen; Drury, Suzanne; James, Chela T; Dziubak, Robert; Ocaka, Louise; Elawad, Mamoun; Beales, Philip; Lench, Nicholas; Uhlig, Holm H; Bacchelli, Chiara; Shah, Neil

2014-11-01

Multiple monogenetic conditions with partially overlapping phenotypes can present with inflammatory bowel disease (IBD)-like intestinal inflammation. With novel genotype-specific therapies emerging, establishing a molecular diagnosis is becoming increasingly important. We have introduced targeted next-generation sequencing (NGS) technology as a prospective screening tool in children with very early onset IBD (VEOIBD). We evaluated the coverage of 40 VEOIBD genes in two separate cohorts undergoing targeted gene panel sequencing (TGPS) (n=25) and whole exome sequencing (WES) (n=20). TGPS revealed causative mutations in four genes (IL10RA, EPCAM, TTC37 and SKIV2L) discovered unexpected phenotypes and directly influenced clinical decision making by supporting as well as avoiding haematopoietic stem cell transplantation. TGPS resulted in significantly higher median coverage when compared with WES, fewer coverage deficiencies and improved variant detection across established VEOIBD genes. Excluding or confirming known VEOIBD genotypes should be considered early in the disease course in all cases of therapy-refractory VEOIBD, as it can have a direct impact on patient management. To combine both described NGS technologies would compensate for the limitations of WES for disease-specific application while offering the opportunity for novel gene discovery in the research setting. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Nonphotosynthetic Pigments as Potential Biosignatures

PubMed Central

Cockell, Charles S.; Meadows, Victoria S.

2015-01-01

Abstract Previous work on possible surface reflectance biosignatures for Earth-like planets has typically focused on analogues to spectral features produced by photosynthetic organisms on Earth, such as the vegetation red edge. Although oxygenic photosynthesis, facilitated by pigments evolved to capture photons, is the dominant metabolism on our planet, pigmentation has evolved for multiple purposes to adapt organisms to their environment. We present an interdisciplinary study of the diversity and detectability of nonphotosynthetic pigments as biosignatures, which includes a description of environments that host nonphotosynthetic biologically pigmented surfaces, and a lab-based experimental analysis of the spectral and broadband color diversity of pigmented organisms on Earth. We test the utility of broadband color to distinguish between Earth-like planets with significant coverage of nonphotosynthetic pigments and those with photosynthetic or nonbiological surfaces, using both 1-D and 3-D spectral models. We demonstrate that, given sufficient surface coverage, nonphotosynthetic pigments could significantly impact the disk-averaged spectrum of a planet. However, we find that due to the possible diversity of organisms and environments, and the confounding effects of the atmosphere and clouds, determination of substantial coverage by biologically produced pigments would be difficult with broadband colors alone and would likely require spectrally resolved data. Key Words: Biosignatures—Exoplanets—Halophiles—Pigmentation—Reflectance spectroscopy—Spectral models. Astrobiology 15, 341–361. PMID:25941875

Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Richard Allen; Bottos, Eric M.; Roy Chowdhury, Taniya

ABSTRACT Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600more » reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “CandidatusPseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundanceAcidobacteriawere highly transcriptionally active, whereas bins corresponding to high-relative-abundanceVerrucomicrobiawere not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities. IMPORTANCESoil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Author Video: Anauthor video summaryof this article is available.« less

Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

PubMed Central

White, Richard Allen; Bottos, Eric M.; Roy Chowdhury, Taniya; Zucker, Jeremy D.; Brislawn, Colin J.; Nicora, Carrie D.; Fansler, Sarah J.; Glaesemann, Kurt R.; Glass, Kevin

2016-01-01

ABSTRACT Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600 reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “Candidatus Pseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundance Acidobacteria were highly transcriptionally active, whereas bins corresponding to high-relative-abundance Verrucomicrobia were not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities. IMPORTANCE Soil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Author Video: An author video summary of this article is available. PMID:27822530

TU-H-CAMPUS-JeP3-05: Adaptive Determination of Needle Sequence HDR Prostate Brachytherapy with Divergent Needle-By-Needle Delivery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borot de Battisti, M; Maenhout, M; Lagendijk, J J W

Purpose: To develop a new method which adaptively determines the optimal needle insertion sequence for HDR prostate brachytherapy involving divergent needle-by-needle dose delivery by e.g. a robotic device. A needle insertion sequence is calculated at the beginning of the intervention and updated after each needle insertion with feedback on needle positioning errors. Methods: Needle positioning errors and anatomy changes may occur during HDR brachytherapy which can lead to errors in the delivered dose. A novel strategy was developed to calculate and update the needle sequence and the dose plan after each needle insertion with feedback on needle positioning errors. Themore » dose plan optimization was performed by numerical simulations. The proposed needle sequence determination optimizes the final dose distribution based on the dose coverage impact of each needle. This impact is predicted stochastically by needle insertion simulations. HDR procedures were simulated with varying number of needle insertions (4 to 12) using 11 patient MR data-sets with PTV, prostate, urethra, bladder and rectum delineated. Needle positioning errors were modeled by random normally distributed angulation errors (standard deviation of 3 mm at the needle’s tip). The final dose parameters were compared in the situations where the needle with the largest vs. the smallest dose coverage impact was selected at each insertion. Results: Over all scenarios, the percentage of clinically acceptable final dose distribution improved when the needle selected had the largest dose coverage impact (91%) compared to the smallest (88%). The differences were larger for few (4 to 6) needle insertions (maximum difference scenario: 79% vs. 60%). The computation time of the needle sequence optimization was below 60s. Conclusion: A new adaptive needle sequence determination for HDR prostate brachytherapy was developed. Coupled to adaptive planning, the selection of the needle with the largest dose coverage impact increases chances of reaching the clinical constraints. M. Borot de Battisti is funded by Philips Medical Systems Nederland B.V.; M. Moerland is principal investigator on a contract funded by Philips Medical Systems Nederland B.V.; G. Hautvast and D. Binnekamp are fulltime employees of Philips Medical Systems Nederland B.V.« less

Comparison of the genomic sequence of the microminipig, a novel breed of swine, with the genomic database for conventional pig.

PubMed

Miura, Naoki; Kucho, Ken-Ichi; Noguchi, Michiko; Miyoshi, Noriaki; Uchiumi, Toshiki; Kawaguchi, Hiroaki; Tanimoto, Akihide

2014-01-01

The microminipig, which weighs less than 10 kg at an early stage of maturity, has been reported as a potential experimental model animal. Its extremely small size and other distinct characteristics suggest the possibility of a number of differences between the genome of the microminipig and that of conventional pigs. In this study, we analyzed the genomes of two healthy microminipigs using a next-generation sequencer SOLiD™ system. We then compared the obtained genomic sequences with a genomic database for the domestic pig (Sus scrofa). The mapping coverage of sequenced tag from the microminipig to conventional pig genomic sequences was greater than 96% and we detected no clear, substantial genomic variance from these data. The results may indicate that the distinct characteristics of the microminipig derive from small-scale alterations in the genome, such as Single Nucleotide Polymorphisms or translational modifications, rather than large-scale deletion or insertion polymorphisms. Further investigation of the entire genomic sequence of the microminipig with methods enabling deeper coverage is required to elucidate the genetic basis of its distinct phenotypic traits. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Approaches to measuring cultural diversity in recreation

Treesearch

Chieh-Lu Li; James D. Absher; Yi-Chung Hsu; Alan R. Graefe

2008-01-01

Measuring cultural diversity in recreation has become an important topic because of the increasing coverage of and interest in ethnicity and cross-cultural aspects of recreation. Introducing theories and methods from established disciplines other than leisure studies/recreation and park studies is necessary to understand this important issue. In this article, we first...

Comparison and quantitative verification of mapping algorithms for whole genome bisulfite sequencing

USDA-ARS?s Scientific Manuscript database

Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitat...

Fast imputation using medium or low-coverage sequence data

USDA-ARS?s Scientific Manuscript database

Accurate genotype imputation can greatly reduce costs and increase benefits by combining whole-genome sequence data of varying read depth and microarray genotypes of varying densities. For large populations, an efficient strategy chooses the two haplotypes most likely to form each genotype and updat...

Role of Mitochondrial Inheritance on Prostate Cancer Outcome in African American Men. Addendum

DTIC Science & Technology

2016-11-01

DNA sequencing technique developed by our collaborator using single amplicon long-range PCR that permits deep coverage (10,000-20,000X on average) of...the mitochondrial genome. We have sequenced 652 samples derived from frozen fully using this technology. The additional DNA samples derived from...paraffin embedded (FFPE) tissue were more challenging, but have now been sequenced . Mapping of DNA variants in our sequenced genomes to mitochondrial

Diverse Bacteria Affiliated with the Genera Microvirga, Phyllobacterium, and Bradyrhizobium Nodulate Lupinus micranthus Growing in Soils of Northern Tunisia

PubMed Central

Msaddak, Abdelhakim; Durán, David; Rejili, Mokhtar; Mars, Mohamed; Ruiz-Argüeso, Tomás; Imperial, Juan; Palacios, José

2017-01-01

ABSTRACT The genetic diversity of bacterial populations nodulating Lupinus micranthus in five geographical sites from northern Tunisia was examined. Phylogenetic analyses of 50 isolates based on partial sequences of recA and gyrB grouped strains into seven clusters, five of which belong to the genus Bradyrhizobium (28 isolates), one to Phyllobacterium (2 isolates), and one, remarkably, to Microvirga (20 isolates). The largest Bradyrhizobium cluster (17 isolates) grouped with the B. lupini species, and the other five clusters were close to different recently defined Bradyrhizobium species. Isolates close to Microvirga were obtained from nodules of plants from four of the five sites sampled. We carried out an in-depth phylogenetic study with representatives of the seven clusters using sequences from housekeeping genes (rrs, recA, glnII, gyrB, and dnaK) and obtained consistent results. A phylogeny based on the sequence of the symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behaviors of the representative strains were tested, and some congruence between symbiovars and symbiotic performance was observed. These data indicate a remarkable diversity of L. micranthus root nodule symbionts in northern Tunisia, including strains from the Bradyrhizobiaceae, Methylobacteriaceae, and Phyllobacteriaceae families, in contrast with those of the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, which are nodulated mostly by Bradyrhizobium strains. IMPORTANCE Lupinus micranthus is a legume broadly distributed in the Mediterranean region and plays an important role in soil fertility and vegetation coverage by fixing nitrogen and solubilizing phosphate in semiarid areas. Direct sowing to extend the distribution of this indigenous legume can contribute to the prevention of soil erosion in pre-Saharan lands of Tunisia. However, rhizobial populations associated with L. micranthus are poorly understood. In this context, the diversity of endosymbionts of this legume was investigated. Most Lupinus species are nodulated by Bradyrhizobium strains. This work showed that about half of the isolates from northern Tunisian soils were in fact Bradyrhizobium symbionts, but the other half were found unexpectedly to be bacteria within the genera Microvirga and Phyllobacterium. These unusual endosymbionts may have a great ecological relevance. Inoculation with the appropriate selected symbiotic bacterial partners will increase L. micranthus survival with consequent advantages for the environment in semiarid areas of Tunisia. PMID:28062461

Diverse Bacteria Affiliated with the Genera Microvirga, Phyllobacterium, and Bradyrhizobium Nodulate Lupinus micranthus Growing in Soils of Northern Tunisia.

