From Conventional to Next Generation Sequencing of Epstein-Barr Virus Genomes.

PubMed

Kwok, Hin; Chiang, Alan Kwok Shing

2016-02-24

Genomic sequences of Epstein-Barr virus (EBV) have been of interest because the virus is associated with cancers, such as nasopharyngeal carcinoma, and conditions such as infectious mononucleosis. The progress of whole-genome EBV sequencing has been limited by the inefficiency and cost of the first-generation sequencing technology. With the advancement of next-generation sequencing (NGS) and target enrichment strategies, increasing number of EBV genomes has been published. These genomes were sequenced using different approaches, either with or without EBV DNA enrichment. This review provides an overview of the EBV genomes published to date, and a description of the sequencing technology and bioinformatic analyses employed in generating these sequences. We further explored ways through which the quality of sequencing data can be improved, such as using DNA oligos for capture hybridization, and longer insert size and read length in the sequencing runs. These advances will enable large-scale genomic sequencing of EBV which will facilitate a better understanding of the genetic variations of EBV in different geographic regions and discovery of potentially pathogenic variants in specific diseases.

Research progress of plant population genomics based on high-throughput sequencing.

PubMed

Wang, Yun-sheng

2016-08-01

Population genomics, a new paradigm for population genetics, combine the concepts and techniques of genomics with the theoretical system of population genetics and improve our understanding of microevolution through identification of site-specific effect and genome-wide effects using genome-wide polymorphic sites genotypeing. With the appearance and improvement of the next generation high-throughput sequencing technology, the numbers of plant species with complete genome sequences increased rapidly and large scale resequencing has also been carried out in recent years. Parallel sequencing has also been done in some plant species without complete genome sequences. These studies have greatly promoted the development of population genomics and deepened our understanding of the genetic diversity, level of linking disequilibium, selection effect, demographical history and molecular mechanism of complex traits of relevant plant population at a genomic level. In this review, I briely introduced the concept and research methods of population genomics and summarized the research progress of plant population genomics based on high-throughput sequencing. I also discussed the prospect as well as existing problems of plant population genomics in order to provide references for related studies.

Genomic Diversity and Evolution of the Lyssaviruses

PubMed Central

Delmas, Olivier; Holmes, Edward C.; Talbi, Chiraz; Larrous, Florence; Dacheux, Laurent; Bouchier, Christiane; Bourhy, Hervé

2008-01-01

Lyssaviruses are RNA viruses with single-strand, negative-sense genomes responsible for rabies-like diseases in mammals. To date, genomic and evolutionary studies have most often utilized partial genome sequences, particularly of the nucleoprotein and glycoprotein genes, with little consideration of genome-scale evolution. Herein, we report the first genomic and evolutionary analysis using complete genome sequences of all recognised lyssavirus genotypes, including 14 new complete genomes of field isolates from 6 genotypes and one genotype that is completely sequenced for the first time. In doing so we significantly increase the extent of genome sequence data available for these important viruses. Our analysis of these genome sequence data reveals that all lyssaviruses have the same genomic organization. A phylogenetic analysis reveals strong geographical structuring, with the greatest genetic diversity in Africa, and an independent origin for the two known genotypes that infect European bats. We also suggest that multiple genotypes may exist within the diversity of viruses currently classified as ‘Lagos Bat’. In sum, we show that rigorous phylogenetic techniques based on full length genome sequence provide the best discriminatory power for genotype classification within the lyssaviruses. PMID:18446239

A novel bioinformatics method for efficient knowledge discovery by BLSOM from big genomic sequence data.

PubMed

Bai, Yu; Iwasaki, Yuki; Kanaya, Shigehiko; Zhao, Yue; Ikemura, Toshimichi

2014-01-01

With remarkable increase of genomic sequence data of a wide range of species, novel tools are needed for comprehensive analyses of the big sequence data. Self-Organizing Map (SOM) is an effective tool for clustering and visualizing high-dimensional data such as oligonucleotide composition on one map. By modifying the conventional SOM, we have previously developed Batch-Learning SOM (BLSOM), which allows classification of sequence fragments according to species, solely depending on the oligonucleotide composition. In the present study, we introduce the oligonucleotide BLSOM used for characterization of vertebrate genome sequences. We first analyzed pentanucleotide compositions in 100 kb sequences derived from a wide range of vertebrate genomes and then the compositions in the human and mouse genomes in order to investigate an efficient method for detecting differences between the closely related genomes. BLSOM can recognize the species-specific key combination of oligonucleotide frequencies in each genome, which is called a "genome signature," and the specific regions specifically enriched in transcription-factor-binding sequences. Because the classification and visualization power is very high, BLSOM is an efficient powerful tool for extracting a wide range of information from massive amounts of genomic sequences (i.e., big sequence data).

MIPS: a database for genomes and protein sequences.

PubMed Central

Mewes, H W; Heumann, K; Kaps, A; Mayer, K; Pfeiffer, F; Stocker, S; Frishman, D

1999-01-01

The Munich Information Center for Protein Sequences (MIPS-GSF), Martinsried near Munich, Germany, develops and maintains genome oriented databases. It is commonplace that the amount of sequence data available increases rapidly, but not the capacity of qualified manual annotation at the sequence databases. Therefore, our strategy aims to cope with the data stream by the comprehensive application of analysis tools to sequences of complete genomes, the systematic classification of protein sequences and the active support of sequence analysis and functional genomics projects. This report describes the systematic and up-to-date analysis of genomes (PEDANT), a comprehensive database of the yeast genome (MYGD), a database reflecting the progress in sequencing the Arabidopsis thaliana genome (MATD), the database of assembled, annotated human EST clusters (MEST), and the collection of protein sequence data within the framework of the PIR-International Protein Sequence Database (described elsewhere in this volume). MIPS provides access through its WWW server (http://www.mips.biochem.mpg.de) to a spectrum of generic databases, including the above mentioned as well as a database of protein families (PROTFAM), the MITOP database, and the all-against-all FASTA database. PMID:9847138

Sockeye: A 3D Environment for Comparative Genomics

PubMed Central

Montgomery, Stephen B.; Astakhova, Tamara; Bilenky, Mikhail; Birney, Ewan; Fu, Tony; Hassel, Maik; Melsopp, Craig; Rak, Marcin; Robertson, A. Gordon; Sleumer, Monica; Siddiqui, Asim S.; Jones, Steven J.M.

2004-01-01

Comparative genomics techniques are used in bioinformatics analyses to identify the structural and functional properties of DNA sequences. As the amount of available sequence data steadily increases, the ability to perform large-scale comparative analyses has become increasingly relevant. In addition, the growing complexity of genomic feature annotation means that new approaches to genomic visualization need to be explored. We have developed a Java-based application called Sockeye that uses three-dimensional (3D) graphics technology to facilitate the visualization of annotation and conservation across multiple sequences. This software uses the Ensembl database project to import sequence and annotation information from several eukaryotic species. A user can additionally import their own custom sequence and annotation data. Individual annotation objects are displayed in Sockeye by using custom 3D models. Ensembl-derived and imported sequences can be analyzed by using a suite of multiple and pair-wise alignment algorithms. The results of these comparative analyses are also displayed in the 3D environment of Sockeye. By using the Java3D API to visualize genomic data in a 3D environment, we are able to compactly display cross-sequence comparisons. This provides the user with a novel platform for visualizing and comparing genomic feature organization. PMID:15123592

Genome sequences of Phytophthora enable translational plant disease management and accelerate research

Treesearch

Niklaus J. Grünwald

2012-01-01

Whole and partial genome sequences are becoming available at an ever-increasing pace. For many plant pathogen systems, we are moving into the era of genome resequencing. The first Phytophthora genomes, P. ramorum and P. sojae, became available in 2004, followed shortly by P. infestans...

Detection of genomic rearrangements in cucumber using genomecmp software

NASA Astrophysics Data System (ADS)

Kulawik, Maciej; Pawełkowicz, Magdalena Ewa; Wojcieszek, Michał; PlÄ der, Wojciech; Nowak, Robert M.

2017-08-01

Comparative genomic by increasing information about the genomes sequences available in the databases is a rapidly evolving science. A simple comparison of the general features of genomes such as genome size, number of genes, and chromosome number presents an entry point into comparative genomic analysis. Here we present the utility of the new tool genomecmp for finding rearrangements across the compared sequences and applications in plant comparative genomics.

The rapid evolution of molecular genetic diagnostics in neuromuscular diseases.

PubMed

Volk, Alexander E; Kubisch, Christian

2017-10-01

The development of massively parallel sequencing (MPS) has revolutionized molecular genetic diagnostics in monogenic disorders. The present review gives a brief overview of different MPS-based approaches used in clinical diagnostics of neuromuscular disorders (NMDs) and highlights their advantages and limitations. MPS-based approaches like gene panel sequencing, (whole) exome sequencing, (whole) genome sequencing, and RNA sequencing have been used to identify the genetic cause in NMDs. Although gene panel sequencing has evolved as a standard test for heterogeneous diseases, it is still debated, mainly because of financial issues and unsolved problems of variant interpretation, whether genome sequencing (and to a lesser extent also exome sequencing) of single patients can already be regarded as routine diagnostics. However, it has been shown that the inclusion of parents and additional family members often leads to a substantial increase in the diagnostic yield in exome-wide/genome-wide MPS approaches. In addition, MPS-based RNA sequencing just enters the research and diagnostic scene. Next-generation sequencing increasingly enables the detection of the genetic cause in highly heterogeneous diseases like NMDs in an efficient and affordable way. Gene panel sequencing and family-based exome sequencing have been proven as potent and cost-efficient diagnostic tools. Although clinical validation and interpretation of genome sequencing is still challenging, diagnostic RNA sequencing represents a promising tool to bypass some hurdles of diagnostics using genomic DNA.

Genome assembly reborn: recent computational challenges

PubMed Central

2009-01-01

Research into genome assembly algorithms has experienced a resurgence due to new challenges created by the development of next generation sequencing technologies. Several genome assemblers have been published in recent years specifically targeted at the new sequence data; however, the ever-changing technological landscape leads to the need for continued research. In addition, the low cost of next generation sequencing data has led to an increased use of sequencing in new settings. For example, the new field of metagenomics relies on large-scale sequencing of entire microbial communities instead of isolate genomes, leading to new computational challenges. In this article, we outline the major algorithmic approaches for genome assembly and describe recent developments in this domain. PMID:19482960

High quality de novo sequencing and assembly of the Saccharomyces arboricolus genome

PubMed Central

2013-01-01

Background Comparative genomics is a formidable tool to identify functional elements throughout a genome. In the past ten years, studies in the budding yeast Saccharomyces cerevisiae and a set of closely related species have been instrumental in showing the benefit of analyzing patterns of sequence conservation. Increasing the number of closely related genome sequences makes the comparative genomics approach more powerful and accurate. Results Here, we report the genome sequence and analysis of Saccharomyces arboricolus, a yeast species recently isolated in China, that is closely related to S. cerevisiae. We obtained high quality de novo sequence and assemblies using a combination of next generation sequencing technologies, established the phylogenetic position of this species and considered its phenotypic profile under multiple environmental conditions in the light of its gene content and phylogeny. Conclusions We suggest that the genome of S. arboricolus will be useful in future comparative genomics analysis of the Saccharomyces sensu stricto yeasts. PMID:23368932

in silico Whole Genome Sequencer & Analyzer (iWGS): A Computational Pipeline to Guide the Design and Analysis of de novo Genome Sequencing Studies

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

Zhou, Xiaofan; Peris, David; Kominek, Jacek

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in nonmodel organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimentalmore » design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects, and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.« less

in silico Whole Genome Sequencer & Analyzer (iWGS): A Computational Pipeline to Guide the Design and Analysis of de novo Genome Sequencing Studies

DOE PAGES

Zhou, Xiaofan; Peris, David; Kominek, Jacek; ...