PubMed

Msaddak, Abdelhakim; Durán, David; Rejili, Mokhtar; Mars, Mohamed; Ruiz-Argüeso, Tomás; Imperial, Juan; Palacios, José; Rey, Luis

2017-03-15

The genetic diversity of bacterial populations nodulating Lupinus micranthus in five geographical sites from northern Tunisia was examined. Phylogenetic analyses of 50 isolates based on partial sequences of recA and gyrB grouped strains into seven clusters, five of which belong to the genus Bradyrhizobium (28 isolates), one to Phyllobacterium (2 isolates), and one, remarkably, to Microvirga (20 isolates). The largest Bradyrhizobium cluster (17 isolates) grouped with the B. lupini species, and the other five clusters were close to different recently defined Bradyrhizobium species. Isolates close to Microvirga were obtained from nodules of plants from four of the five sites sampled. We carried out an in-depth phylogenetic study with representatives of the seven clusters using sequences from housekeeping genes ( rrs , recA , glnII , gyrB , and dnaK ) and obtained consistent results. A phylogeny based on the sequence of the symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behaviors of the representative strains were tested, and some congruence between symbiovars and symbiotic performance was observed. These data indicate a remarkable diversity of L. micranthus root nodule symbionts in northern Tunisia, including strains from the Bradyrhizobiaceae , Methylobacteriaceae , and Phyllobacteriaceae families, in contrast with those of the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, which are nodulated mostly by Bradyrhizobium strains. IMPORTANCE Lupinus micranthus is a legume broadly distributed in the Mediterranean region and plays an important role in soil fertility and vegetation coverage by fixing nitrogen and solubilizing phosphate in semiarid areas. Direct sowing to extend the distribution of this indigenous legume can contribute to the prevention of soil erosion in pre-Saharan lands of Tunisia. However, rhizobial populations associated with L. micranthus are poorly understood. In this context, the diversity of endosymbionts of this legume was investigated. Most Lupinus species are nodulated by Bradyrhizobium strains. This work showed that about half of the isolates from northern Tunisian soils were in fact Bradyrhizobium symbionts, but the other half were found unexpectedly to be bacteria within the genera Microvirga and Phyllobacterium These unusual endosymbionts may have a great ecological relevance. Inoculation with the appropriate selected symbiotic bacterial partners will increase L. micranthus survival with consequent advantages for the environment in semiarid areas of Tunisia. Copyright © 2017 American Society for Microbiology.

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

PubMed Central

Harris, R. Alan; Wang, Ting; Coarfa, Cristian; Nagarajan, Raman P.; Hong, Chibo; Downey, Sara L.; Johnson, Brett E.; Fouse, Shaun D.; Delaney, Allen; Zhao, Yongjun; Olshen, Adam; Ballinger, Tracy; Zhou, Xin; Forsberg, Kevin J.; Gu, Junchen; Echipare, Lorigail; O’Geen, Henriette; Lister, Ryan; Pelizzola, Mattia; Xi, Yuanxin; Epstein, Charles B.; Bernstein, Bradley E.; Hawkins, R. David; Ren, Bing; Chung, Wen-Yu; Gu, Hongcang; Bock, Christoph; Gnirke, Andreas; Zhang, Michael Q.; Haussler, David; Ecker, Joseph; Li, Wei; Farnham, Peggy J.; Waterland, Robert A.; Meissner, Alexander; Marra, Marco A.; Hirst, Martin; Milosavljevic, Aleksandar; Costello, Joseph F.

2010-01-01

Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression. PMID:20852635

DOE Office of Scientific and Technical Information (OSTI.GOV)

Labonté, Jessica M.; Swan, Brandon K.; Poulos, Bonnie

Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. Furthermore, a combination of comparative genomics, metagenomic fragmentmore » recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. This study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities.« less

Single-cell genomics-based analysis of virus–host interactions in marine surface bacterioplankton

DOE PAGES

Labonté, Jessica M.; Swan, Brandon K.; Poulos, Bonnie; ...

2015-04-07

Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus–host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. Furthermore, a combination of comparative genomics, metagenomic fragmentmore » recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus–host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage–host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. This study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host–virus interactions in complex microbial communities.« less

It's all relative: ranking the diversity of aquatic bacterial communities.

PubMed

Shaw, Allison K; Halpern, Aaron L; Beeson, Karen; Tran, Bao; Venter, J Craig; Martiny, Jennifer B H

2008-09-01

The study of microbial diversity patterns is hampered by the enormous diversity of microbial communities and the lack of resources to sample them exhaustively. For many questions about richness and evenness, however, one only needs to know the relative order of diversity among samples rather than total diversity. We used 16S libraries from the Global Ocean Survey to investigate the ability of 10 diversity statistics (including rarefaction, non-parametric, parametric, curve extrapolation and diversity indices) to assess the relative diversity of six aquatic bacterial communities. Overall, we found that the statistics yielded remarkably similar rankings of the samples for a given sequence similarity cut-off. This correspondence, despite the different underlying assumptions of the statistics, suggests that diversity statistics are a useful tool for ranking samples of microbial diversity. In addition, sequence similarity cut-off influenced the diversity ranking of the samples, demonstrating that diversity statistics can also be used to detect differences in phylogenetic structure among microbial communities. Finally, a subsampling analysis suggests that further sequencing from these particular clone libraries would not have substantially changed the richness rankings of the samples.

Quality scores for 32,000 genomes

DOE PAGES

Land, Miriam L.; Hyatt, Doug; Jun, Se-Ran; ...

2014-12-08

More than 80% of the microbial genomes in GenBank are of ‘draft’ quality (12,553 draft vs. 2,679 finished, as of October, 2013). In this study, we have examined all the microbial DNA sequences available for complete, draft, and Sequence Read Archive genomes in GenBank as well as three other major public databases, and assigned quality scores for more than 30,000 prokaryotic genome sequences. Scores were assigned using four categories: the completeness of the assembly, the presence of full-length rRNA genes, tRNA composition and the presence of a set of 102 conserved genes in prokaryotes. Most (~88%) of the genomes hadmore » quality scores of 0.8 or better and can be safely used for standard comparative genomics analysis. We compared genomes across factors that may influence the score. We found that although sequencing depth coverage of over 100x did not ensure a better score, sequencing read length was a better indicator of sequencing quality. With few exceptions, most of the 30,000 genomes have nearly all the 102 essential genes. The score can be used to set thresholds for screening data when analyzing “all published genomes” and reference data is either not available or not applicable. The scores highlighted organisms for which commonly used tools do not perform well. This information can be used to improve tools and to serve a broad group of users as more diverse organisms are sequenced. Finally and unexpectedly, the comparison of predicted tRNAs across 15,000 high quality genomes showed that anticodons beginning with an ‘A’ (codons ending with a ‘U’) are almost non-existent, with the exception of one arginine codon (CGU); this has been noted previously in the literature for a few genomes, but not with the depth found here.« less

Quality scores for 32,000 genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Land, Miriam L.; Hyatt, Doug; Jun, Se-Ran

More than 80% of the microbial genomes in GenBank are of ‘draft’ quality (12,553 draft vs. 2,679 finished, as of October, 2013). In this study, we have examined all the microbial DNA sequences available for complete, draft, and Sequence Read Archive genomes in GenBank as well as three other major public databases, and assigned quality scores for more than 30,000 prokaryotic genome sequences. Scores were assigned using four categories: the completeness of the assembly, the presence of full-length rRNA genes, tRNA composition and the presence of a set of 102 conserved genes in prokaryotes. Most (~88%) of the genomes hadmore » quality scores of 0.8 or better and can be safely used for standard comparative genomics analysis. We compared genomes across factors that may influence the score. We found that although sequencing depth coverage of over 100x did not ensure a better score, sequencing read length was a better indicator of sequencing quality. With few exceptions, most of the 30,000 genomes have nearly all the 102 essential genes. The score can be used to set thresholds for screening data when analyzing “all published genomes” and reference data is either not available or not applicable. The scores highlighted organisms for which commonly used tools do not perform well. This information can be used to improve tools and to serve a broad group of users as more diverse organisms are sequenced. Finally and unexpectedly, the comparison of predicted tRNAs across 15,000 high quality genomes showed that anticodons beginning with an ‘A’ (codons ending with a ‘U’) are almost non-existent, with the exception of one arginine codon (CGU); this has been noted previously in the literature for a few genomes, but not with the depth found here.« less

The genetic diversity of merozoite surface antigen 1 (MSA-1) among Babesia bovis detected from cattle populations in Thailand, Brazil and Ghana.

PubMed

Nagano, Daisuke; Sivakumar, Thillaiampalam; De De Macedo, Alane Caine Costa; Inpankaew, Tawin; Alhassan, Andy; Igarashi, Ikuo; Yokoyama, Naoaki

2013-11-01

In the present study, we screened blood DNA samples obtained from cattle bred in Brazil (n=164) and Ghana (n=80) for Babesia bovis using a diagnostic PCR assay and found prevalences of 14.6% and 46.3%, respectively. Subsequently, the genetic diversity of B. bovis in Thailand, Brazil and Ghana was analyzed, based on the DNA sequence of merozoite surface antigen-1 (MSA-1). In Thailand, MSA-1 sequences were relatively conserved and found in a single clade of the phylogram, while Brazilian MSA-1 sequences showed high genetic diversity and were dispersed across three different clades. In contrast, the sequences from Ghanaian samples were detected in two different clades, one of which contained only a single Ghanaian sequence. The identities among the MSA-1 sequences from Thailand, Brazil and Ghana were 99.0-100%, 57.5-99.4% and 60.3-100%, respectively, while the similarities among the deduced MSA-1 amino acid sequences within the respective countries were 98.4-100%, 59.4-99.7% and 58.7-100%, respectively. These observations suggested that the genetic diversity of B. bovis based on MSA-1 sequences was higher in Brazil and Ghana than in Thailand. The current data highlight the importance of conducting extensive studies on the genetic diversity of B. bovis before designing immune control strategies in each surveyed country.

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PubMed

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

PubMed Central

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology. PMID:25423096

Coprolites as a source of information on the genome and diet of the cave hyena

PubMed Central

Bon, Céline; Berthonaud, Véronique; Maksud, Frédéric; Labadie, Karine; Poulain, Julie; Artiguenave, François; Wincker, Patrick; Aury, Jean-Marc; Elalouf, Jean-Marc

2012-01-01

We performed high-throughput sequencing of DNA from fossilized faeces to evaluate this material as a source of information on the genome and diet of Pleistocene carnivores. We analysed coprolites derived from the extinct cave hyena (Crocuta crocuta spelaea), and sequenced 90 million DNA fragments from two specimens. The DNA reads enabled a reconstruction of the cave hyena mitochondrial genome with up to a 158-fold coverage. This genome, and those sequenced from extant spotted (Crocuta crocuta) and striped (Hyaena hyaena) hyena specimens, allows for the establishment of a robust phylogeny that supports a close relationship between the cave and the spotted hyena. We also demonstrate that high-throughput sequencing yields data for cave hyena multi-copy and single-copy nuclear genes, and that about 50 per cent of the coprolite DNA can be ascribed to this species. Analysing the data for additional species to indicate the cave hyena diet, we retrieved abundant sequences for the red deer (Cervus elaphus), and characterized its mitochondrial genome with up to a 3.8-fold coverage. In conclusion, we have demonstrated the presence of abundant ancient DNA in the coprolites surveyed. Shotgun sequencing of this material yielded a wealth of DNA sequences for a Pleistocene carnivore and allowed unbiased identification of diet. PMID:22456883

Sequence Analysis of the Genome of Carnation (Dianthus caryophyllus L.)

PubMed Central

Yagi, Masafumi; Kosugi, Shunichi; Hirakawa, Hideki; Ohmiya, Akemi; Tanase, Koji; Harada, Taro; Kishimoto, Kyutaro; Nakayama, Masayoshi; Ichimura, Kazuo; Onozaki, Takashi; Yamaguchi, Hiroyasu; Sasaki, Nobuhiro; Miyahara, Taira; Nishizaki, Yuzo; Ozeki, Yoshihiro; Nakamura, Noriko; Suzuki, Takamasa; Tanaka, Yoshikazu; Sato, Shusei; Shirasawa, Kenta; Isobe, Sachiko; Miyamura, Yoshinori; Watanabe, Akiko; Nakayama, Shinobu; Kishida, Yoshie; Kohara, Mitsuyo; Tabata, Satoshi

2014-01-01

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. ‘Francesco’ was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568 887 315 bp, consisting of 45 088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16 644 bp and 60 737 bp, respectively, and the longest scaffold was 1 287 144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp. PMID:24344172

The genome sequence of the outbreeding globe artichoke constructed de novo incorporating a phase-aware low-pass sequencing strategy of F1 progeny

PubMed Central

Scaglione, Davide; Reyes-Chin-Wo, Sebastian; Acquadro, Alberto; Froenicke, Lutz; Portis, Ezio; Beitel, Christopher; Tirone, Matteo; Mauro, Rosario; Lo Monaco, Antonino; Mauromicale, Giovanni; Faccioli, Primetta; Cattivelli, Luigi; Rieseberg, Loren; Michelmore, Richard; Lanteri, Sergio

2016-01-01

Globe artichoke (Cynara cardunculus var. scolymus) is an out-crossing, perennial, multi-use crop species that is grown worldwide and belongs to the Compositae, one of the most successful Angiosperm families. We describe the first genome sequence of globe artichoke. The assembly, comprising of 13,588 scaffolds covering 725 of the 1,084 Mb genome, was generated using ~133-fold Illumina sequencing data and encodes 26,889 predicted genes. Re-sequencing (30×) of globe artichoke and cultivated cardoon (C. cardunculus var. altilis) parental genotypes and low-coverage (0.5 to 1×) genotyping-by-sequencing of 163 F1 individuals resulted in 73% of the assembled genome being anchored in 2,178 genetic bins ordered along 17 chromosomal pseudomolecules. This was achieved using a novel pipeline, SOILoCo (Scaffold Ordering by Imputation with Low Coverage), to detect heterozygous regions and assign parental haplotypes with low sequencing read depth and of unknown phase. SOILoCo provides a powerful tool for de novo genome analysis of outcrossing species. Our data will enable genome-scale analyses of evolutionary processes among crops, weeds, and wild species within and beyond the Compositae, and will facilitate the identification of economically important genes from related species. PMID:26786968

On the Power and the Systematic Biases of the Detection of Chromosomal Inversions by Paired-End Genome Sequencing

PubMed Central

Lucas Lledó, José Ignacio; Cáceres, Mario

2013-01-01

One of the most used techniques to study structural variation at a genome level is paired-end mapping (PEM). PEM has the advantage of being able to detect balanced events, such as inversions and translocations. However, inversions are still quite difficult to predict reliably, especially from high-throughput sequencing data. We simulated realistic PEM experiments with different combinations of read and library fragment lengths, including sequencing errors and meaningful base-qualities, to quantify and track down the origin of false positives and negatives along sequencing, mapping, and downstream analysis. We show that PEM is very appropriate to detect a wide range of inversions, even with low coverage data. However, % of inversions located between segmental duplications are expected to go undetected by the most common sequencing strategies. In general, longer DNA libraries improve the detectability of inversions far better than increments of the coverage depth or the read length. Finally, we review the performance of three algorithms to detect inversions —SVDetect, GRIAL, and VariationHunter—, identify common pitfalls, and reveal important differences in their breakpoint precisions. These results stress the importance of the sequencing strategy for the detection of structural variants, especially inversions, and offer guidelines for the design of future genome sequencing projects. PMID:23637806

Estimating Bacterial Diversity for Ecological Studies: Methods, Metrics, and Assumptions

PubMed Central

Birtel, Julia; Walser, Jean-Claude; Pichon, Samuel; Bürgmann, Helmut; Matthews, Blake

2015-01-01

Methods to estimate microbial diversity have developed rapidly in an effort to understand the distribution and diversity of microorganisms in natural environments. For bacterial communities, the 16S rRNA gene is the phylogenetic marker gene of choice, but most studies select only a specific region of the 16S rRNA to estimate bacterial diversity. Whereas biases derived from from DNA extraction, primer choice and PCR amplification are well documented, we here address how the choice of variable region can influence a wide range of standard ecological metrics, such as species richness, phylogenetic diversity, β-diversity and rank-abundance distributions. We have used Illumina paired-end sequencing to estimate the bacterial diversity of 20 natural lakes across Switzerland derived from three trimmed variable 16S rRNA regions (V3, V4, V5). Species richness, phylogenetic diversity, community composition, β-diversity, and rank-abundance distributions differed significantly between 16S rRNA regions. Overall, patterns of diversity quantified by the V3 and V5 regions were more similar to one another than those assessed by the V4 region. Similar results were obtained when analyzing the datasets with different sequence similarity thresholds used during sequences clustering and when the same analysis was used on a reference dataset of sequences from the Greengenes database. In addition we also measured species richness from the same lake samples using ARISA Fingerprinting, but did not find a strong relationship between species richness estimated by Illumina and ARISA. We conclude that the selection of 16S rRNA region significantly influences the estimation of bacterial diversity and species distributions and that caution is warranted when comparing data from different variable regions as well as when using different sequencing techniques. PMID:25915756

Overlap and diversity in antimicrobial peptide databases: compiling a non-redundant set of sequences.

PubMed

Aguilera-Mendoza, Longendri; Marrero-Ponce, Yovani; Tellez-Ibarra, Roberto; Llorente-Quesada, Monica T; Salgado, Jesús; Barigye, Stephen J; Liu, Jun

2015-08-01

The large variety of antimicrobial peptide (AMP) databases developed to date are characterized by a substantial overlap of data and similarity of sequences. Our goals are to analyze the levels of redundancy for all available AMP databases and use this information to build a new non-redundant sequence database. For this purpose, a new software tool is introduced. A comparative study of 25 AMP databases reveals the overlap and diversity among them and the internal diversity within each database. The overlap analysis shows that only one database (Peptaibol) contains exclusive data, not present in any other, whereas all sequences in the LAMP_Patent database are included in CAMP_Patent. However, the majority of databases have their own set of unique sequences, as well as some overlap with other databases. The complete set of non-duplicate sequences comprises 16 990 cases, which is almost half of the total number of reported peptides. On the other hand, the diversity analysis identifies the most and least diverse databases and proves that all databases exhibit some level of redundancy. Finally, we present a new parallel-free software, named Dover Analyzer, developed to compute the overlap and diversity between any number of databases and compile a set of non-redundant sequences. These results are useful for selecting or building a suitable representative set of AMPs, according to specific needs. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Differential sequence diversity at merozoite surface protein-1 locus of Plasmodium knowlesi from humans and macaques in Thailand.