2016-09-16

The availability of genomes across the tree of life is highly biased toward vertebrates, pathogens, human disease models, and organisms with relatively small and simple genomes. Recent progress in genomics has enabled the de novo decoding of the genome of virtually any organism, greatly expanding its potential for understanding the biology and evolution of the full spectrum of biodiversity. The increasing diversity of sequencing technologies, assays, and de novo assembly algorithms have augmented the complexity of de novo genome sequencing projects in nonmodel organisms. To reduce the costs and challenges in de novo genome sequencing projects and streamline their experimentalmore » design and analysis, we developed iWGS (in silico Whole Genome Sequencer and Analyzer), an automated pipeline for guiding the choice of appropriate sequencing strategy and assembly protocols. iWGS seamlessly integrates the four key steps of a de novo genome sequencing project: data generation (through simulation), data quality control, de novo assembly, and assembly evaluation and validation. The last three steps can also be applied to the analysis of real data. iWGS is designed to enable the user to have great flexibility in testing the range of experimental designs available for genome sequencing projects, and supports all major sequencing technologies and popular assembly tools. Three case studies illustrate how iWGS can guide the design of de novo genome sequencing projects, and evaluate the performance of a wide variety of user-specified sequencing strategies and assembly protocols on genomes of differing architectures. iWGS, along with a detailed documentation, is freely available at https://github.com/zhouxiaofan1983/iWGS.« less

Rapid and accurate pyrosequencing of angiosperm plastid genomes

PubMed Central

Moore, Michael J; Dhingra, Amit; Soltis, Pamela S; Shaw, Regina; Farmerie, William G; Folta, Kevin M; Soltis, Douglas E

2006-01-01

Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20) System (454 Life Sciences Corporation), to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae). Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy observed in the GS 20 plastid genome sequence was generated for a significant reduction in time and cost over traditional shotgun-based genome sequencing techniques, although with approximately half the coverage of previously reported GS 20 de novo genome sequence. The GS 20 should be broadly applicable to angiosperm plastid genome sequencing, and therefore promises to expand the scale of plant genetic and phylogenetic research dramatically. PMID:16934154

The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies.

PubMed

Yoshida, Catherine E; Kruczkiewicz, Peter; Laing, Chad R; Lingohr, Erika J; Gannon, Victor P J; Nash, John H E; Taboada, Eduardo N

2016-01-01

For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-genome sequencing (WGS) promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR), a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence-based methods of sub-typing allows for continuity with historical serotyping data as we transition towards the increasing adoption of genomic analyses in epidemiology. The SISTR platform is freely available on the web at https://lfz.corefacility.ca/sistr-app/.

Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies.

PubMed

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.

Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies

USGS Publications Warehouse

Card, Daren C.; Schield, Drew R.; Reyes-Velasco, Jacobo; Fujita, Matthre 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.

Swine transcriptome characterization by combined Iso-Seq and RNA-seq for annotating the emerging long read-based reference genome

USDA-ARS?s Scientific Manuscript database

PacBio long-read sequencing technology is increasingly popular in genome sequence assembly and transcriptome cataloguing. Recently, a new-generation pig reference genome was assembled based on long reads from this technology. To finely annotate this genome assembly, transcriptomes of nine tissues fr...

Novel Insights into Tree Biology and Genome Evolution as Revealed Through Genomics.

PubMed

Neale, David B; Martínez-García, Pedro J; De La Torre, Amanda R; Montanari, Sara; Wei, Xiao-Xin

2017-04-28

Reference genome sequences are the key to the discovery of genes and gene families that determine traits of interest. Recent progress in sequencing technologies has enabled a rapid increase in genome sequencing of tree species, allowing the dissection of complex characters of economic importance, such as fruit and wood quality and resistance to biotic and abiotic stresses. Although the number of reference genome sequences for trees lags behind those for other plant species, it is not too early to gain insight into the unique features that distinguish trees from nontree plants. Our review of the published data suggests that, although many gene families are conserved among herbaceous and tree species, some gene families, such as those involved in resistance to biotic and abiotic stresses and in the synthesis and transport of sugars, are often expanded in tree genomes. As the genomes of more tree species are sequenced, comparative genomics will further elucidate the complexity of tree genomes and how this relates to traits unique to trees.

The 'dark matter' in the plant genomes: non-coding and unannotated DNA sequences associated with open chromatin.

PubMed

Jiang, Jiming

2015-04-01

Sequencing of complete plant genomes has become increasingly more routine since the advent of the next-generation sequencing technology. Identification and annotation of large amounts of noncoding but functional DNA sequences, including cis-regulatory DNA elements (CREs), have become a new frontier in plant genome research. Genomic regions containing active CREs bound to regulatory proteins are hypersensitive to DNase I digestion and are called DNase I hypersensitive sites (DHSs). Several recent DHS studies in plants illustrate that DHS datasets produced by DNase I digestion followed by next-generation sequencing (DNase-seq) are highly valuable for the identification and characterization of CREs associated with plant development and responses to environmental cues. DHS-based genomic profiling has opened a door to identify and annotate the 'dark matter' in sequenced plant genomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sequencing and comparing whole mitochondrial genomes ofanimals

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

Boore, Jeffrey L.; Macey, J. Robert; Medina, Monica

2005-04-22

Comparing complete animal mitochondrial genome sequences is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. Not only are they much more informative than shorter sequences of individual genes for inferring evolutionary relatedness, but these data also provide sets of genome-level characters, such as the relative arrangements of genes, that can be especially powerful. We describe here the protocols commonly used for physically isolating mtDNA, for amplifying these by PCR or RCA, for cloning,sequencing, assembly, validation, and gene annotation, and for comparing both sequences and gene arrangements. On several topics, we offer general observations based onmore » our experiences to date with determining and comparing complete mtDNA sequences.« less

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.

Illustrative case studies in the return of exome and genome sequencing results

PubMed Central

Amendola, Laura M; Lautenbach, Denise; Scollon, Sarah; Bernhardt, Barbara; Biswas, Sawona; East, Kelly; Everett, Jessica; Gilmore, Marian J; Himes, Patricia; Raymond, Victoria M; Wynn, Julia; Hart, Ragan; Jarvik, Gail P

2015-01-01

Whole genome and exome sequencing tests are increasingly being ordered in clinical practice, creating a need for research exploring the return of results from these tests. A goal of the Clinical Sequencing and Exploratory Research (CSER) consortium is to gain experience with this process to develop best practice recommendations for offering exome and genome testing and returning results. Genetic counselors in the CSER consortium have an integral role in the return of results from these genomic sequencing tests and have gained valuable insight. We present seven emerging themes related to return of exome and genome sequencing results accompanied by case descriptions illustrating important lessons learned, counseling challenges specific to these tests and considerations for future research and practice. PMID:26478737

Goodbye genome paper, hello genome report: the increasing popularity of 'genome announcements' and their impact on science.

PubMed

Smith, David Roy

2017-05-01

Next-generation sequencing technologies have revolutionized genomics and altered the scientific publication landscape. Life-science journals abound with genome papers-peer-reviewed descriptions of newly sequenced chromosomes. Although they once filled the pages of Nature and Science, genome papers are now mostly relegated to journals with low-impact factors. Some have forecast the death of the genome paper and argued that they are using up valuable resources and not advancing science. However, the publication rate of genome papers is on the rise. This increase is largely because some journals have created a new category of manuscript called genome reports, which are short, fast-tracked papers describing a chromosome sequence(s), its GenBank accession number and little else. In 2015, for example, more than 2000 genome reports were published, and 2016 is poised to bring even more. Here, I highlight the growing popularity of genome reports and discuss their merits, drawbacks and impact on science and the academic publication infrastructure. Genome reports can be excellent assets for the research community, but they are also being used as quick and easy routes to a publication, and in some instances they are not peer reviewed. One of the best arguments for genome reports is that they are a citable, user-generated genomic resource providing essential methodological and biological information, which may not be present in the sequence database. But they are expensive and time-consuming avenues for achieving such a goal. © The Author 2016. Published by Oxford University Press.

The Saccharomyces Genome Database Variant Viewer

PubMed Central

Sheppard, Travis K.; Hitz, Benjamin C.; Engel, Stacia R.; Song, Giltae; Balakrishnan, Rama; Binkley, Gail; Costanzo, Maria C.; Dalusag, Kyla S.; Demeter, Janos; Hellerstedt, Sage T.; Karra, Kalpana; Nash, Robert S.; Paskov, Kelley M.; Skrzypek, Marek S.; Weng, Shuai; Wong, Edith D.; Cherry, J. Michael

2016-01-01

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is the authoritative community resource for the Saccharomyces cerevisiae reference genome sequence and its annotation. In recent years, we have moved toward increased representation of sequence variation and allelic differences within S. cerevisiae. The publication of numerous additional genomes has motivated the creation of new tools for their annotation and analysis. Here we present the Variant Viewer: a dynamic open-source web application for the visualization of genomic and proteomic differences. Multiple sequence alignments have been constructed across high quality genome sequences from 11 different S. cerevisiae strains and stored in the SGD. The alignments and summaries are encoded in JSON and used to create a two-tiered dynamic view of the budding yeast pan-genome, available at http://www.yeastgenome.org/variant-viewer. PMID:26578556

Harnessing Whole Genome Sequencing in Medical Mycology.

PubMed

Cuomo, Christina A

2017-01-01

Comparative genome sequencing studies of human fungal pathogens enable identification of genes and variants associated with virulence and drug resistance. This review describes current approaches, resources, and advances in applying whole genome sequencing to study clinically important fungal pathogens. Genomes for some important fungal pathogens were only recently assembled, revealing gene family expansions in many species and extreme gene loss in one obligate species. The scale and scope of species sequenced is rapidly expanding, leveraging technological advances to assemble and annotate genomes with higher precision. By using iteratively improved reference assemblies or those generated de novo for new species, recent studies have compared the sequence of isolates representing populations or clinical cohorts. Whole genome approaches provide the resolution necessary for comparison of closely related isolates, for example, in the analysis of outbreaks or sampled across time within a single host. Genomic analysis of fungal pathogens has enabled both basic research and diagnostic studies. The increased scale of sequencing can be applied across populations, and new metagenomic methods allow direct analysis of complex samples.

KGCAK: a K-mer based database for genome-wide phylogeny and complexity evaluation.

PubMed

Wang, Dapeng; Xu, Jiayue; Yu, Jun

2015-09-16

The K-mer approach, treating genomic sequences as simple characters and counting the relative abundance of each string upon a fixed K, has been extensively applied to phylogeny inference for genome assembly, annotation, and comparison. To meet increasing demands for comparing large genome sequences and to promote the use of the K-mer approach, we develop a versatile database, KGCAK ( http://kgcak.big.ac.cn/KGCAK/ ), containing ~8,000 genomes that include genome sequences of diverse life forms (viruses, prokaryotes, protists, animals, and plants) and cellular organelles of eukaryotic lineages. It builds phylogeny based on genomic elements in an alignment-free fashion and provides in-depth data processing enabling users to compare the complexity of genome sequences based on K-mer distribution. We hope that KGCAK becomes a powerful tool for exploring relationship within and among groups of species in a tree of life based on genomic data.

Complete genome sequence of Flavobacterium psychrophilum strain CSF259-93 used to select rainbow trout for increased genetic resistance against bacterial cold water disease

USDA-ARS?s Scientific Manuscript database

The genome sequence of Flavobacterium psychrophilum strain CSF259-93, isolated from rainbow trout (Oncorhynchus mykiss), consists of a single circular genome of 2,900,735 bp and 2,701 predicted open reading frames (ORFs). Strain CSF259-93 has been used to select a line of rainbow trout with increase...

Single molecule sequencing of the M13 virus genome without amplification

PubMed Central

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X.; Yan, Qin; Deem, Michael W.; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias. PMID:29253901

Single molecule sequencing of the M13 virus genome without amplification.

PubMed

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X; Yan, Qin; Deem, Michael W; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.

Use of sequence-independent-single-primer-amplification (SISPA) for whole genome sequencing using illumina MiSeq platform for avian influenza virus, Newcastle disease virus, and infectious bronchitis virus

USDA-ARS?s Scientific Manuscript database

Over the past decade, Next Generation Sequencing (NGS) technologies, also called deep sequencing, have continued to evolve, increasing capacity and lower the cost necessary for large genome sequencing projects. The one of the advantage of NGS platforms is the possibility to sequence the samples with...

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

PubMed Central

Nowrousian, Minou; Stajich, Jason E.; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D.; Pöggeler, Stefanie; Read, Nick D.; Seiler, Stephan; Smith, Kristina M.; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-01-01

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology. PMID:20386741

De novo assembly of a 40 Mb eukaryotic genome from short sequence reads: Sordaria macrospora, a model organism for fungal morphogenesis.

PubMed

Nowrousian, Minou; Stajich, Jason E; Chu, Meiling; Engh, Ines; Espagne, Eric; Halliday, Karen; Kamerewerd, Jens; Kempken, Frank; Knab, Birgit; Kuo, Hsiao-Che; Osiewacz, Heinz D; Pöggeler, Stefanie; Read, Nick D; Seiler, Stephan; Smith, Kristina M; Zickler, Denise; Kück, Ulrich; Freitag, Michael

2010-04-08

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

APPLaUD: access for patients and participants to individual level uninterpreted genomic data.

PubMed

Thorogood, Adrian; Bobe, Jason; Prainsack, Barbara; Middleton, Anna; Scott, Erick; Nelson, Sarah; Corpas, Manuel; Bonhomme, Natasha; Rodriguez, Laura Lyman; Murtagh, Madeleine; Kleiderman, Erika

2018-02-17

There is a growing support for the stance that patients and research participants should have better and easier access to their raw (uninterpreted) genomic sequence data in both clinical and research contexts. We review legal frameworks and literature on the benefits, risks, and practical barriers of providing individuals access to their data. We also survey genomic sequencing initiatives that provide or plan to provide individual access. Many patients and research participants expect to be able to access their health and genomic data. Individuals have a legal right to access their genomic data in some countries and contexts. Moreover, increasing numbers of participatory research projects, direct-to-consumer genetic testing companies, and now major national sequencing initiatives grant individuals access to their genomic sequence data upon request. Drawing on current practice and regulatory analysis, we outline legal, ethical, and practical guidance for genomic sequencing initiatives seeking to offer interested patients and participants access to their raw genomic data.

The origins and impact of primate segmental duplications.

PubMed

Marques-Bonet, Tomas; Girirajan, Santhosh; Eichler, Evan E

2009-10-01

Duplicated sequences are substrates for the emergence of new genes and are an important source of genetic instability associated with rare and common diseases. Analyses of primate genomes have shown an increase in the proportion of interspersed segmental duplications (SDs) within the genomes of humans and great apes. This contrasts with other mammalian genomes that seem to have their recently duplicated sequences organized in a tandem configuration. In this review, we focus on the mechanistic origin and impact of this difference with respect to evolution, genetic diversity and primate phenotype. Although many genomes will be sequenced in the future, resolution of this aspect of genomic architecture still requires high quality sequences and detailed analyses.

CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences

PubMed Central

2012-01-01

Background The complete sequences of chloroplast genomes provide wealthy information regarding the evolutionary history of species. With the advance of next-generation sequencing technology, the number of completely sequenced chloroplast genomes is expected to increase exponentially, powerful computational tools annotating the genome sequences are in urgent need. Results We have developed a web server CPGAVAS. The server accepts a complete chloroplast genome sequence as input. First, it predicts protein-coding and rRNA genes based on the identification and mapping of the most similar, full-length protein, cDNA and rRNA sequences by integrating results from Blastx, Blastn, protein2genome and est2genome programs. Second, tRNA genes and inverted repeats (IR) are identified using tRNAscan, ARAGORN and vmatch respectively. Third, it calculates the summary statistics for the annotated genome. Fourth, it generates a circular map ready for publication. Fifth, it can create a Sequin file for GenBank submission. Last, it allows the extractions of protein and mRNA sequences for given list of genes and species. The annotation results in GFF3 format can be edited using any compatible annotation editing tools. The edited annotations can then be uploaded to CPGAVAS for update and re-analyses repeatedly. Using known chloroplast genome sequences as test set, we show that CPGAVAS performs comparably to another application DOGMA, while having several superior functionalities. Conclusions CPGAVAS allows the semi-automatic and complete annotation of a chloroplast genome sequence, and the visualization, editing and analysis of the annotation results. It will become an indispensible tool for researchers studying chloroplast genomes. The software is freely accessible from http://www.herbalgenomics.org/cpgavas. PMID:23256920

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

Progress in Understanding and Sequencing the Genome of Brassica rapa

PubMed Central

Hong, Chang Pyo; Kwon, Soo-Jin; Kim, Jung Sun; Yang, Tae-Jin; Park, Beom-Seok; Lim, Yong Pyo

2008-01-01

Brassica rapa, which is closely related to Arabidopsis thaliana, is an important crop and a model plant for studying genome evolution via polyploidization. We report the current understanding of the genome structure of B. rapa and efforts for the whole-genome sequencing of the species. The tribe Brassicaceae, which comprises ca. 240 species, descended from a common hexaploid ancestor with a basic genome similar to that of Arabidopsis. Chromosome rearrangements, including fusions and/or fissions, resulted in the present-day “diploid” Brassica species with variation in chromosome number and phenotype. Triplicated genomic segments of B. rapa are collinear to those of A. thaliana with InDels. The genome triplication has led to an approximately 1.7-fold increase in the B. rapa gene number compared to that of A. thaliana. Repetitive DNA of B. rapa has also been extensively amplified and has diverged from that of A. thaliana. For its whole-genome sequencing, the Brassica rapa Genome Sequencing Project (BrGSP) consortium has developed suitable genomic resources and constructed genetic and physical maps. Ten chromosomes of B. rapa are being allocated to BrGSP consortium participants, and each chromosome will be sequenced by a BAC-by-BAC approach. Genome sequencing of B. rapa will offer a new perspective for plant biology and evolution in the context of polyploidization. PMID:18288250

Salmonella Typhi genomics: envisaging the future of typhoid eradication.

PubMed

Yap, Kien-Pong; Thong, Kwai Lin

2017-08-01

Next-generation whole-genome sequencing has revolutionised the study of infectious diseases in recent years. The availability of genome sequences and its understanding have transformed the field of molecular microbiology, epidemiology, infection treatments and vaccine developments. We review the key findings of the publicly accessible genomes of Salmonella enterica serovar Typhi since the first complete genome to the most recent release of thousands of Salmonella Typhi genomes, which remarkably shape the genomic research of S. Typhi and other pathogens. Important new insights acquired from the genome sequencing of S. Typhi, pertaining to genomic variations, evolution, population structure, antibiotic resistance, virulence, pathogenesis, disease surveillance/investigation and disease control are discussed. As the numbers of sequenced genomes are increasing at an unprecedented rate, fine variations in the gene pool of S. Typhi are captured in high resolution, allowing deeper understanding of the pathogen's evolutionary trends and its pathogenesis, paving the way to bringing us closer to eradication of typhoid through effective vaccine/treatment development. © 2017 John Wiley & Sons Ltd.

Design of multiple sequence alignment algorithms on parallel, distributed memory supercomputers.

PubMed

Church, Philip C; Goscinski, Andrzej; Holt, Kathryn; Inouye, Michael; Ghoting, Amol; Makarychev, Konstantin; Reumann, Matthias

2011-01-01

The challenge of comparing two or more genomes that have undergone recombination and substantial amounts of segmental loss and gain has recently been addressed for small numbers of genomes. However, datasets of hundreds of genomes are now common and their sizes will only increase in the future. Multiple sequence alignment of hundreds of genomes remains an intractable problem due to quadratic increases in compute time and memory footprint. To date, most alignment algorithms are designed for commodity clusters without parallelism. Hence, we propose the design of a multiple sequence alignment algorithm on massively parallel, distributed memory supercomputers to enable research into comparative genomics on large data sets. Following the methodology of the sequential progressiveMauve algorithm, we design data structures including sequences and sorted k-mer lists on the IBM Blue Gene/P supercomputer (BG/P). Preliminary results show that we can reduce the memory footprint so that we can potentially align over 250 bacterial genomes on a single BG/P compute node. We verify our results on a dataset of E.coli, Shigella and S.pneumoniae genomes. Our implementation returns results matching those of the original algorithm but in 1/2 the time and with 1/4 the memory footprint for scaffold building. In this study, we have laid the basis for multiple sequence alignment of large-scale datasets on a massively parallel, distributed memory supercomputer, thus enabling comparison of hundreds instead of a few genome sequences within reasonable time.

MBGD update 2015: microbial genome database for flexible ortholog analysis utilizing a diverse set of genomic data.

PubMed

Uchiyama, Ikuo; Mihara, Motohiro; Nishide, Hiroyo; Chiba, Hirokazu

2015-01-01

The microbial genome database for comparative analysis (MBGD) (available at http://mbgd.genome.ad.jp/) is a comprehensive ortholog database for flexible comparative analysis of microbial genomes, where the users are allowed to create an ortholog table among any specified set of organisms. Because of the rapid increase in microbial genome data owing to the next-generation sequencing technology, it becomes increasingly challenging to maintain high-quality orthology relationships while allowing the users to incorporate the latest genomic data available into an analysis. Because many of the recently accumulating genomic data are draft genome sequences for which some complete genome sequences of the same or closely related species are available, MBGD now stores draft genome data and allows the users to incorporate them into a user-specific ortholog database using the MyMBGD functionality. In this function, draft genome data are incorporated into an existing ortholog table created only from the complete genome data in an incremental manner to prevent low-quality draft data from affecting clustering results. In addition, to provide high-quality orthology relationships, the standard ortholog table containing all the representative genomes, which is first created by the rapid classification program DomClust, is now refined using DomRefine, a recently developed program for improving domain-level clustering using multiple sequence alignment information. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Expanding probe repertoire and improving reproducibility in human genomic hybridization

PubMed Central

Dorman, Stephanie N.; Shirley, Ben C.; Knoll, Joan H. M.; Rogan, Peter K.

2013-01-01

Diagnostic DNA hybridization relies on probes composed of single copy (sc) genomic sequences. Sc sequences in probe design ensure high specificity and avoid cross-hybridization to other regions of the genome, which could lead to ambiguous results that are difficult to interpret. We examine how the distribution and composition of repetitive sequences in the genome affects sc probe performance. A divide and conquer algorithm was implemented to design sc probes. With this approach, sc probes can include divergent repetitive elements, which hybridize to unique genomic targets under higher stringency experimental conditions. Genome-wide custom probe sets were created for fluorescent in situ hybridization (FISH) and microarray genomic hybridization. The scFISH probes were developed for detection of copy number changes within small tumour suppressor genes and oncogenes. The microarrays demonstrated increased reproducibility by eliminating cross-hybridization to repetitive sequences adjacent to probe targets. The genome-wide microarrays exhibited lower median coefficients of variation (17.8%) for two HapMap family trios. The coefficients of variations of commercial probes within 300 nt of a repetitive element were 48.3% higher than the nearest custom probe. Furthermore, the custom microarray called a chromosome 15q11.2q13 deletion more consistently. This method for sc probe design increases probe coverage for FISH and lowers variability in genomic microarrays. PMID:23376933

Real-time, portable genome sequencing for Ebola surveillance.