PubMed

Putaporntip, Chaturong; Thongaree, Siriporn; Jongwutiwes, Somchai

2013-08-01

To determine the genetic diversity and potential transmission routes of Plasmodium knowlesi, we analyzed the complete nucleotide sequence of the gene encoding the merozoite surface protein-1 of this simian malaria (Pkmsp-1), an asexual blood-stage vaccine candidate, from naturally infected humans and macaques in Thailand. Analysis of Pkmsp-1 sequences from humans (n=12) and monkeys (n=12) reveals five conserved and four variable domains. Most nucleotide substitutions in conserved domains were dimorphic whereas three of four variable domains contained complex repeats with extensive sequence and size variation. Besides purifying selection in conserved domains, evidence of intragenic recombination scattering across Pkmsp-1 was detected. The number of haplotypes, haplotype diversity, nucleotide diversity and recombination sites of human-derived sequences exceeded that of monkey-derived sequences. Phylogenetic networks based on concatenated conserved sequences of Pkmsp-1 displayed a character pattern that could have arisen from sampling process or the presence of two independent routes of P. knowlesi transmission, i.e. from macaques to human and from human to humans in Thailand. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterizing novel endogenous retroviruses from genetic variation inferred from short sequence reads

PubMed Central

Mourier, Tobias; Mollerup, Sarah; Vinner, Lasse; Hansen, Thomas Arn; Kjartansdóttir, Kristín Rós; Guldberg Frøslev, Tobias; Snogdal Boutrup, Torsten; Nielsen, Lars Peter; Willerslev, Eske; Hansen, Anders J.

2015-01-01

From Illumina sequencing of DNA from brain and liver tissue from the lion, Panthera leo, and tumor samples from the pike-perch, Sander lucioperca, we obtained two assembled sequence contigs with similarity to known retroviruses. Phylogenetic analyses suggest that the pike-perch retrovirus belongs to the epsilonretroviruses, and the lion retrovirus to the gammaretroviruses. To determine if these novel retroviral sequences originate from an endogenous retrovirus or from a recently integrated exogenous retrovirus, we assessed the genetic diversity of the parental sequences from which the short Illumina reads are derived. First, we showed by simulations that we can robustly infer the level of genetic diversity from short sequence reads. Second, we find that the measures of nucleotide diversity inferred from our retroviral sequences significantly exceed the level observed from Human Immunodeficiency Virus infections, prompting us to conclude that the novel retroviruses are both of endogenous origin. Through further simulations, we rule out the possibility that the observed elevated levels of nucleotide diversity are the result of co-infection with two closely related exogenous retroviruses. PMID:26493184

Analysis of genetic diversity using SNP markers in oat

USDA-ARS?s Scientific Manuscript database

A large-scale single nucleotide polymorphism (SNP) discovery was carried out in cultivated oat using Roche 454 sequencing methods. DNA sequences were generated from cDNAs originating from a panel of 20 diverse oat cultivars, and from Diversity Array Technology (DArT) genomic complexity reductions fr...

Developing spectral, structural, and phenological diversity proxies for monitoring biodiversity change across space and time using ESA's Sentinel satellites

NASA Astrophysics Data System (ADS)

Ma, X.; Mahecha, M. D.; Migliavacca, M.; Luo, Y.; Urban, M.; Bohn, F. J.; Huth, A.; Reichstein, M.

2017-12-01

A key challenge for monitoring biodiversity change is the lack of consistent measures of biodiversity across space and time. This challenge may be addressed by exploring the potentials provided by novel remote sensing observations. By continuously observing broad-scale patterns of vegetation and land surface parameters, remote sensing can complement the restricted coverage afforded by field measurements. Here we develop methods to infer spatial patterns of biodiversity at ecosystem level from ESA's next-generation Sentinel sensors (Sentinel-1: C-band radar & Sentinel-2: multispectral). Both satellites offer very high spatial (10 m) and temporal resolutions (5 days) measurements with global coverage. We propose and test several ecosystem biodiversity proxies, including landscape spectral diversity, phenological diversity, and canopy structural diversity. These diversity proxies are highly related to some key aspects of essential biodiversity variables (EBVs) as defined by GEO-BON, such as habitat structure, community composition, ecosystem function and structure. We verify spaceborne retrievals of these biodiversity proxies with in situ measurements from drone (spectral diversity), phenocam (phenological diversity), and airborne LiDAR (canopy structural diversity) over multiple flux tower sites within the Mediterranean region. We further compare our remote sensing retrievals of biodiversity proxies against several biodiversity indices as derived from field measurements (incl. ⍺-/β- diversity and Shannon-index) to explore the limitations and potentials of extending the RS proxies to a greater spatial extent. We expect the new concept as to maximize the potential of remote sensing information might help to monitor key aspects of EBVs on a global scale.

Enhancing genome assemblies by integrating non-sequence based data

PubMed Central

2011-01-01

Introduction Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. Methods The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Results Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. Conclusions We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses. PMID:21554765

Enhancing genome assemblies by integrating non-sequence based data.

PubMed

Heider, Thomas N; Lindsay, James; Wang, Chenwei; O'Neill, Rachel J; Pask, Andrew J

2011-05-28

Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses.

Novel Degenerate PCR Method for Whole-Genome Amplification Applied to Peru Margin (ODP Leg 201) Subsurface Samples

PubMed Central

Martino, Amanda J.; Rhodes, Matthew E.; Biddle, Jennifer F.; Brandt, Leah D.; Tomsho, Lynn P.; House, Christopher H.

2011-01-01

A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3′ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples. PMID:22319519

Discovery of Influenza A Virus Sequence Pairs and Their Combinations for Simultaneous Heterosubtypic Targeting that Hedge against Antiviral Resistance

PubMed Central

Lin, Jing; Pramono, Zacharias Aloysius Dwi; Maurer-Stroh, Sebastian

2016-01-01

The multiple circulating human influenza A virus subtypes coupled with the perpetual genomic mutations and segment reassortment events challenge the development of effective therapeutics. The capacity to drug most RNAs motivates the investigation on viral RNA targets. 123,060 segment sequences from 35,938 strains of the most prevalent subtypes also infecting humans–H1N1, 2009 pandemic H1N1, H3N2, H5N1 and H7N9, were used to identify 1,183 conserved RNA target sequences (≥15-mer) in the internal segments. 100% theoretical coverage in simultaneous heterosubtypic targeting is achieved by pairing specific sequences from the same segment (“Duals”) or from two segments (“Doubles”); 1,662 Duals and 28,463 Doubles identified. By combining specific Duals and/or Doubles to form a target graph wherein an edge connecting two vertices (target sequences) represents a Dual or Double, it is possible to hedge against antiviral resistance besides maintaining 100% heterosubtypic coverage. To evaluate the hedging potential, we define the hedge-factor as the minimum number of resistant target sequences that will render the graph to become resistant i.e. eliminate all the edges therein; a target sequence or a graph is considered resistant when it cannot achieve 100% heterosubtypic coverage. In an n-vertices graph (n ≥ 3), the hedge-factor is maximal (= n– 1) when it is a complete graph i.e. every distinct pair in a graph is either a Dual or Double. Computational analyses uncover an extensive number of complete graphs of different sizes. Monte Carlo simulations show that the mutation counts and time elapsed for a target graph to become resistant increase with the hedge-factor. Incidentally, target sequences which were reported to reduce virus titre in experiments are included in our target graphs. The identity of target sequence pairs for heterosubtypic targeting and their combinations for hedging antiviral resistance are useful toolkits to construct target graphs for different therapeutic objectives. PMID:26771381

Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

PubMed Central

Haas, Brian J.; Gevers, Dirk; Earl, Ashlee M.; Feldgarden, Mike; Ward, Doyle V.; Giannoukos, Georgia; Ciulla, Dawn; Tabbaa, Diana; Highlander, Sarah K.; Sodergren, Erica; Methé, Barbara; DeSantis, Todd Z.; Petrosino, Joseph F.; Knight, Rob; Birren, Bruce W.

2011-01-01

Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys. PMID:21212162

Forest fire impact on bird habitat in a mixed oak-pine forest in Puebla, Mexico

Treesearch

Laura P. Ponce-Calderón Ponce-Calderón; Dante A. Rodríguez-Trejo; Beatriz C. Aguilar-Váldez; Elvia López-Pérez

2013-01-01

To assess the impact of different-severity wildfires on bird habitat, habitat quality was determined by analyzing the degree of richness association, abundance and diversity of bird species and vegetation structure (richness, abundance, diversity and coverage). These attributes were quantified with four sampling sites for birds and five for quadrant-centered points...

School Experiences of Transgender and Gender Diverse Students in Australia

ERIC Educational Resources Information Center

Jones, Tiffany; Smith, Elizabeth; Ward, Roz; Dixon, Jennifer; Hillier, Lynne; Mitchell, Anne

2016-01-01

Over the last decade, there has been an increase in global and local policy protections on the basis of gender identity and expression in education and a recent spate of coverage of transgender students on Australian television and news media. This paper explores the school experiences of Australian transgender and gender diverse students, with…

Genotyping-by-sequencing (GBS) revealed molecular genetic diversity of Iranian wheat landraces and cultivars

USDA-ARS?s Scientific Manuscript database

Genetic diversity is an essential resource for breeders to improve new cultivars with desirable characteristics. Recently genotyping-by-sequencing (GBS), a next generation sequencing (NGS) based technology that can simplify complex genomes, has been used as a high-throughput and cost-effective molec...