PubMed

Quick, Joshua; Loman, Nicholas J; Duraffour, Sophie; Simpson, Jared T; Severi, Ettore; Cowley, Lauren; Bore, Joseph Akoi; Koundouno, Raymond; Dudas, Gytis; Mikhail, Amy; Ouédraogo, Nobila; Afrough, Babak; Bah, Amadou; Baum, Jonathan Hj; Becker-Ziaja, Beate; Boettcher, Jan-Peter; Cabeza-Cabrerizo, Mar; Camino-Sanchez, Alvaro; Carter, Lisa L; Doerrbecker, Juiliane; Enkirch, Theresa; Dorival, Isabel Graciela García; Hetzelt, Nicole; Hinzmann, Julia; Holm, Tobias; Kafetzopoulou, Liana Eleni; Koropogui, Michel; Kosgey, Abigail; Kuisma, Eeva; Logue, Christopher H; Mazzarelli, Antonio; Meisel, Sarah; Mertens, Marc; Michel, Janine; Ngabo, Didier; Nitzsche, Katja; Pallash, Elisa; Patrono, Livia Victoria; Portmann, Jasmine; Repits, Johanna Gabriella; Rickett, Natasha Yasmin; Sachse, Andrea; Singethan, Katrin; Vitoriano, Inês; Yemanaberhan, Rahel L; Zekeng, Elsa G; Trina, Racine; Bello, Alexander; Sall, Amadou Alpha; Faye, Ousmane; Faye, Oumar; Magassouba, N'Faly; Williams, Cecelia V; Amburgey, Victoria; Winona, Linda; Davis, Emily; Gerlach, Jon; Washington, Franck; Monteil, Vanessa; Jourdain, Marine; Bererd, Marion; Camara, Alimou; Somlare, Hermann; Camara, Abdoulaye; Gerard, Marianne; Bado, Guillaume; Baillet, Bernard; Delaune, Déborah; Nebie, Koumpingnin Yacouba; Diarra, Abdoulaye; Savane, Yacouba; Pallawo, Raymond Bernard; Gutierrez, Giovanna Jaramillo; Milhano, Natacha; Roger, Isabelle; Williams, Christopher J; Yattara, Facinet; Lewandowski, Kuiama; Taylor, Jamie; Rachwal, Philip; Turner, Daniel; Pollakis, Georgios; Hiscox, Julian A; Matthews, David A; O'Shea, Matthew K; Johnston, Andrew McD; Wilson, Duncan; Hutley, Emma; Smit, Erasmus; Di Caro, Antonino; Woelfel, Roman; Stoecker, Kilian; Fleischmann, Erna; Gabriel, Martin; Weller, Simon A; Koivogui, Lamine; Diallo, Boubacar; Keita, Sakoba; Rambaut, Andrew; Formenty, Pierre; Gunther, Stephan; Carroll, Miles W

2016-02-11

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10(-3) and 1.42 × 10(-3) mutations per site per year. This is equivalent to 16-27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

Whole-genome sequencing and genetic variant analysis of a Quarter Horse mare.

PubMed

Doan, Ryan; Cohen, Noah D; Sawyer, Jason; Ghaffari, Noushin; Johnson, Charlie D; Dindot, Scott V

2012-02-17

The catalog of genetic variants in the horse genome originates from a few select animals, the majority originating from the Thoroughbred mare used for the equine genome sequencing project. The purpose of this study was to identify genetic variants, including single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (INDELs), and copy number variants (CNVs) in the genome of an individual Quarter Horse mare sequenced by next-generation sequencing. Using massively parallel paired-end sequencing, we generated 59.6 Gb of DNA sequence from a Quarter Horse mare resulting in an average of 24.7X sequence coverage. Reads were mapped to approximately 97% of the reference Thoroughbred genome. Unmapped reads were de novo assembled resulting in 19.1 Mb of new genomic sequence in the horse. Using a stringent filtering method, we identified 3.1 million SNPs, 193 thousand INDELs, and 282 CNVs. Genetic variants were annotated to determine their impact on gene structure and function. Additionally, we genotyped this Quarter Horse for mutations of known diseases and for variants associated with particular traits. Functional clustering analysis of genetic variants revealed that most of the genetic variation in the horse's genome was enriched in sensory perception, signal transduction, and immunity and defense pathways. This is the first sequencing of a horse genome by next-generation sequencing and the first genomic sequence of an individual Quarter Horse mare. We have increased the catalog of genetic variants for use in equine genomics by the addition of novel SNPs, INDELs, and CNVs. The genetic variants described here will be a useful resource for future studies of genetic variation regulating performance traits and diseases in equids.

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.

GI-SVM: A sensitive method for predicting genomic islands based on unannotated sequence of a single genome.

PubMed

Lu, Bingxin; Leong, Hon Wai

2016-02-01

Genomic islands (GIs) are clusters of functionally related genes acquired by lateral genetic transfer (LGT), and they are present in many bacterial genomes. GIs are extremely important for bacterial research, because they not only promote genome evolution but also contain genes that enhance adaption and enable antibiotic resistance. Many methods have been proposed to predict GI. But most of them rely on either annotations or comparisons with other closely related genomes. Hence these methods cannot be easily applied to new genomes. As the number of newly sequenced bacterial genomes rapidly increases, there is a need for methods to detect GI based solely on sequences of a single genome. In this paper, we propose a novel method, GI-SVM, to predict GIs given only the unannotated genome sequence. GI-SVM is based on one-class support vector machine (SVM), utilizing composition bias in terms of k-mer content. From our evaluations on three real genomes, GI-SVM can achieve higher recall compared with current methods, without much loss of precision. Besides, GI-SVM allows flexible parameter tuning to get optimal results for each genome. In short, GI-SVM provides a more sensitive method for researchers interested in a first-pass detection of GI in newly sequenced genomes.

Assessing Diversity of DNA Structure-Related Sequence Features in Prokaryotic Genomes

PubMed Central

Huang, Yongjie; Mrázek, Jan

2014-01-01

Prokaryotic genomes are diverse in terms of their nucleotide and oligonucleotide composition as well as presence of various sequence features that can affect physical properties of the DNA molecule. We present a survey of local sequence patterns which have a potential to promote non-canonical DNA conformations (i.e. different from standard B-DNA double helix) and interpret the results in terms of relationships with organisms' habitats, phylogenetic classifications, and other characteristics. Our present work differs from earlier similar surveys not only by investigating a wider range of sequence patterns in a large number of genomes but also by using a more realistic null model to assess significant deviations. Our results show that simple sequence repeats and Z-DNA-promoting patterns are generally suppressed in prokaryotic genomes, whereas palindromes and inverted repeats are over-represented. Representation of patterns that promote Z-DNA and intrinsic DNA curvature increases with increasing optimal growth temperature (OGT), and decreases with increasing oxygen requirement. Additionally, representations of close direct repeats, palindromes and inverted repeats exhibit clear negative trends with increasing OGT. The observed relationships with environmental characteristics, particularly OGT, suggest possible evolutionary scenarios of structural adaptation of DNA to particular environmental niches. PMID:24408877

IMGD: an integrated platform supporting comparative genomics and phylogenetics of insect mitochondrial genomes

PubMed Central

Lee, Wonhoon; Park, Jongsun; Choi, Jaeyoung; Jung, Kyongyong; Park, Bongsoo; Kim, Donghan; Lee, Jaeyoung; Ahn, Kyohun; Song, Wonho; Kang, Seogchan; Lee, Yong-Hwan; Lee, Seunghwan

2009-01-01

Background Sequences and organization of the mitochondrial genome have been used as markers to investigate evolutionary history and relationships in many taxonomic groups. The rapidly increasing mitochondrial genome sequences from diverse insects provide ample opportunities to explore various global evolutionary questions in the superclass Hexapoda. To adequately support such questions, it is imperative to establish an informatics platform that facilitates the retrieval and utilization of available mitochondrial genome sequence data. Results The Insect Mitochondrial Genome Database (IMGD) is a new integrated platform that archives the mitochondrial genome sequences from 25,747 hexapod species, including 112 completely sequenced and 20 nearly completed genomes and 113,985 partially sequenced mitochondrial genomes. The Species-driven User Interface (SUI) of IMGD supports data retrieval and diverse analyses at multi-taxon levels. The Phyloviewer implemented in IMGD provides three methods for drawing phylogenetic trees and displays the resulting trees on the web. The SNP database incorporated to IMGD presents the distribution of SNPs and INDELs in the mitochondrial genomes of multiple isolates within eight species. A newly developed comparative SNU Genome Browser supports the graphical presentation and interactive interface for the identified SNPs/INDELs. Conclusion The IMGD provides a solid foundation for the comparative mitochondrial genomics and phylogenetics of insects. All data and functions described here are available at the web site . PMID:19351385

Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes

Treesearch

Matthew Parks; Richard Cronn; Aaron Liston

2009-01-01

We reconstruct the infrageneric phylogeny of Pinus from 37 nearly-complete chloroplast genomes (average 109 kilobases each of an approximately 120 kilobase genome) generated using multiplexed massively parallel sequencing. We found that 30/33 ingroup nodes resolved wlth > 95-percent bootstrap support; this is a substantial improvement relative...

GenomePeek—an online tool for prokaryotic genome and metagenome analysis

DOE PAGES

McNair, Katelyn; Edwards, Robert A.

2015-06-16

As increases in prokaryotic sequencing take place, a method to quickly and accurately analyze this data is needed. Previous tools are mainly designed for metagenomic analysis and have limitations; such as long runtimes and significant false positive error rates. The online tool GenomePeek (edwards.sdsu.edu/GenomePeek) was developed to analyze both single genome and metagenome sequencing files, quickly and with low error rates. GenomePeek uses a sequence assembly approach where reads to a set of conserved genes are extracted, assembled and then aligned against the highly specific reference database. GenomePeek was found to be faster than traditional approaches while still keeping errormore » rates low, as well as offering unique data visualization options.« less

The Saccharomyces Genome Database Variant Viewer.

PubMed

Sheppard, Travis K; Hitz, Benjamin C; Engel, Stacia R; Song, Giltae; Balakrishnan, Rama; Binkley, Gail; Costanzo, Maria C; Dalusag, Kyla S; Demeter, Janos; Hellerstedt, Sage T; Karra, Kalpana; Nash, Robert S; Paskov, Kelley M; Skrzypek, Marek S; Weng, Shuai; Wong, Edith D; Cherry, J Michael

2016-01-04

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is the authoritative community resource for the Saccharomyces cerevisiae reference genome sequence and its annotation. In recent years, we have moved toward increased representation of sequence variation and allelic differences within S. cerevisiae. The publication of numerous additional genomes has motivated the creation of new tools for their annotation and analysis. Here we present the Variant Viewer: a dynamic open-source web application for the visualization of genomic and proteomic differences. Multiple sequence alignments have been constructed across high quality genome sequences from 11 different S. cerevisiae strains and stored in the SGD. The alignments and summaries are encoded in JSON and used to create a two-tiered dynamic view of the budding yeast pan-genome, available at http://www.yeastgenome.org/variant-viewer. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development of Mycoplasma synoviae (MS) core genome multilocus sequence typing (cgMLST) scheme.

PubMed

Ghanem, Mostafa; El-Gazzar, Mohamed

2018-05-01

Mycoplasma synoviae (MS) is a poultry pathogen with reported increased prevalence and virulence in recent years. MS strain identification is essential for prevention, control efforts and epidemiological outbreak investigations. Multiple multilocus based sequence typing schemes have been developed for MS, yet the resolution of these schemes could be limited for outbreak investigation. The cost of whole genome sequencing became close to that of sequencing the seven MLST targets; however, there is no standardized method for typing MS strains based on whole genome sequences. In this paper, we propose a core genome multilocus sequence typing (cgMLST) scheme as a standardized and reproducible method for typing MS based whole genome sequences. A diverse set of 25 MS whole genome sequences were used to identify 302 core genome genes as cgMLST targets (35.5% of MS genome) and 44 whole genome sequences of MS isolates from six countries in four continents were used for typing applying this scheme. cgMLST based phylogenetic trees displayed a high degree of agreement with core genome SNP based analysis and available epidemiological information. cgMLST allowed evaluation of two conventional MLST schemes of MS. The high discriminatory power of cgMLST allowed differentiation between samples of the same conventional MLST type. cgMLST represents a standardized, accurate, highly discriminatory, and reproducible method for differentiation between MS isolates. Like conventional MLST, it provides stable and expandable nomenclature, allowing for comparing and sharing the typing results between different laboratories worldwide. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Recurrence time statistics: versatile tools for genomic DNA sequence analysis.

PubMed

Cao, Yinhe; Tung, Wen-Wen; Gao, J B

2004-01-01

With the completion of the human and a few model organisms' genomes, and the genomes of many other organisms waiting to be sequenced, it has become increasingly important to develop faster computational tools which are capable of easily identifying the structures and extracting features from DNA sequences. One of the more important structures in a DNA sequence is repeat-related. Often they have to be masked before protein coding regions along a DNA sequence are to be identified or redundant expressed sequence tags (ESTs) are to be sequenced. Here we report a novel recurrence time based method for sequence analysis. The method can conveniently study all kinds of periodicity and exhaustively find all repeat-related features from a genomic DNA sequence. An efficient codon index is also derived from the recurrence time statistics, which has the salient features of being largely species-independent and working well on very short sequences. Efficient codon indices are key elements of successful gene finding algorithms, and are particularly useful for determining whether a suspected EST belongs to a coding or non-coding region. We illustrate the power of the method by studying the genomes of E. coli, the yeast S. cervisivae, the nematode worm C. elegans, and the human, Homo sapiens. Computationally, our method is very efficient. It allows us to carry out analysis of genomes on the whole genomic scale by a PC.

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.

Analysis of BAC end sequences in oak, a keystone forest tree species, providing insight into the composition of its genome

PubMed Central

2011-01-01

Background One of the key goals of oak genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of forests to increase their health and productivity. Deep-coverage large-insert genomic libraries are a crucial tool for attaining this objective. We report herein the construction of a BAC library for Quercus robur, its characterization and an analysis of BAC end sequences. Results The EcoRI library generated consisted of 92,160 clones, 7% of which had no insert. Levels of chloroplast and mitochondrial contamination were below 3% and 1%, respectively. Mean clone insert size was estimated at 135 kb. The library represents 12 haploid genome equivalents and, the likelihood of finding a particular oak sequence of interest is greater than 99%. Genome coverage was confirmed by PCR screening of the library with 60 unique genetic loci sampled from the genetic linkage map. In total, about 20,000 high-quality BAC end sequences (BESs) were generated by sequencing 15,000 clones. Roughly 5.88% of the combined BAC end sequence length corresponded to known retroelements while ab initio repeat detection methods identified 41 additional repeats. Collectively, characterized and novel repeats account for roughly 8.94% of the genome. Further analysis of the BESs revealed 1,823 putative genes suggesting at least 29,340 genes in the oak genome. BESs were aligned with the genome sequences of Arabidopsis thaliana, Vitis vinifera and Populus trichocarpa. One putative collinear microsyntenic region encoding an alcohol acyl transferase protein was observed between oak and chromosome 2 of V. vinifera. Conclusions This BAC library provides a new resource for genomic studies, including SSR marker development, physical mapping, comparative genomics and genome sequencing. BES analysis provided insight into the structure of the oak genome. These sequences will be used in the assembly of a future genome sequence for oak. PMID:21645357

Draft genome sequence of Enterococcus faecium strain LMG 8148.

PubMed

Michiels, Joran E; Van den Bergh, Bram; Fauvart, Maarten; Michiels, Jan

2016-01-01

Enterococcus faecium, traditionally considered a harmless gut commensal, is emerging as an important nosocomial pathogen showing increasing rates of multidrug resistance. We report the draft genome sequence of E. faecium strain LMG 8148, isolated in 1968 from a human in Gothenburg, Sweden. The draft genome has a total length of 2,697,490 bp, a GC-content of 38.3 %, and 2,402 predicted protein-coding sequences. The isolation of this strain predates the emergence of E. faecium as a nosocomial pathogen. Consequently, its genome can be useful in comparative genomic studies investigating the evolution of E. faecium as a pathogen.

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.

Lessons learnt on the analysis of large sequence data in animal genomics.

PubMed

Biscarini, F; Cozzi, P; Orozco-Ter Wengel, P

2018-04-06

The 'omics revolution has made a large amount of sequence data available to researchers and the industry. This has had a profound impact in the field of bioinformatics, stimulating unprecedented advancements in this discipline. Mostly, this is usually looked at from the perspective of human 'omics, in particular human genomics. Plant and animal genomics, however, have also been deeply influenced by next-generation sequencing technologies, with several genomics applications now popular among researchers and the breeding industry. Genomics tends to generate huge amounts of data, and genomic sequence data account for an increasing proportion of big data in biological sciences, due largely to decreasing sequencing and genotyping costs and to large-scale sequencing and resequencing projects. The analysis of big data poses a challenge to scientists, as data gathering currently takes place at a faster pace than does data processing and analysis, and the associated computational burden is increasingly taxing, making even simple manipulation, visualization and transferring of data a cumbersome operation. The time consumed by the processing and analysing of huge data sets may be at the expense of data quality assessment and critical interpretation. Additionally, when analysing lots of data, something is likely to go awry-the software may crash or stop-and it can be very frustrating to track the error. We herein review the most relevant issues related to tackling these challenges and problems, from the perspective of animal genomics, and provide researchers that lack extensive computing experience with guidelines that will help when processing large genomic data sets. © 2018 Stichting International Foundation for Animal Genetics.

UV Decontamination of MDA Reagents for Single Cell Genomics

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

Lee, Janey; Tighe, Damon; Sczyrba, Alexander

2011-03-18

Single cell genomics, the amplification and sequencing of genomes from single cells, can provide a glimpse into the genetic make-up and thus life style of the vast majority of uncultured microbial cells, making it an immensely powerful and increasingly popular tool. This is accomplished by use of multiple displacement amplification (MDA), which can generate billions of copies of a single bacterial genome producing microgram-range DNA required for shotgun sequencing. Here, we address a key challenge inherent to this approach and propose a solution for the improved recovery of single cell genomes. While DNA-free reagents for the amplification of a singlemore » cell genome are a prerequisite for successful single cell sequencing and analysis, DNA contamination has been detected in various reagents, which poses a considerable challenge. Our study demonstrates the effect of UV irradiation in efficient elimination of exogenous contaminant DNA found in MDA reagents, while maintaining Phi29 activity. Consequently, we also find that increased UV exposure to Phi29 does not adversely affect genome coverage of MDA amplified single cells. While additional challenges in single cell genomics remain to be resolved, the proposed methodology is relatively quick and simple and we believe that its application will be of high value for future single cell sequencing projects.« less

G-Anchor: a novel approach for whole-genome comparative mapping utilizing evolutionary conserved DNA sequences.

PubMed

Lenis, Vasileios Panagiotis E; Swain, Martin; Larkin, Denis M

2018-05-01

Cross-species whole-genome sequence alignment is a critical first step for genome comparative analyses, ranging from the detection of sequence variants to studies of chromosome evolution. Animal genomes are large and complex, and whole-genome alignment is a computationally intense process, requiring expensive high-performance computing systems due to the need to explore extensive local alignments. With hundreds of sequenced animal genomes available from multiple projects, there is an increasing demand for genome comparative analyses. Here, we introduce G-Anchor, a new, fast, and efficient pipeline that uses a strictly limited but highly effective set of local sequence alignments to anchor (or map) an animal genome to another species' reference genome. G-Anchor makes novel use of a databank of highly conserved DNA sequence elements. We demonstrate how these elements may be aligned to a pair of genomes, creating anchors. These anchors enable the rapid mapping of scaffolds from a de novo assembled genome to chromosome assemblies of a reference species. Our results demonstrate that G-Anchor can successfully anchor a vertebrate genome onto a phylogenetically related reference species genome using a desktop or laptop computer within a few hours and with comparable accuracy to that achieved by a highly accurate whole-genome alignment tool such as LASTZ. G-Anchor thus makes whole-genome comparisons accessible to researchers with limited computational resources. G-Anchor is a ready-to-use tool for anchoring a pair of vertebrate genomes. It may be used with large genomes that contain a significant fraction of evolutionally conserved DNA sequences and that are not highly repetitive, polypoid, or excessively fragmented. G-Anchor is not a substitute for whole-genome aligning software but can be used for fast and accurate initial genome comparisons. G-Anchor is freely available and a ready-to-use tool for the pairwise comparison of two genomes.

Independent assessment and improvement of wheat genome sequence assemblies using Fosill jumping libraries.

PubMed

Lu, Fu-Hao; McKenzie, Neil; Kettleborough, George; Heavens, Darren; Clark, Matthew D; Bevan, Michael W

2018-05-01

The accurate sequencing and assembly of very large, often polyploid, genomes remains a challenging task, limiting long-range sequence information and phased sequence variation for applications such as plant breeding. The 15-Gb hexaploid bread wheat (Triticum aestivum) genome has been particularly challenging to sequence, and several different approaches have recently generated long-range assemblies. Mapping and understanding the types of assembly errors are important for optimising future sequencing and assembly approaches and for comparative genomics. Here we use a Fosill 38-kb jumping library to assess medium and longer-range order of different publicly available wheat genome assemblies. Modifications to the Fosill protocol generated longer Illumina sequences and enabled comprehensive genome coverage. Analyses of two independent Bacterial Artificial Chromosome (BAC)-based chromosome-scale assemblies, two independent Illumina whole genome shotgun assemblies, and a hybrid Single Molecule Real Time (SMRT-PacBio) and short read (Illumina) assembly were carried out. We revealed a surprising scale and variety of discrepancies using Fosill mate-pair mapping and validated several of each class. In addition, Fosill mate-pairs were used to scaffold a whole genome Illumina assembly, leading to a 3-fold increase in N50 values. Our analyses, using an independent means to validate different wheat genome assemblies, show that whole genome shotgun assemblies based solely on Illumina sequences are significantly more accurate by all measures compared to BAC-based chromosome-scale assemblies and hybrid SMRT-Illumina approaches. Although current whole genome assemblies are reasonably accurate and useful, additional improvements will be needed to generate complete assemblies of wheat genomes using open-source, computationally efficient, and cost-effective methods.

DNA Data Bank of Japan (DDBJ) for genome scale research in life science

PubMed Central

Tateno, Y.; Imanishi, T.; Miyazaki, S.; Fukami-Kobayashi, K.; Saitou, N.; Sugawara, H.; Gojobori, T.

2002-01-01

The DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp) has made an effort to collect as much data as possible mainly from Japanese researchers. The increase rates of the data we collected, annotated and released to the public in the past year are 43% for the number of entries and 52% for the number of bases. The increase rates are accelerated even after the human genome was sequenced, because sequencing technology has been remarkably advanced and simplified, and research in life science has been shifted from the gene scale to the genome scale. In addition, we have developed the Genome Information Broker (GIB, http://gib.genes.nig.ac.jp) that now includes more than 50 complete microbial genome and Arabidopsis genome data. We have also developed a database of the human genome, the Human Genomics Studio (HGS, http://studio.nig.ac.jp). HGS provides one with a set of sequences being as continuous as possible in any one of the 24 chromosomes. Both GIB and HGS have been updated incorporating newly available data and retrieval tools. PMID:11752245

The genome of melon (Cucumis melo L.)

PubMed Central

Garcia-Mas, Jordi; Benjak, Andrej; Sanseverino, Walter; Bourgeois, Michael; Mir, Gisela; González, Víctor M.; Hénaff, Elizabeth; Câmara, Francisco; Cozzuto, Luca; Lowy, Ernesto; Alioto, Tyler; Capella-Gutiérrez, Salvador; Blanca, Jose; Cañizares, Joaquín; Ziarsolo, Pello; Gonzalez-Ibeas, Daniel; Rodríguez-Moreno, Luis; Droege, Marcus; Du, Lei; Alvarez-Tejado, Miguel; Lorente-Galdos, Belen; Melé, Marta; Yang, Luming; Weng, Yiqun; Navarro, Arcadi; Marques-Bonet, Tomas; Aranda, Miguel A.; Nuez, Fernando; Picó, Belén; Gabaldón, Toni; Roma, Guglielmo; Guigó, Roderic; Casacuberta, Josep M.; Arús, Pere; Puigdomènech, Pere

2012-01-01

We report the genome sequence of melon, an important horticultural crop worldwide. We assembled 375 Mb of the double-haploid line DHL92, representing 83.3% of the estimated melon genome. We predicted 27,427 protein-coding genes, which we analyzed by reconstructing 22,218 phylogenetic trees, allowing mapping of the orthology and paralogy relationships of sequenced plant genomes. We observed the absence of recent whole-genome duplications in the melon lineage since the ancient eudicot triplication, and our data suggest that transposon amplification may in part explain the increased size of the melon genome compared with the close relative cucumber. A low number of nucleotide-binding site–leucine-rich repeat disease resistance genes were annotated, suggesting the existence of specific defense mechanisms in this species. The DHL92 genome was compared with that of its parental lines allowing the quantification of sequence variability in the species. The use of the genome sequence in future investigations will facilitate the understanding of evolution of cucurbits and the improvement of breeding strategies. PMID:22753475

ABACAS: algorithm-based automatic contiguation of assembled sequences

PubMed Central

Assefa, Samuel; Keane, Thomas M.; Otto, Thomas D.; Newbold, Chris; Berriman, Matthew

2009-01-01

Summary: Due to the availability of new sequencing technologies, we are now increasingly interested in sequencing closely related strains of existing finished genomes. Recently a number of de novo and mapping-based assemblers have been developed to produce high quality draft genomes from new sequencing technology reads. New tools are necessary to take contigs from a draft assembly through to a fully contiguated genome sequence. ABACAS is intended as a tool to rapidly contiguate (align, order, orientate), visualize and design primers to close gaps on shotgun assembled contigs based on a reference sequence. The input to ABACAS is a set of contigs which will be aligned to the reference genome, ordered and orientated, visualized in the ACT comparative browser, and optimal primer sequences are automatically generated. Availability and Implementation: ABACAS is implemented in Perl and is freely available for download from http://abacas.sourceforge.net Contact: sa4@sanger.ac.uk PMID:19497936

Illumina GA IIx& HiSeq 2000 Production Sequenccing and QC Analysis Pipelines at the DOE Joint Genome Institute

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

Daum, Christopher; Zane, Matthew; Han, James

2011-01-31

The U.S. Department of Energy (DOE) Joint Genome Institute's (JGI) Production Sequencing group is committed to the generation of high-quality genomic DNA sequence to support the mission areas of renewable energy generation, global carbon management, and environmental characterization and clean-up. Within the JGI's Production Sequencing group, a robust Illumina Genome Analyzer and HiSeq pipeline has been established. Optimization of the sesequencer pipelines has been ongoing with the aim of continual process improvement of the laboratory workflow, reducing operational costs and project cycle times to increases ample throughput, and improving the overall quality of the sequence generated. A sequence QC analysismore » pipeline has been implemented to automatically generate read and assembly level quality metrics. The foremost of these optimization projects, along with sequencing and operational strategies, throughput numbers, and sequencing quality results will be presented.« less

Draft genome sequence of multidrug-resistant Staphylococcus haemolyticus IPK_TSA25 harbouring a Staphylococcus aureus plasmid, pS0385-1.