Transcriptome sequencing of diverse peanut (arachis) wild species and the cultivated species reveals a wealth of untapped genetic variability

USDA-ARS?s Scientific Manuscript database

Next generation sequencing technologies and improved bioinformatics methods have provided opportunities to study sequence variability in complex polyploid transcriptomes. In this study, we used a diverse panel of twenty-two Arachis accessions representing seven Arachis hypogaea market classes, A-, B...

The need for an assembly pilot project

USDA-ARS?s Scientific Manuscript database

Progress has been rapid since the June 2008 start of the cacao genome sequencing project with the completion of the physical map and the accumulation of approximately 10x coverage of the genome with Titanium 454 sequence data of Matina1-6, the highly homozygous Amelonado tree chosen for the project....

Fast imputation using medium- or low-coverage sequence data

USDA-ARS?s Scientific Manuscript database

Direct imputation from raw sequence reads can be more accurate than calling genotypes first and then imputing, especially if read depth is low or error rates high, but different imputation strategies are required than those used for data from genotyping chips. A fast algorithm to impute from lower t...

Transcriptome assembly, gene annotation and tissue gene expression atlas of the rainbow trout

USDA-ARS?s Scientific Manuscript database

Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complimented by transcriptome information that will enhance genome assembly and annotation. Previously, we reported a transcriptome reference sequence using a 19X coverage of Sanger and 454-pyrosequencing dat...

Dominant frames in legacy and social media coverage of the IPCC Fifth Assessment Report

NASA Astrophysics Data System (ADS)

O'Neill, Saffron; Williams, Hywel T. P.; Kurz, Tim; Wiersma, Bouke; Boykoff, Maxwell

2015-04-01

The media are powerful agents that translate information across the science-policy interface, framing it for audiences. Yet frames are never neutral: they define an issue, identify causes, make moral judgements and shape proposed solutions. Here, we show how the IPCC Fifth Assessment Report (AR5) was framed in UK and US broadcast and print coverage, and on Twitter. Coverage of IPCC Working Group I (WGI) was contested and politicized, employing the `Settled Science, Uncertain Science, Political or Ideological Struggle and Role of Science’ frames. WGII coverage commonly used Disaster or Security. More diverse frames were employed for WGII and WGIII, including Economics and Morality and Ethics. Framing also varied by media institution: for example, the BBC used Uncertain Science, whereas Channel 4 did not. Coverage varied by working group, with WGIII gaining far less coverage than WGI or WGII. We suggest that media coverage and framing of AR5 was influenced by its sequential three-part structure and by the availability of accessible narratives and visuals. We recommend that these communication lessons be applied to future climate science reports.

Recombination between Poliovirus and Coxsackie A Viruses of Species C: A Model of Viral Genetic Plasticity and Emergence

PubMed Central

Combelas, Nicolas; Holmblat, Barbara; Joffret, Marie-Line; Colbère-Garapin, Florence; Delpeyroux, Francis

2011-01-01

Genetic recombination in RNA viruses was discovered many years ago for poliovirus (PV), an enterovirus of the Picornaviridae family, and studied using PV or other picornaviruses as models. Recently, recombination was shown to be a general phenomenon between different types of enteroviruses of the same species. In particular, the interest for this mechanism of genetic plasticity was renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses (cVDPVs), which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. Most of these cVDPVs had mosaic genomes constituted of mutated poliovaccine capsid sequences and part or all of the non-structural sequences from other human enteroviruses of species C (HEV-C), in particular coxsackie A viruses. A study in Madagascar showed that recombinant cVDPVs had been co-circulating in a small population of children with many different HEV-C types. This viral ecosystem showed a surprising and extensive biodiversity associated to several types and recombinant genotypes, indicating that intertypic genetic recombination was not only a mechanism of evolution for HEV-C, but an usual mode of genetic plasticity shaping viral diversity. Results suggested that recombination may be, in conjunction with mutations, implicated in the phenotypic diversity of enterovirus strains and in the emergence of new pathogenic strains. Nevertheless, little is known about the rules and mechanisms which govern genetic exchanges between HEV-C types, as well as about the importance of intertypic recombination in generating phenotypic variation. This review summarizes our current knowledge of the mechanisms of evolution of PV, in particular recombination events leading to the emergence of recombinant cVDPVs. PMID:21994791

Recombination between poliovirus and coxsackie A viruses of species C: a model of viral genetic plasticity and emergence.

PubMed

Combelas, Nicolas; Holmblat, Barbara; Joffret, Marie-Line; Colbère-Garapin, Florence; Delpeyroux, Francis

2011-08-01

Genetic recombination in RNA viruses was discovered many years ago for poliovirus (PV), an enterovirus of the Picornaviridae family, and studied using PV or other picornaviruses as models. Recently, recombination was shown to be a general phenomenon between different types of enteroviruses of the same species. In particular, the interest for this mechanism of genetic plasticity was renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses (cVDPVs), which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. Most of these cVDPVs had mosaic genomes constituted of mutated poliovaccine capsid sequences and part or all of the non-structural sequences from other human enteroviruses of species C (HEV-C), in particular coxsackie A viruses. A study in Madagascar showed that recombinant cVDPVs had been co-circulating in a small population of children with many different HEV-C types. This viral ecosystem showed a surprising and extensive biodiversity associated to several types and recombinant genotypes, indicating that intertypic genetic recombination was not only a mechanism of evolution for HEV-C, but an usual mode of genetic plasticity shaping viral diversity. Results suggested that recombination may be, in conjunction with mutations, implicated in the phenotypic diversity of enterovirus strains and in the emergence of new pathogenic strains. Nevertheless, little is known about the rules and mechanisms which govern genetic exchanges between HEV-C types, as well as about the importance of intertypic recombination in generating phenotypic variation. This review summarizes our current knowledge of the mechanisms of evolution of PV, in particular recombination events leading to the emergence of recombinant cVDPVs.

Diversity and population structure of northern switchgrass as revealed through exome capture sequencing

DOE PAGES

Evans, Joseph; Crisovan, Emily; Barry, Kerrie; ...

2015-10-01

Panicum virgatum L. (switchgrass) is a polyploid, perennial grass species that is native to North America, and is being developed as a future biofuel feedstock crop. Switchgrass is present primarily in two ecotypes: a northern upland ecotype, composed of tetraploid and octoploid accessions, and a southern lowland ecotype, composed of primarily tetraploid accessions. We employed high-coverage exome capture sequencing (~2.4 Tb) to genotype 537 individuals from 45 upland and 21 lowland populations. From these data, we identified ~27 million single-nucleotide polymorphisms (SNPs), of which 1 590 653 high-confidence SNPs were used in downstream analyses of diversity within and between themore » populations. From the 66 populations, we identified five primary population groups within the upland and lowland ecotypes, a result that was further supported through genetic distance analysis. We identified conserved, ecotype-restricted, non-synonymous SNPs that are predicted to affect the protein function of CONSTANS (CO) and EARLY HEADING DATE 1 (EHD1), key genes involved in flowering, which may contribute to the phenotypic differences between the two ecotypes. We also identified, relative to the near-reference Kanlow population, 17 228 genes present in more copies than in the reference genome (up-CNVs), 112 630 genes present in fewer copies than in the reference genome (down-CNVs) and 14 430 presence/absence variants (PAVs), affecting a total of 9979 genes, including two upland-specific CNV clusters. In total, 45 719 genes were affected by an SNP, CNV, or PAV across the panel, providing a firm foundation to identify functional variation associated with phenotypic traits of interest for biofuel feedstock production.« less

Diversity and population structure of northern switchgrass as revealed through exome capture sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evans, Joseph; Crisovan, Emily; Barry, Kerrie

Panicum virgatum L. (switchgrass) is a polyploid, perennial grass species that is native to North America, and is being developed as a future biofuel feedstock crop. Switchgrass is present primarily in two ecotypes: a northern upland ecotype, composed of tetraploid and octoploid accessions, and a southern lowland ecotype, composed of primarily tetraploid accessions. We employed high-coverage exome capture sequencing (~2.4 Tb) to genotype 537 individuals from 45 upland and 21 lowland populations. From these data, we identified ~27 million single-nucleotide polymorphisms (SNPs), of which 1 590 653 high-confidence SNPs were used in downstream analyses of diversity within and between themore » populations. From the 66 populations, we identified five primary population groups within the upland and lowland ecotypes, a result that was further supported through genetic distance analysis. We identified conserved, ecotype-restricted, non-synonymous SNPs that are predicted to affect the protein function of CONSTANS (CO) and EARLY HEADING DATE 1 (EHD1), key genes involved in flowering, which may contribute to the phenotypic differences between the two ecotypes. We also identified, relative to the near-reference Kanlow population, 17 228 genes present in more copies than in the reference genome (up-CNVs), 112 630 genes present in fewer copies than in the reference genome (down-CNVs) and 14 430 presence/absence variants (PAVs), affecting a total of 9979 genes, including two upland-specific CNV clusters. In total, 45 719 genes were affected by an SNP, CNV, or PAV across the panel, providing a firm foundation to identify functional variation associated with phenotypic traits of interest for biofuel feedstock production.« less

Twenty-one genome sequences from Pseudomonas species and 19 genome sequences from diverse bacteria isolated from the rhizosphere and endosphere of Populus deltoides.

PubMed

Brown, Steven D; Utturkar, Sagar M; Klingeman, Dawn M; Johnson, Courtney M; Martin, Stanton L; Land, Miriam L; Lu, Tse-Yuan S; Schadt, Christopher W; Doktycz, Mitchel J; Pelletier, Dale A

2012-11-01

To aid in the investigation of the Populus deltoides microbiome, we generated draft genome sequences for 21 Pseudomonas strains and 19 other diverse bacteria isolated from Populus deltoides roots. Genome sequences for isolates similar to Acidovorax, Bradyrhizobium, Brevibacillus, Caulobacter, Chryseobacterium, Flavobacterium, Herbaspirillum, Novosphingobium, Pantoea, Phyllobacterium, Polaromonas, Rhizobium, Sphingobium, and Variovorax were generated.

Global sequence diversity of the lactate dehydrogenase gene in Plasmodium falciparum.