PubMed

Kim, Hyung Jun; Jang, Soojin

2017-12-01

Staphylococcus haemolyticus is the second most frequently isolated coagulase-negative staphylococci from blood cultures. Moreover, multidrug resistance associated with the genome flexibility of S. haemolyticus has been increasingly reported worldwide. Here we report the draft genome sequence of multidrug-resistant S. haemolyticus IPK_TSA25 isolated from a building surface in South Korea. Genomic DNA of S. haemolyticus IPK_TSA25 was sequenced using the PacBio RS II sequencing platform. Generated reads were assembled using PacBio SMRT Analysis 2.3.0. The draft genome was annotated and antibiotic resistance genes were identified. The genome of 2517398bp contains various antibiotic resistance genes associated with resistance to β-lactams, aminoglycosides and macrolides. Genome analysis also revealed chromosomal integration of the full-length Staphylococcus aureus plasmid pS0385-1 containing a tetracycline resistance gene. The genome sequence reported in this study will provide valuable information to understand the flexibility of the S. haemolyticus genome, which facilitates acquisition of antibiotic resistance genes and contributes to the dissemination of antibiotic resistance by this emerging pathogen. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Crossing the LINE toward genomic instability: LINE-1 retrotransposition in cancer

NASA Astrophysics Data System (ADS)

Kemp, Jacqueline; Longworth, Michelle

2015-12-01

Retrotransposons are repetitive DNA sequences that are positioned throughout the human genome. Retrotransposons are capable of copying themselves and mobilizing new copies to novel genomic locations in a process called retrotransposition. While most retrotransposon sequences in the human genome are incomplete and incapable of mobilization, the LINE-1 retrotransposon, which comprises approximately 17% of the human genome, remains active. The disruption of cellular mechanisms that suppress retrotransposon activity is linked to the generation of aneuploidy, a potential driver of tumor development. When retrotransposons insert into a novel genomic region, they have the potential to disrupt the coding sequence of endogenous genes and alter gene expression, which can lead to deleterious consequences for the organism. Additionally, increased LINE-1 copy numbers provide more chances for recombination events to occur between retrotransposons, which can lead to chromosomal breaks and rearrangements. LINE-1 activity is increased in various cancer cell lines and in patient tissues resected from primary tumors. LINE-1 activity also correlates with increased cancer metastasis. This review aims to give a brief overview of the connections between LINE-1 retrotransposition and the loss of genome stability. We will also discuss the mechanisms that repress retrotransposition in human cells and their links to cancer.

A programmable method for massively parallel targeted sequencing

PubMed Central

Hopmans, Erik S.; Natsoulis, Georges; Bell, John M.; Grimes, Susan M.; Sieh, Weiva; Ji, Hanlee P.

2014-01-01

We have developed a targeted resequencing approach referred to as Oligonucleotide-Selective Sequencing. In this study, we report a series of significant improvements and novel applications of this method whereby the surface of a sequencing flow cell is modified in situ to capture specific genomic regions of interest from a sample and then sequenced. These improvements include a fully automated targeted sequencing platform through the use of a standard Illumina cBot fluidics station. Targeting optimization increased the yield of total on-target sequencing data 2-fold compared to the previous iteration, while simultaneously increasing the percentage of reads that could be mapped to the human genome. The described assays cover up to 1421 genes with a total coverage of 5.5 Megabases (Mb). We demonstrate a 10-fold abundance uniformity of greater than 90% in 1 log distance from the median and a targeting rate of up to 95%. We also sequenced continuous genomic loci up to 1.5 Mb while simultaneously genotyping SNPs and genes. Variants with low minor allele fraction were sensitively detected at levels of 5%. Finally, we determined the exact breakpoint sequence of cancer rearrangements. Overall, this approach has high performance for selective sequencing of genome targets, configuration flexibility and variant calling accuracy. PMID:24782526

Preferences for learning different types of genome sequencing results among young breast cancer patients: Role of psychological and clinical factors.

PubMed

Kaphingst, Kimberly A; Ivanovich, Jennifer; Lyons, Sarah; Biesecker, Barbara; Dresser, Rebecca; Elrick, Ashley; Matsen, Cindy; Goodman, Melody

2018-01-29

The growing importance of genome sequencing means that patients will increasingly face decisions regarding what results they would like to learn. The present study examined psychological and clinical factors that might affect these preferences. 1,080 women diagnosed with breast cancer at age 40 or younger completed an online survey. We assessed their interest in learning various types of genome sequencing results: risk of preventable disease or unpreventable disease, cancer treatment response, uncertain meaning, risk to relatives' health, and ancestry/physical traits. Multivariable logistic regression was used to examine whether being "very" interested in each result type was associated with clinical factors: BRCA1/2 mutation status, prior genetic testing, family history of breast cancer, and psychological factors: cancer recurrence worry, genetic risk worry, future orientation, health information orientation, and genome sequencing knowledge. The proportion of respondents who were very interested in learning each type of result ranged from 16% to 77%. In all multivariable models, those who were very interested in learning a result type had significantly higher knowledge about sequencing benefits, greater genetic risks worry, and stronger health information orientation compared to those with less interest (p-values < .05). Our findings indicate that high interest in return of various types of genome sequencing results was more closely related to psychological factors. Shared decision-making approaches that increase knowledge about genome sequencing and incorporate patient preferences for health information and learning about genetic risks may help support patients' informed choices about learning different types of sequencing results. © Society of Behavioral Medicine 2018.

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

PubMed Central

Carpenter, Meredith L.; Buenrostro, Jason D.; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E.; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M. Thomas P.; Willerslev, Eske; Greenleaf, William J.; Bustamante, Carlos D.

2013-01-01

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples. PMID:24568772

Implementing Genome-Driven Oncology

PubMed Central

Hyman, David M.; Taylor, Barry S.; Baselga, José

2017-01-01

Early successes in identifying and targeting individual oncogenic drivers, together with the increasing feasibility of sequencing tumor genomes, have brought forth the promise of genome-driven oncology care. As we expand the breadth and depth of genomic analyses, the biological and clinical complexity of its implementation will be unparalleled. Challenges include target credentialing and validation, implementing drug combinations, clinical trial designs, targeting tumor heterogeneity, and deploying technologies beyond DNA sequencing, among others. We review how contemporary approaches are tackling these challenges and will ultimately serve as an engine for biological discovery and increase our insight into cancer and its treatment. PMID:28187282

Advances in Cryptococcus genomics: insights into the evolution of pathogenesis.

PubMed

Cuomo, Christina A; Rhodes, Johanna; Desjardins, Christopher A

2018-01-01

Cryptococcus species are the causative agents of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals. Initial work on the molecular epidemiology of this fungal pathogen utilized genotyping approaches to describe the genetic diversity and biogeography of two species, Cryptococcus neoformans and Cryptococcus gattii. Whole genome sequencing of representatives of both species resulted in reference assemblies enabling a wide array of downstream studies and genomic resources. With the increasing availability of whole genome sequencing, both species have now had hundreds of individual isolates sequenced, providing fine-scale insight into the evolution and diversification of Cryptococcus and allowing for the first genome-wide association studies to identify genetic variants associated with human virulence. Sequencing has also begun to examine the microevolution of isolates during prolonged infection and to identify variants specific to outbreak lineages, highlighting the potential role of hyper-mutation in evolving within short time scales. We can anticipate that further advances in sequencing technology and sequencing microbial genomes at scale, including metagenomics approaches, will continue to refine our view of how the evolution of Cryptococcus drives its success as a pathogen.

Genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of Loci encoding increased protein quality control mechanisms

USDA-ARS?s Scientific Manuscript database

Salmonella enterica subsp. enterica bacteria are important foodborne pathogens with major economic impact. Some isolates exhibit increased heat tolerance, a concern for food safety. Analysis of a finished-quality genome sequence of an isolate commonly used in heat resistance studies, S. enterica sub...

The future is now: single-cell genomics of bacteria and archaea

PubMed Central

Blainey, Paul C.

2013-01-01

Interest in the expanding catalog of uncultivated microorganisms, increasing recognition of heterogeneity among seemingly similar cells, and technological advances in whole-genome amplification and single-cell manipulation are driving considerable progress in single-cell genomics. Here, the spectrum of applications for single-cell genomics, key advances in the development of the field, and emerging methodology for single-cell genome sequencing are reviewed by example with attention to the diversity of approaches and their unique characteristics. Experimental strategies transcending specific methodologies are identified and organized as a road map for future studies in single-cell genomics of environmental microorganisms. Over the next decade, increasingly powerful tools for single-cell genome sequencing and analysis will play key roles in accessing the genomes of uncultivated organisms, determining the basis of microbial community functions, and fundamental aspects of microbial population biology. PMID:23298390

ReprDB and panDB: minimalist databases with maximal microbial representation.

PubMed

Zhou, Wei; Gay, Nicole; Oh, Julia

2018-01-18

Profiling of shotgun metagenomic samples is hindered by a lack of unified microbial reference genome databases that (i) assemble genomic information from all open access microbial genomes, (ii) have relatively small sizes, and (iii) are compatible to various metagenomic read mapping tools. Moreover, computational tools to rapidly compile and update such databases to accommodate the rapid increase in new reference genomes do not exist. As a result, database-guided analyses often fail to profile a substantial fraction of metagenomic shotgun sequencing reads from complex microbiomes. We report pipelines that efficiently traverse all open access microbial genomes and assemble non-redundant genomic information. The pipelines result in two species-resolution microbial reference databases of relatively small sizes: reprDB, which assembles microbial representative or reference genomes, and panDB, for which we developed a novel iterative alignment algorithm to identify and assemble non-redundant genomic regions in multiple sequenced strains. With the databases, we managed to assign taxonomic labels and genome positions to the majority of metagenomic reads from human skin and gut microbiomes, demonstrating a significant improvement over a previous database-guided analysis on the same datasets. reprDB and panDB leverage the rapid increases in the number of open access microbial genomes to more fully profile metagenomic samples. Additionally, the databases exclude redundant sequence information to avoid inflated storage or memory space and indexing or analyzing time. Finally, the novel iterative alignment algorithm significantly increases efficiency in pan-genome identification and can be useful in comparative genomic analyses.

Update on Genomic Databases and Resources at the National Center for Biotechnology Information.

PubMed

Tatusova, Tatiana

2016-01-01

The National Center for Biotechnology Information (NCBI), as a primary public repository of genomic sequence data, collects and maintains enormous amounts of heterogeneous data. Data for genomes, genes, gene expressions, gene variation, gene families, proteins, and protein domains are integrated with the analytical, search, and retrieval resources through the NCBI website, text-based search and retrieval system, provides a fast and easy way to navigate across diverse biological databases.Comparative genome analysis tools lead to further understanding of evolution processes quickening the pace of discovery. Recent technological innovations have ignited an explosion in genome sequencing that has fundamentally changed our understanding of the biology of living organisms. This huge increase in DNA sequence data presents new challenges for the information management system and the visualization tools. New strategies have been designed to bring an order to this genome sequence shockwave and improve the usability of associated data.

RSAT 2015: Regulatory Sequence Analysis Tools

PubMed Central

Medina-Rivera, Alejandra; Defrance, Matthieu; Sand, Olivier; Herrmann, Carl; Castro-Mondragon, Jaime A.; Delerce, Jeremy; Jaeger, Sébastien; Blanchet, Christophe; Vincens, Pierre; Caron, Christophe; Staines, Daniel M.; Contreras-Moreira, Bruno; Artufel, Marie; Charbonnier-Khamvongsa, Lucie; Hernandez, Céline; Thieffry, Denis; Thomas-Chollier, Morgane; van Helden, Jacques

2015-01-01

RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/. PMID:25904632

VCFtoTree: a user-friendly tool to construct locus-specific alignments and phylogenies from thousands of anthropologically relevant genome sequences.

PubMed

Xu, Duo; Jaber, Yousef; Pavlidis, Pavlos; Gokcumen, Omer

2017-09-26

Constructing alignments and phylogenies for a given locus from large genome sequencing studies with relevant outgroups allow novel evolutionary and anthropological insights. However, no user-friendly tool has been developed to integrate thousands of recently available and anthropologically relevant genome sequences to construct complete sequence alignments and phylogenies. Here, we provide VCFtoTree, a user friendly tool with a graphical user interface that directly accesses online databases to download, parse and analyze genome variation data for regions of interest. Our pipeline combines popular sequence datasets and tree building algorithms with custom data parsing to generate accurate alignments and phylogenies using all the individuals from the 1000 Genomes Project, Neanderthal and Denisovan genomes, as well as reference genomes of Chimpanzee and Rhesus Macaque. It can also be applied to other phased human genomes, as well as genomes from other species. The output of our pipeline includes an alignment in FASTA format and a tree file in newick format. VCFtoTree fulfills the increasing demand for constructing alignments and phylogenies for a given loci from thousands of available genomes. Our software provides a user friendly interface for a wider audience without prerequisite knowledge in programming. VCFtoTree can be accessed from https://github.com/duoduoo/VCFtoTree_3.0.0 .

Preliminary Genomic Characterization of Ten Hardwood Tree Species from Multiplexed Low Coverage Whole Genome Sequencing

PubMed Central

Staton, Margaret; Best, Teodora; Khodwekar, Sudhir; Owusu, Sandra; Xu, Tao; Xu, Yi; Jennings, Tara; Cronn, Richard; Arumuganathan, A. Kathiravetpilla; Coggeshall, Mark; Gailing, Oliver; Liang, Haiying; Romero-Severson, Jeanne; Schlarbaum, Scott; Carlson, John E.

2015-01-01

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence. PMID:26698853

Genomic prediction using imputed whole-genome sequence data in Holstein Friesian cattle.

PubMed

van Binsbergen, Rianne; Calus, Mario P L; Bink, Marco C A M; van Eeuwijk, Fred A; Schrooten, Chris; Veerkamp, Roel F

2015-09-17

In contrast to currently used single nucleotide polymorphism (SNP) panels, the use of whole-genome sequence data is expected to enable the direct estimation of the effects of causal mutations on a given trait. This could lead to higher reliabilities of genomic predictions compared to those based on SNP genotypes. Also, at each generation of selection, recombination events between a SNP and a mutation can cause decay in reliability of genomic predictions based on markers rather than on the causal variants. Our objective was to investigate the use of imputed whole-genome sequence genotypes versus high-density SNP genotypes on (the persistency of) the reliability of genomic predictions using real cattle data. Highly accurate phenotypes based on daughter performance and Illumina BovineHD Beadchip genotypes were available for 5503 Holstein Friesian bulls. The BovineHD genotypes (631,428 SNPs) of each bull were used to impute whole-genome sequence genotypes (12,590,056 SNPs) using the Beagle software. Imputation was done using a multi-breed reference panel of 429 sequenced individuals. Genomic estimated breeding values for three traits were predicted using a Bayesian stochastic search variable selection (BSSVS) model and a genome-enabled best linear unbiased prediction model (GBLUP). Reliabilities of predictions were based on 2087 validation bulls, while the other 3416 bulls were used for training. Prediction reliabilities ranged from 0.37 to 0.52. BSSVS performed better than GBLUP in all cases. Reliabilities of genomic predictions were slightly lower with imputed sequence data than with BovineHD chip data. Also, the reliabilities tended to be lower for both sequence data and BovineHD chip data when relationships between training animals were low. No increase in persistency of prediction reliability using imputed sequence data was observed. Compared to BovineHD genotype data, using imputed sequence data for genomic prediction produced no advantage. To investigate the putative advantage of genomic prediction using (imputed) sequence data, a training set with a larger number of individuals that are distantly related to each other and genomic prediction models that incorporate biological information on the SNPs or that apply stricter SNP pre-selection should be considered.

A field ornithologist’s guide to genomics: Practical considerations for ecology and conservation

USGS Publications Warehouse

Oyler-McCance, Sara J.; Oh, Kevin; Langin, Kathryn; Aldridge, Cameron L.

2016-01-01

Vast improvements in sequencing technology have made it practical to simultaneously sequence millions of nucleotides distributed across the genome, opening the door for genomic studies in virtually any species. Ornithological research stands to benefit in three substantial ways. First, genomic methods enhance our ability to parse and simultaneously analyze both neutral and non-neutral genomic regions, thus providing insight into adaptive evolution and divergence. Second, the sheer quantity of sequence data generated by current sequencing platforms allows increased precision and resolution in analyses. Third, high-throughput sequencing can benefit applications that focus on a small number of loci that are otherwise prohibitively expensive, time-consuming, and technically difficult using traditional sequencing methods. These advances have improved our ability to understand evolutionary processes like speciation and local adaptation, but they also offer many practical applications in the fields of population ecology, migration tracking, conservation planning, diet analyses, and disease ecology. This review provides a guide for field ornithologists interested in incorporating genomic approaches into their research program, with an emphasis on techniques related to ecology and conservation. We present a general overview of contemporary genomic approaches and methods, as well as important considerations when selecting a genomic technique. We also discuss research questions that are likely to benefit from utilizing high-throughput sequencing instruments, highlighting select examples from recent avian studies.

Genotyping-by-sequencing for Populus population genomics: An assessment of genome sampling patterns and filtering approaches

Treesearch

Martin P. Schilling; Paul G. Wolf; Aaron M. Duffy; Hardeep S. Rai; Carol A. Rowe; Bryce A. Richardson; Karen E. Mock

2014-01-01

Continuing advances in nucleotide sequencing technology are inspiring a suite of genomic approaches in studies of natural populations. Researchers are faced with data management and analytical scales that are increasing by orders of magnitude. With such dramatic advances comes a need to understand biases and error rates, which can be propagated and magnified in large-...

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

Economic evaluation of genomic sequencing in the paediatric population: a critical review.

PubMed

Alam, Khurshid; Schofield, Deborah

2018-05-24

Systematic evidence is critical to the formulation of national health policy to provide public funding for the integration of genomic sequencing into routine clinical care. The purpose of this review is to present systematic evidence on the economic evaluation of genomic sequencing conducted for paediatric patients in clinical care, and to identify any gaps in the methodology of economic evaluations. We undertook a critical review of the empirical evidence from economic evaluations of genomic sequencing among paediatric patients searching five electronic databases. Our inclusion criteria were limited to literature published in the English language between 2010 and 2017 in OECD countries. Articles that met our inclusion criteria were assessed using a recognised checklist for a well-designed economic evaluation. We found 11 full-text articles that met our inclusion criteria. Our analysis found that genomic sequencing markedly increased the diagnostic rate to 16-79%, but lowered the cost by 11-64% compared to the standard diagnostic pathway. Only five recent studies in paediatric clinical cohorts met most of the criteria for a well-designed economic evaluation and demonstrated cost-effectiveness of genomic sequencing in paediatric clinical cohorts of patients. Our review identified the need for improvement in the rigour of the methodologies used to provide robust evidence for the formulation of health policy on public funding to integrate genomic sequencing into routine clinical care. Nonetheless, there is emerging evidence of the cost-effectiveness of genomic sequencing over usual care for paediatric patients.

VCGDB: a dynamic genome database of the Chinese population

PubMed Central

2014-01-01

Background The data released by the 1000 Genomes Project contain an increasing number of genome sequences from different nations and populations with a large number of genetic variations. As a result, the focus of human genome studies is changing from single and static to complex and dynamic. The currently available human reference genome (GRCh37) is based on sequencing data from 13 anonymous Caucasian volunteers, which might limit the scope of genomics, transcriptomics, epigenetics, and genome wide association studies. Description We used the massive amount of sequencing data published by the 1000 Genomes Project Consortium to construct the Virtual Chinese Genome Database (VCGDB), a dynamic genome database of the Chinese population based on the whole genome sequencing data of 194 individuals. VCGDB provides dynamic genomic information, which contains 35 million single nucleotide variations (SNVs), 0.5 million insertions/deletions (indels), and 29 million rare variations, together with genomic annotation information. VCGDB also provides a highly interactive user-friendly virtual Chinese genome browser (VCGBrowser) with functions like seamless zooming and real-time searching. In addition, we have established three population-specific consensus Chinese reference genomes that are compatible with mainstream alignment software. Conclusions VCGDB offers a feasible strategy for processing big data to keep pace with the biological data explosion by providing a robust resource for genomics studies; in particular, studies aimed at finding regions of the genome associated with diseases. PMID:24708222

Evaluation of next generation mtGenome sequencing using the Ion Torrent Personal Genome Machine (PGM)☆

PubMed Central

Parson, Walther; Strobl, Christina; Huber, Gabriela; Zimmermann, Bettina; Gomes, Sibylle M.; Souto, Luis; Fendt, Liane; Delport, Rhena; Langit, Reina; Wootton, Sharon; Lagacé, Robert; Irwin, Jodi

2013-01-01

Insights into the human mitochondrial phylogeny have been primarily achieved by sequencing full mitochondrial genomes (mtGenomes). In forensic genetics (partial) mtGenome information can be used to assign haplotypes to their phylogenetic backgrounds, which may, in turn, have characteristic geographic distributions that would offer useful information in a forensic case. In addition and perhaps even more relevant in the forensic context, haplogroup-specific patterns of mutations form the basis for quality control of mtDNA sequences. The current method for establishing (partial) mtDNA haplotypes is Sanger-type sequencing (STS), which is laborious, time-consuming, and expensive. With the emergence of Next Generation Sequencing (NGS) technologies, the body of available mtDNA data can potentially be extended much more quickly and cost-efficiently. Customized chemistries, laboratory workflows and data analysis packages could support the community and increase the utility of mtDNA analysis in forensics. We have evaluated the performance of mtGenome sequencing using the Personal Genome Machine (PGM) and compared the resulting haplotypes directly with conventional Sanger-type sequencing. A total of 64 mtGenomes (>1 million bases) were established that yielded high concordance with the corresponding STS haplotypes (<0.02% differences). About two-thirds of the differences were observed in or around homopolymeric sequence stretches. In addition, the sequence alignment algorithm employed to align NGS reads played a significant role in the analysis of the data and the resulting mtDNA haplotypes. Further development of alignment software would be desirable to facilitate the application of NGS in mtDNA forensic genetics. PMID:23948325

High-throughput sequencing of three Lemnoideae (duckweeds) chloroplast genomes from total DNA.

PubMed

Wang, Wenqin; Messing, Joachim

2011-01-01

Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

High-Throughput Sequencing of Three Lemnoideae (Duckweeds) Chloroplast Genomes from Total DNA

PubMed Central

Wang, Wenqin; Messing, Joachim

2011-01-01

Background Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. Methods We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. Conclusions This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power. PMID:21931804

The eukaryotic genome is structurally and functionally more like a social insect colony than a book.