PubMed

Simpalipan, Phumin; Pattaradilokrat, Sittiporn; Harnyuttanakorn, Pongchai

2018-01-09

Antigen-detecting rapid diagnostic tests (RDTs) have been recommended by the World Health Organization for use in remote areas to improve malaria case management. Lactate dehydrogenase (LDH) of Plasmodium falciparum is one of the main parasite antigens employed by various commercial RDTs. It has been hypothesized that the poor detection of LDH-based RDTs is attributed in part to the sequence diversity of the gene. To test this, the present study aimed to investigate the genetic diversity of the P. falciparum ldh gene in Thailand and to construct the map of LDH sequence diversity in P. falciparum populations worldwide. The ldh gene was sequenced for 50 P. falciparum isolates in Thailand and compared with hundreds of sequences from P. falciparum populations worldwide. Several indices of molecular variation were calculated, including the proportion of polymorphic sites, the average nucleotide diversity index (π), and the haplotype diversity index (H). Tests of positive selection and neutrality tests were performed to determine signatures of natural selection on the gene. Mean genetic distance within and between species of Plasmodium ldh was analysed to infer evolutionary relationships. Nucleotide sequences of P. falciparum ldh could be classified into 9 alleles, encoding 5 isoforms of LDH. L1a was the most common allelic type and was distributed in P. falciparum populations worldwide. Plasmodium falciparum ldh sequences were highly conserved, with haplotype and nucleotide diversity values of 0.203 and 0.0004, respectively. The extremely low genetic diversity was maintained by purifying selection, likely due to functional constraints. Phylogenetic analysis inferred the close genetic relationship of P. falciparum to malaria parasites of great apes, rather than to other human malaria parasites. This study revealed the global genetic variation of the ldh gene in P. falciparum, providing knowledge for improving detection of LDH-based RDTs and supporting the candidacy of LDH as a therapeutic drug target.

HIV-1 sequence variation between isolates from mother-infant transmission pairs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wike, C.M.; Daniels, M.R.; Furtado, M.

1991-12-31

To examine the sequence diversity of human immunodeficiency virus type 1 (HIV-1) between known transmission sets, sequences from the V3 and V4-V5 region of the env gene from 4 mother-infant pairs were analyzed. The mean interpatient sequence variation between isolates from linked mother-infant pairs was comparable to the sequence diversity found between isolates from other close contacts. The mean intrapatient variation was significantly less in the infants` isolates then the isolates from both their mothers and other characterized intrapatient sequence sets. In addition, a distinct and characteristic difference in the glycosylation pattern preceding the V3 loop was found between eachmore » linked transmission pair. These findings indicate that selection of specific genotypic variants, which may play a role in some direct transmission sets, and the duration of infection are important factors in the degree of diversity seen between the sequence sets.« less

αβ T cell receptors as predictors of health and disease

PubMed Central

Attaf, Meriem; Huseby, Eric; Sewell, Andrew K

2015-01-01

The diversity of antigen receptors and the specificity it underlies are the hallmarks of the cellular arm of the adaptive immune system. T and B lymphocytes are indeed truly unique in their ability to generate receptors capable of recognizing virtually any pathogen. It has been known for several decades that T lymphocytes recognize short peptides derived from degraded proteins presented by major histocompatibility complex (MHC) molecules at the cell surface. Interaction between peptide-MHC (pMHC) and the T cell receptor (TCR) is central to both thymic selection and peripheral antigen recognition. It is widely assumed that TCR diversity is required, or at least highly desirable, to provide sufficient immune coverage. However, a number of immune responses are associated with the selection of predictable, narrow, or skewed repertoires and public TCR chains. Here, we summarize the current knowledge on the formation of the TCR repertoire and its maintenance in health and disease. We also outline the various molecular mechanisms that govern the composition of the pre-selection, naive and antigen-specific TCR repertoires. Finally, we suggest that with the development of high-throughput sequencing, common TCR ‘signatures' raised against specific antigens could provide important diagnostic biomarkers and surrogate predictors of disease onset, progression and outcome. PMID:25619506

Whole-genome sequencing and analyses identify high genetic heterogeneity, diversity and endemicity of rotavirus genotype P[6] strains circulating in Africa.

PubMed

Nyaga, Martin M; Tan, Yi; Seheri, Mapaseka L; Halpin, Rebecca A; Akopov, Asmik; Stucker, Karla M; Fedorova, Nadia B; Shrivastava, Susmita; Duncan Steele, A; Mwenda, Jason M; Pickett, Brett E; Das, Suman R; Jeffrey Mphahlele, M

2018-05-18

Rotavirus A (RVA) exhibits a wide genotype diversity globally. Little is known about the genetic composition of genotype P[6] from Africa. This study investigated possible evolutionary mechanisms leading to genetic diversity of genotype P[6] VP4 sequences. Phylogenetic analyses on 167 P[6] VP4 full-length sequences were conducted, which included six porcine-origin sequences. Of the 167 sequences, 57 were newly acquired through whole genome sequencing as part of this study. The other 110 sequences were all publicly-available global P[6] VP4 full-length sequences downloaded from GenBank. The strength of association between the phenotypic features and the phylogeny was also determined. A number of reassortment and mixed infections of RVA genotype P[6] strains were observed in this study. Phylogenetic analyses demostrated the extensive genetic diversity that exists among human P[6] strains, porcine-like strains, their concomitant clades/subclades and estimated that P[6] VP4 gene has a higher substitution rate with the mean of 1.05E-3 substitutions/site/year. Further, the phylogenetic analyses indicated that genotype P[6] strains were endemic in Africa, characterised by an extensive genetic diversity and long-time local evolution of the viruses. This was also supported by phylogeographic clustering and G-genotype clustering of the P[6] strains when Bayesian Tip-association Significance testing (BaTS) was applied, clearly supporting that the viruses evolved locally in Africa instead of spatial mixing among different regions. Overall, the results demonstrated that multiple mechanisms such as reassortment events, various mutations and possibly interspecies transmission account for the enormous diversity of genotype P[6] strains in Africa. These findings highlight the need for continued global surveillance of rotavirus diversity. Copyright © 2018 Elsevier B.V. All rights reserved.

Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula

PubMed Central

Macas, Jiří; Neumann, Pavel; Navrátilová, Alice

2007-01-01

Background Extraordinary size variation of higher plant nuclear genomes is in large part caused by differences in accumulation of repetitive DNA. This makes repetitive DNA of great interest for studying the molecular mechanisms shaping architecture and function of complex plant genomes. However, due to methodological constraints of conventional cloning and sequencing, a global description of repeat composition is available for only a very limited number of higher plants. In order to provide further data required for investigating evolutionary patterns of repeated DNA within and between species, we used a novel approach based on massive parallel sequencing which allowed a comprehensive repeat characterization in our model species, garden pea (Pisum sativum). Results Analysis of 33.3 Mb sequence data resulted in quantification and partial sequence reconstruction of major repeat families occurring in the pea genome with at least thousands of copies. Our results showed that the pea genome is dominated by LTR-retrotransposons, estimated at 140,000 copies/1C. Ty3/gypsy elements are less diverse and accumulated to higher copy numbers than Ty1/copia. This is in part due to a large population of Ogre-like retrotransposons which alone make up over 20% of the genome. In addition to numerous types of mobile elements, we have discovered a set of novel satellite repeats and two additional variants of telomeric sequences. Comparative genome analysis revealed that there are only a few repeat sequences conserved between pea and soybean genomes. On the other hand, all major families of pea mobile elements are well represented in M. truncatula. Conclusion We have demonstrated that even in a species with a relatively large genome like pea, where a single 454-sequencing run provided only 0.77% coverage, the generated sequences were sufficient to reconstruct and analyze major repeat families corresponding to a total of 35–48% of the genome. These data provide a starting point for further investigations of legume plant genomes based on their global comparative analysis and for the development of more sophisticated approaches for data mining. PMID:18031571

Investigation into the sequence structure of 23 Y chromosomal STR loci using massively parallel sequencing.

PubMed

Kwon, So Yeun; Lee, Hwan Young; Kim, Eun Hye; Lee, Eun Young; Shin, Kyoung-Jin

2016-11-01

Next-generation sequencing (NGS) can produce massively parallel sequencing (MPS) data for many targeted regions with a high depth of coverage, suggesting its successful application to the amplicons of forensic genetic markers. In the present study, we evaluated the practical utility of MPS in Y-chromosome short tandem repeat (Y-STR) analysis using a multiplex polymerase chain reaction (PCR) system. The multiplex PCR system simultaneously amplified 24 Y-chromosomal markers, including the PowerPlex ® Y23 loci (DYS19, DYS385ab, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and YGATAH4) and the M175 marker with the small-sized amplicons ranging from 85 to 253bp. The barcoded libraries for the amplicons of the 24 Y-chromosomal markers were produced using a simplified PCR-based library preparation method and successfully sequenced using MPS on a MiSeq ® System with samples from 250 unrelated Korean males. The genotyping concordance between MPS and the capillary electrophoresis (CE) method, as well as the sequence structure of the 23 Y-STRs, were investigated. Three samples exhibited discordance between the MPS and CE results at DYS385, DYS439, and DYS576. There were 12 Y-STR loci that showed sequence variations in the alleles by a fragment size determination, and the most varied alleles occurred in DYS389II with a different sequence structure in the repeat region. The largest increase in gene diversity between the CE and MPS results was in DYS437 at +34.41%. Single nucleotide polymorphisms (SNPs), insertions, and deletions (indels) were observed in the flanking regions of DYS481, DYS576, and DYS385, respectively. Stutter and noise ratios of the 23 Y-STRs using the developed MPS system were also investigated. Based on these results, the MPS analysis system used in this study could facilitate the investigation into the sequences of the 23 Y-STRs in forensic genetics laboratories. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Alternative Enzymes Lead to Improvements in Sequence Coverage and PTM Analysis

PubMed Central

Hooper, Kyle; Rosenblatt, Michael; Urh, Marjeta; Saveliev, Sergei; Hosfield, Chris; Kobs, Gary; Ford, Michael; Jones, Richard; Amunugama, Ravi; Allen, David; Brazas, Robert

2013-01-01

The profiling of proteins using biological mass spectrometry (bottom up proteomics) most commonly requires trypsin. Trypsin is advantageous in that it produces peptides of optimal charge and size. However, for applications in which the proteins under investigation are part of a complex mixture or not isolated at high levels (i.e. low ng from an immunoprecipitation), sequence coverage is rarely complete. In addition, we have found that in several cases, like phosphorylation, acetylation, and methylation, alternative proteases are required to prepare peptides suitable for MS detection. This poster will provide specific examples which demonstrate this observation. For example, the application of a combined Trypsin/ Lys-C mixture reduces the number of missed cleavages by more than 3-fold producing samples with lower CV's (for biological replicates). The mixture is also well-suited for the complete proteolysis of hydrophobic, compact proteins. The addition of chymotrypsin and elastase has been found to be useful for identifying phosphorylation sites on proteins, especially on sequences where the site of phosphorylation inhibits trypsin (i.e. proximal to K or R). Many epigenetic applications have focused on histone modifications, like lysine acetylation and arginine methylation. Alternative proteases like Asp-N, Glu-C, and chymotrypsin have been especially useful given the fact that the modified K and R residues are resistant to c-terminal cleavage by trypsin. Finally, in the case of serum profiling, the addition of the endoglycosidase, PNGase F has been found to improve sequence coverage due to the removal of N-linked glycans.

ECHO: A reference-free short-read error correction algorithm

PubMed Central

Kao, Wei-Chun; Chan, Andrew H.; Song, Yun S.

2011-01-01

Developing accurate, scalable algorithms to improve data quality is an important computational challenge associated with recent advances in high-throughput sequencing technology. In this study, a novel error-correction algorithm, called ECHO, is introduced for correcting base-call errors in short-reads, without the need of a reference genome. Unlike most previous methods, ECHO does not require the user to specify parameters of which optimal values are typically unknown a priori. ECHO automatically sets the parameters in the assumed model and estimates error characteristics specific to each sequencing run, while maintaining a running time that is within the range of practical use. ECHO is based on a probabilistic model and is able to assign a quality score to each corrected base. Furthermore, it explicitly models heterozygosity in diploid genomes and provides a reference-free method for detecting bases that originated from heterozygous sites. On both real and simulated data, ECHO is able to improve the accuracy of previous error-correction methods by several folds to an order of magnitude, depending on the sequence coverage depth and the position in the read. The improvement is most pronounced toward the end of the read, where previous methods become noticeably less effective. Using a whole-genome yeast data set, it is demonstrated here that ECHO is capable of coping with nonuniform coverage. Also, it is shown that using ECHO to perform error correction as a preprocessing step considerably facilitates de novo assembly, particularly in the case of low-to-moderate sequence coverage depth. PMID:21482625

Low-coverage, whole-genome sequencing of Artocarpus camansi (Moraceae) for phylogenetic marker development and gene discovery1

PubMed Central

Gardner, Elliot M.; Johnson, Matthew G.; Ragone, Diane; Wickett, Norman J.; Zerega, Nyree J. C.

2016-01-01

Premise of the study: We used moderately low-coverage (17×) whole-genome sequencing of Artocarpus camansi (Moraceae) to develop genomic resources for Artocarpus and Moraceae. Methods and Results: A de novo assembly of Illumina short reads (251,378,536 pairs, 2 × 100 bp) accounted for 93% of the predicted genome size. Predicted coding regions were used in a three-way orthology search with published genomes of Morus notabilis and Cannabis sativa. Phylogenetic markers for Moraceae were developed from 333 inferred single-copy exons. Ninety-eight putative MADS-box genes were identified. Analysis of all predicted coding regions resulted in preliminary annotation of 49,089 genes. An analysis of synonymous substitutions for pairs of orthologs (Ks analysis) in M. notabilis and A. camansi strongly suggested a lineage-specific whole-genome duplication in Artocarpus. Conclusions: This study substantially increases the genomic resources available for Artocarpus and Moraceae and demonstrates the value of low-coverage de novo assemblies for nonmodel organisms with moderately large genomes. PMID:27437173

Transcriptome sequencing reveals high isoform diversity in the ant Formica exsecta

PubMed Central

Paviala, Jenni; Morandin, Claire; Wheat, Christopher; Sundström, Liselotte; Helanterä, Heikki

2017-01-01

Transcriptome resources for social insects have the potential to provide new insight into polyphenism, i.e., how divergent phenotypes arise from the same genome. Here we present a transcriptome based on paired-end RNA sequencing data for the ant Formica exsecta (Formicidae, Hymenoptera). The RNA sequencing libraries were constructed from samples of several life stages of both sexes and female castes of queens and workers, in order to maximize representation of expressed genes. We first compare the performance of common assembly and scaffolding software (Trinity, Velvet-Oases, and SOAPdenovo-trans), in producing de novo assemblies. Second, we annotate the resulting expressed contigs to the currently published genomes of ants, and other insects, including the honeybee, to filter genes that have annotation evidence of being true genes. Our pipeline resulted in a final assembly of altogether 39,262 mRNA transcripts, with an average coverage of >300X, belonging to 17,496 unique genes with annotation in the related ant species. From these genes, 536 genes were unique to one caste or sex only, highlighting the importance of comprehensive sampling. Our final assembly also showed expression of several splice variants in 6,975 genes, and we show that accounting for splice variants affects the outcome of downstream analyses such as gene ontologies. Our transcriptome provides an outstanding resource for future genetic studies on F. exsecta and other ant species, and the presented transcriptome assembly can be adapted to any non-model species that has genomic resources available from a related taxon. PMID:29177112

Diversity of Termitomyces associated with fungus-farming termites assessed by cultural and culture-independent methods.

PubMed

Makonde, Huxley M; Boga, Hamadi I; Osiemo, Zipporah; Mwirichia, Romano; Stielow, J Benjamin; Göker, Markus; Klenk, Hans-Peter

2013-01-01

Fungus-cultivating termites make use of an obligate mutualism with fungi from the genus Termitomyces, which are acquired through either vertical transmission via reproductive alates or horizontally transmitted during the formation of new mounds. Termitomyces taxonomy, and thus estimating diversity and host specificity of these fungi, is challenging because fruiting bodies are rarely found. Molecular techniques can be applied but need not necessarily yield the same outcome than morphological identification. Culture-dependent and culture-independent methods were used to comprehensively assess host specificity and gut fungal diversity. Termites were identified using mitochondrial cytochrome oxidase II (COII) genes. Twenty-three Termitomyces cultures were isolated from fungal combs. Internal transcribed spacer (ITS) clone libraries were constructed from termite guts. Presence of Termitomyces was confirmed using specific and universal primers. Termitomyces species boundaries were estimated by cross-comparison of macromorphological and sequence features, and ITS clustering parameters accordingly optimized. The overall trends in coverage of Termitomyces diversity and host associations were estimated using Genbank data. Results indicate a monoculture of Termitomyces in the guts as well as the isolation sources (fungal combs). However, cases of more than one Termitomyces strains per mound were observed since mounds can contain different termite colonies. The newly found cultures, as well as the clustering analysis of GenBank data indicate that there are on average between one and two host genera per Termitomyces species. Saturation does not appear to have been reached, neither for the total number of known Termitomyces species nor for the number of Termitomyces species per host taxon, nor for the number of known hosts per Termitomyces species. Considering the rarity of Termitomyces fruiting bodies, it is suggested to base the future taxonomy of the group mainly on well-characterized and publicly accessible cultures.

Microbial Diversity in Deep-sea Methane Seep Sediments Presented by SSU rRNA Gene Tag Sequencing

PubMed Central

Nunoura, Takuro; Takaki, Yoshihiro; Kazama, Hiromi; Hirai, Miho; Ashi, Juichiro; Imachi, Hiroyuki; Takai, Ken

2012-01-01

Microbial community structures in methane seep sediments in the Nankai Trough were analyzed by tag-sequencing analysis for the small subunit (SSU) rRNA gene using a newly developed primer set. The dominant members of Archaea were Deep-sea Hydrothermal Vent Euryarchaeotic Group 6 (DHVEG 6), Marine Group I (MGI) and Deep Sea Archaeal Group (DSAG), and those in Bacteria were Alpha-, Gamma-, Delta- and Epsilonproteobacteria, Chloroflexi, Bacteroidetes, Planctomycetes and Acidobacteria. Diversity and richness were examined by 8,709 and 7,690 tag-sequences from sediments at 5 and 25 cm below the seafloor (cmbsf), respectively. The estimated diversity and richness in the methane seep sediment are as high as those in soil and deep-sea hydrothermal environments, although the tag-sequences obtained in this study were not sufficient to show whole microbial diversity in this analysis. We also compared the diversity and richness of each taxon/division between the sediments from the two depths, and found that the diversity and richness of some taxa/divisions varied significantly along with the depth. PMID:22510646

Liver imaging at 3.0 T: diffusion-induced black-blood echo-planar imaging with large anatomic volumetric coverage as an alternative for specific absorption rate-intensive echo-train spin-echo sequences: feasibility study.

PubMed

van den Bos, Indra C; Hussain, Shahid M; Krestin, Gabriel P; Wielopolski, Piotr A

2008-07-01

Institutional Review Board approval and signed informed consent were obtained by all participants for an ongoing sequence optimization project at 3.0 T. The purpose of this study was to evaluate breath-hold diffusion-induced black-blood echo-planar imaging (BBEPI) as a potential alternative for specific absorption rate (SAR)-intensive spin-echo sequences, in particular, the fast spin-echo (FSE) sequences, at 3.0 T. Fourteen healthy volunteers (seven men, seven women; mean age +/- standard deviation, 32.7 years +/- 6.8) were imaged for this purpose. Liver coverage (20 cm, z-axis) was always performed in one 25-second breath hold. Imaging parameters were varied interactively with regard to echo time, diffusion b value, and voxel size. Images were evaluated and compared with fat-suppressed T2-weighted FSE images for image quality, liver delineation, geometric distortions, fat suppression, suppression of the blood signal, contrast-to-noise ratio (CNR), and signal-to-noise ratio (SNR). An optimized short- (25 msec) and long-echo (80 msec) BBEPI provided full anatomic, single breath-hold liver coverage (100 and 50 sections, respectively), with resulting voxel sizes of 3.3 x 2.7 x 2.0 mm and 3.3 x 2.7 x 4.0 mm, respectively. Repetition time was 6300 msec, matrix size was 160 x 192, and an acceleration factor of 2.00 was used. b Values of more than 20 sec/mm(2) showed better suppression of the blood signal but b values of 10 sec/mm(2) provided improved volume coverage and signal consistency. Compared with fat-suppressed T2-weighted FSE, the optimized BBEPI sequence provided (a) comparable image quality and liver delineation, (b) acceptable geometric distortions, (c) improved suppression of fat and blood signals, and (d) high CNR and SNR. BBEPI is feasible for fast, low-SAR, thin-section morphologic imaging of the entire liver in a single breath hold at 3.0 T. (c) RSNA, 2008.

Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing.

PubMed

Zhang, Jin; Ruhlman, Tracey A; Mower, Jeffrey P; Jansen, Robert K

2013-12-29

Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling RNA from four tissue types rather than from a single tissue. Finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants.

Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing

PubMed Central

2013-01-01

Background Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Results Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. Conclusions The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling RNA from four tissue types rather than from a single tissue. Finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants. PMID:24373163

Genetic Diversity of Bacterial Communities and Gene Transfer Agents in Northern South China Sea

PubMed Central

Sun, Fu-Lin; Wang, You-Shao; Wu, Mei-Lin; Jiang, Zhao-Yu; Sun, Cui-Ci; Cheng, Hao

2014-01-01

Pyrosequencing of the 16S ribosomal RNA gene (rDNA) amplicons was performed to investigate the unique distribution of bacterial communities in northern South China Sea (nSCS) and evaluate community structure and spatial differences of bacterial diversity. Cyanobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes constitute the majority of bacteria. The taxonomic description of bacterial communities revealed that more Chroococcales, SAR11 clade, Acidimicrobiales, Rhodobacterales, and Flavobacteriales are present in the nSCS waters than other bacterial groups. Rhodobacterales were less abundant in tropical water (nSCS) than in temperate and cold waters. Furthermore, the diversity of Rhodobacterales based on the gene transfer agent (GTA) major capsid gene (g5) was investigated. Four g5 gene clone libraries were constructed from samples representing different regions and yielded diverse sequences. Fourteen g5 clusters could be identified among 197 nSCS clones. These clusters were also related to known g5 sequences derived from genome-sequenced Rhodobacterales. The composition of g5 sequences in surface water varied with the g5 sequences in the sampling sites; this result indicated that the Rhodobacterales population could be highly diverse in nSCS. Phylogenetic tree analysis result indicated distinguishable diversity patterns among tropical (nSCS), temperate, and cold waters, thereby supporting the niche adaptation of specific Rhodobacterales members in unique environments. PMID:25364820

Phylogenetic diversity and biogeography of the Mamiellophyceae lineage of eukaryotic phytoplankton across the oceans.

PubMed

Monier, Adam; Worden, Alexandra Z; Richards, Thomas A

2016-08-01

High-throughput diversity amplicon sequencing of marine microbial samples has revealed that members of the Mamiellophyceae lineage are successful phytoplankton in many oceanic habitats. Indeed, these eukaryotic green algae can dominate the picoplanktonic biomass, however, given the broad expanses of the oceans, their geographical distributions and the phylogenetic diversity of some groups remain poorly characterized. As these algae play a foundational role in marine food webs, it is crucial to assess their global distribution in order to better predict potential changes in abundance and community structure. To this end, we analyzed the V9-18S small subunit rDNA sequences deposited from the Tara Oceans expedition to evaluate the diversity and biogeography of these phytoplankton. Our results show that the phylogenetic composition of Mamiellophyceae communities is in part determined by geographical provenance, and do not appear to be influenced - in the samples recovered - by water depth, at least at the resolution possible with the V9-18S. Phylogenetic classification of Mamiellophyceae sequences revealed that the Dolichomastigales order encompasses more sequence diversity than other orders in this lineage. These results indicate that a large fraction of the Mamiellophyceae diversity has been hitherto overlooked, likely because of a combination of size fraction, sequencing and geographical limitations. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

The positive relationships between plant coverage, species richness, and aboveground biomass are ubiquitous across plant growth forms in semi-steppe rangelands.

PubMed

Sanaei, Anvar; Ali, Arshad; Chahouki, Mohammad Ali Zare

2018-01-01

The positive relationships between biodiversity and aboveground biomass are important for biodiversity conservation and greater ecosystem functioning and services that humans depend on. However, the interaction effects of plant coverage and biodiversity on aboveground biomass across plant growth forms (shrubs, forbs and grasses) in natural rangelands are poorly studied. Here, we hypothesized that, while accounting for environmental factors and disturbance intensities, the positive relationships between plant coverage, biodiversity, and aboveground biomass are ubiquitous across plant growth forms in natural rangelands. We applied structural equation models (SEMs) using data from 735 quadrats across 35 study sites in semi-steppe rangelands in Iran. The combination of plant coverage and species richness rather than Shannon's diversity or species diversity (a latent variable of species richness and evenness) substantially enhance aboveground biomass across plant growth forms. In all selected SEMs, plant coverage had a strong positive direct effect on aboveground biomass (β = 0.72 for shrubs, 0.84 for forbs and 0.80 for grasses), followed by a positive effect of species richness (β = 0.26 for shrubs, 0.05 for forbs and 0.09 for grasses), and topographic factors. Disturbance intensity had a negative effect on plant coverage, whereas it had a variable effect on species richness across plant growth forms. Plant coverage had a strong positive total effect on aboveground biomass (β = 0.84 for shrubs, 0.88 for forbs, and 0.85 for grasses), followed by a positive effect of species richness, and a negative effect of disturbance intensity across plant growth forms. Our results shed light on the management of rangelands that is high plant coverage can significantly improve species richness and aboveground biomass across plant growth forms. We also found that high disturbance intensity due to heavy grazing has a strong negative effect on plant coverage rather than species richness in semi-steppe rangelands. This study suggests that proper grazing systems (e.g. rotational system) based on carrying capacity and stocking rate of a rangeland may be helpful for biodiversity conservation, better grazing of livestock, improvement of plant coverage and enhancement of aboveground biomass. Copyright © 2017 Elsevier Ltd. All rights reserved.

Urbanization reduces and homogenizes trait diversity in stream macroinvertebrate communities.

PubMed

Barnum, Thomas R; Weller, Donald E; Williams, Meghan

2017-12-01

More than one-half of the world's population lives in urban areas, so quantifying the effects of urbanization on ecological communities is important for understanding whether anthropogenic stressors homogenize communities across environmental and climatic gradients. We examined the relationship of impervious surface coverage (a marker of urbanization) and the structure of stream macroinvertebrate communities across the state of Maryland and within each of Maryland's three ecoregions: Coastal Plain, Piedmont, and Appalachian, which differ in stream geomorphology and community composition. We considered three levels of trait organization: individual traits, unique combinations of traits, and community metrics (functional richness, functional evenness, and functional divergence) and three levels of impervious surface coverage (low [<2.5%], medium [2.5% to 10%], and high [>10%]). The prevalence of an individual trait differed very little between low impervious surface and high impervious surface sites. The arrangement of trait combinations in community trait space for each ecoregion differed when impervious surface coverage was low, but the arrangement became more similar among ecoregions as impervious surface coverage increased. Furthermore, trait combinations that occurred only at low or medium impervious surface coverage were clustered in a subset of the community trait space, indicating that impervious surface affected the presence of only a subset of trait combinations. Functional richness declined with increasing impervious surface, providing evidence for environmental filtering. Community metrics that include abundance were also sensitive to increasing impervious surface coverage: functional divergence decreased while functional evenness increased. These changes demonstrate that increasing impervious surface coverage homogenizes the trait diversity of macroinvertebrate communities in streams, despite differences in initial community composition and stream geomorphology among ecoregions. Community metrics were also more sensitive to changes in the abundance rather than the gain or loss of trait combinations, showing the potential for trait-based approaches to serve as early warning indicators of environmental stress for monitoring and biological assessment programs. © 2017 by the Ecological Society of America.

New Insight Into the Diversity of SemiSWEET Sugar Transporters and the Homologs in Prokaryotes

PubMed Central

Jia, Baolei; Hao, Lujiang; Xuan, Yuan Hu; Jeon, Che Ok

2018-01-01

Sugars will eventually be exported transporters (SWEETs) and SemiSWEETs represent a family of sugar transporters in eukaryotes and prokaryotes, respectively. SWEETs contain seven transmembrane helices (TMHs), while SemiSWEETs contain three. The functions of SemiSWEETs are less studied. In this perspective article, we analyzed the diversity and conservation of SemiSWEETs and further proposed the possible functions. 1,922 SemiSWEET homologs were retrieved from the UniProt database, which is not proportional to the sequenced prokaryotic genomes. However, these proteins are very diverse in sequences and can be classified into 19 clusters when >50% sequence identity is required. Moreover, a gene context analysis indicated that several SemiSWEETs are located in the operons that are related to diverse carbohydrate metabolism. Several proteins with seven TMHs can be found in bacteria, and sequence alignment suggested that these proteins in bacteria may be formed by the duplication and fusion. Multiple sequence alignments showed that the amino acids for sugar translocation are still conserved and coevolved, although the sequences show diversity. Among them, the functions of a few amino acids are still not clear. These findings highlight the challenges that exist in SemiSWEETs and provide future researchers the foundation to explore these uncharted areas. PMID:29872447