PubMed

Qiu, Guo-Hua; Yang, Xiaoyan; Zheng, Xintian; Huang, Cuiqin

2017-11-01

Traditionally, the genome has been described as the 'book of life'. However, the metaphor of a book may not reflect the dynamic nature of the structure and function of the genome. In the eukaryotic genome, the number of centrally located protein-coding sequences is relatively constant across species, but the amount of noncoding DNA increases considerably with the increase of organismal evolutional complexity. Therefore, it has been hypothesized that the abundant peripheral noncoding DNA protects the genome and the central protein-coding sequences in the eukaryotic genome. Upon comparison with the habitation, sociality and defense mechanisms of a social insect colony, it is found that the genome is similar to a social insect colony in various aspects. A social insect colony may thus be a better metaphor than a book to describe the spatial organization and physical functions of the genome. The potential implications of the metaphor are also discussed.

Carnivore-specific SINEs (Can-SINEs): distribution, evolution, and genomic impact.

PubMed

Walters-Conte, Kathryn B; Johnson, Diana L E; Allard, Marc W; Pecon-Slattery, Jill

2011-01-01

Short interspersed nuclear elements (SINEs) are a type of class 1 transposable element (retrotransposon) with features that allow investigators to resolve evolutionary relationships between populations and species while providing insight into genome composition and function. Characterization of a Carnivora-specific SINE family, Can-SINEs, has, has aided comparative genomic studies by providing rare genomic changes, and neutral sequence variants often needed to resolve difficult evolutionary questions. In addition, Can-SINEs constitute a significant source of functional diversity with Carnivora. Publication of the whole-genome sequence of domestic dog, domestic cat, and giant panda serves as a valuable resource in comparative genomic inferences gleaned from Can-SINEs. In anticipation of forthcoming studies bolstered by new genomic data, this review describes the discovery and characterization of Can-SINE motifs as well as describes composition, distribution, and effect on genome function. As the contribution of noncoding sequences to genomic diversity becomes more apparent, SINEs and other transposable elements will play an increasingly large role in mammalian comparative genomics.

Carnivore-Specific SINEs (Can-SINEs): Distribution, Evolution, and Genomic Impact

PubMed Central

Johnson, Diana L.E.; Allard, Marc W.; Pecon-Slattery, Jill

2011-01-01

Short interspersed nuclear elements (SINEs) are a type of class 1 transposable element (retrotransposon) with features that allow investigators to resolve evolutionary relationships between populations and species while providing insight into genome composition and function. Characterization of a Carnivora-specific SINE family, Can-SINEs, has, has aided comparative genomic studies by providing rare genomic changes, and neutral sequence variants often needed to resolve difficult evolutionary questions. In addition, Can-SINEs constitute a significant source of functional diversity with Carnivora. Publication of the whole-genome sequence of domestic dog, domestic cat, and giant panda serves as a valuable resource in comparative genomic inferences gleaned from Can-SINEs. In anticipation of forthcoming studies bolstered by new genomic data, this review describes the discovery and characterization of Can-SINE motifs as well as describes composition, distribution, and effect on genome function. As the contribution of noncoding sequences to genomic diversity becomes more apparent, SINEs and other transposable elements will play an increasingly large role in mammalian comparative genomics. PMID:21846743

The Genome 10K Project: a way forward.

PubMed

Koepfli, Klaus-Peter; Paten, Benedict; O'Brien, Stephen J

2015-01-01

The Genome 10K Project was established in 2009 by a consortium of biologists and genome scientists determined to facilitate the sequencing and analysis of the complete genomes of 10,000 vertebrate species. Since then the number of selected and initiated species has risen from ∼26 to 277 sequenced or ongoing with funding, an approximately tenfold increase in five years. Here we summarize the advances and commitments that have occurred by mid-2014 and outline the achievements and present challenges of reaching the 10,000-species goal. We summarize the status of known vertebrate genome projects, recommend standards for pronouncing a genome as sequenced or completed, and provide our present and future vision of the landscape of Genome 10K. The endeavor is ambitious, bold, expensive, and uncertain, but together the Genome 10K Consortium of Scientists and the worldwide genomics community are moving toward their goal of delivering to the coming generation the gift of genome empowerment for many vertebrate species.

The Genome 10K Project: A Way Forward

PubMed Central

Koepfli, Klaus-Peter; Paten, Benedict; O’Brien, Stephen J.

2017-01-01

The Genome 10K Project was established in 2009 by a consortium of biologists and genome scientists determined to facilitate the sequencing and analysis of the complete genomes of 10,000 vertebrate species. Since then the number of selected and initiated species has risen from ~26 to 277 sequenced or ongoing with funding, an approximately tenfold increase in five years. Here we summarize the advances and commitments that have occurred by mid-2014 and outline the achievements and present challenges of reaching the 10,000-species goal. We summarize the status of known vertebrate genome projects, recommend standards for pronouncing a genome as sequenced or completed, and provide our present and future vision of the landscape of Genome 10K. The endeavor is ambitious, bold, expensive, and uncertain, but together the Genome 10K Consortium of Scientists and the worldwide genomics community are moving toward their goal of delivering to the coming generation the gift of genome empowerment for many vertebrate species. PMID:25689317

An improved genome assembly uncovers prolific tandem repeats in Atlantic cod.

PubMed

Tørresen, Ole K; Star, Bastiaan; Jentoft, Sissel; Reinar, William B; Grove, Harald; Miller, Jason R; Walenz, Brian P; Knight, James; Ekholm, Jenny M; Peluso, Paul; Edvardsen, Rolf B; Tooming-Klunderud, Ave; Skage, Morten; Lien, Sigbjørn; Jakobsen, Kjetill S; Nederbragt, Alexander J

2017-01-18

The first Atlantic cod (Gadus morhua) genome assembly published in 2011 was one of the early genome assemblies exclusively based on high-throughput 454 pyrosequencing. Since then, rapid advances in sequencing technologies have led to a multitude of assemblies generated for complex genomes, although many of these are of a fragmented nature with a significant fraction of bases in gaps. The development of long-read sequencing and improved software now enable the generation of more contiguous genome assemblies. By combining data from Illumina, 454 and the longer PacBio sequencing technologies, as well as integrating the results of multiple assembly programs, we have created a substantially improved version of the Atlantic cod genome assembly. The sequence contiguity of this assembly is increased fifty-fold and the proportion of gap-bases has been reduced fifteen-fold. Compared to other vertebrates, the assembly contains an unusual high density of tandem repeats (TRs). Indeed, retrospective analyses reveal that gaps in the first genome assembly were largely associated with these TRs. We show that 21% of the TRs across the assembly, 19% in the promoter regions and 12% in the coding sequences are heterozygous in the sequenced individual. The inclusion of PacBio reads combined with the use of multiple assembly programs drastically improved the Atlantic cod genome assembly by successfully resolving long TRs. The high frequency of heterozygous TRs within or in the vicinity of genes in the genome indicate a considerable standing genomic variation in Atlantic cod populations, which is likely of evolutionary importance.

The value of new genome references.

PubMed

Worley, Kim C; Richards, Stephen; Rogers, Jeffrey

2017-09-15

Genomic information has become a ubiquitous and almost essential aspect of biological research. Over the last 10-15 years, the cost of generating sequence data from DNA or RNA samples has dramatically declined and our ability to interpret those data increased just as remarkably. Although it is still possible for biologists to conduct interesting and valuable research on species for which genomic data are not available, the impact of having access to a high quality whole genome reference assembly for a given species is nothing short of transformational. Research on a species for which we have no DNA or RNA sequence data is restricted in fundamental ways. In contrast, even access to an initial draft quality genome (see below for definitions) opens a wide range of opportunities that are simply not available without that reference genome assembly. Although a complete discussion of the impact of genome sequencing and assembly is beyond the scope of this short paper, the goal of this review is to summarize the most common and highest impact contributions that whole genome sequencing and assembly has had on comparative and evolutionary biology. Copyright © 2016. Published by Elsevier Inc.

Substantial genome synteny preservation among woody angiosperm species: comparative genomics of Chinese chestnut (Castanea mollissima) and plant reference genomes.

PubMed

Staton, Margaret; Zhebentyayeva, Tetyana; Olukolu, Bode; Fang, Guang Chen; Nelson, Dana; Carlson, John E; Abbott, Albert G

2015-10-05

Chinese chestnut (Castanea mollissima) has emerged as a model species for the Fagaceae family with extensive genomic resources including a physical map, a dense genetic map and quantitative trait loci (QTLs) for chestnut blight resistance. These resources enable comparative genomics analyses relative to model plants. We assessed the degree of conservation between the chestnut genome and other well annotated and assembled plant genomic sequences, focusing on the QTL regions of most interest to the chestnut breeding community. The integrated physical and genetic map of Chinese chestnut has been improved to now include 858 shared sequence-based markers. The utility of the integrated map has also been improved through the addition of 42,970 BAC (bacterial artificial chromosome) end sequences spanning over 26 million bases of the estimated 800 Mb chestnut genome. Synteny between chestnut and ten model plant species was conducted on a macro-syntenic scale using sequences from both individual probes and BAC end sequences across the chestnut physical map. Blocks of synteny with chestnut were found in all ten reference species, with the percent of the chestnut physical map that could be aligned ranging from 10 to 39 %. The integrated genetic and physical map was utilized to identify BACs that spanned the three previously identified QTL regions conferring blight resistance. The clones were pooled and sequenced, yielding 396 sequence scaffolds covering 13.9 Mbp. Comparative genomic analysis on a microsytenic scale, using the QTL-associated genomic sequence, identified synteny from chestnut to other plant genomes ranging from 5.4 to 12.9 % of the genome sequences aligning. On both the macro- and micro-synteny levels, the peach, grape and poplar genomes were found to be the most structurally conserved with chestnut. Interestingly, these results did not strictly follow the expectation that decreased phylogenetic distance would correspond to increased levels of genome preservation, but rather suggest the additional influence of life-history traits on preservation of synteny. The regions of synteny that were detected provide an important tool for defining and cataloging genes in the QTL regions for advancing chestnut blight resistance research.

Complete Genome Sequence of the Crohn's Disease-Associated Adherent-Invasive Escherichia coliStrain HM605▿

PubMed Central

Clarke, David J.; Chaudhuri, Roy R.; Martin, Helen M.; Campbell, Barry J.; Rhodes, Jonathan M.; Constantinidou, Chrystala; Pallen, Mark J.; Loman, Nicholas J.; Cunningham, Adam F.; Browning, Douglas F.; Henderson, Ian R.

2011-01-01

Adherent-invasive Escherichia colistrains are increasingly being associated with intestinal pathologies. Here we present the genome sequence of E. coliHM605, a strain isolated from colonic biopsy specimens of a patient with Crohn's disease. PMID:21705601

Similar Ratios of Introns to Intergenic Sequence across Animal Genomes

PubMed Central

Wörheide, Gert

2017-01-01

Abstract One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought. PMID:28633296

The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110

PubMed Central

2012-01-01

Background Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. Results Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element. Conclusions The complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest. PMID:22443545

The complete genome sequence of the acarbose producer Actinoplanes sp. SE50/110.

PubMed

Schwientek, Patrick; Szczepanowski, Rafael; Rückert, Christian; Kalinowski, Jörn; Klein, Andreas; Selber, Klaus; Wehmeier, Udo F; Stoye, Jens; Pühler, Alfred

2012-03-23

Actinoplanes sp. SE50/110 is known as the wild type producer of the alpha-glucosidase inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes mellitus. As the incidence of diabetes is rapidly rising worldwide, an ever increasing demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently, derived Actinoplanes strains with increased acarbose yields are being used in large scale industrial batch fermentation since 1990 and were continuously optimized by conventional mutagenesis and screening experiments. This strategy reached its limits and is generally superseded by modern genetic engineering approaches. As a prerequisite for targeted genetic modifications, the complete genome sequence of the organism has to be known. Here, we present the complete genome sequence of Actinoplanes sp. SE50/110 [GenBank:CP003170], the first publicly available genome of the genus Actinoplanes, comprising various producers of pharmaceutically and economically important secondary metabolites. The genome features a high mean G + C content of 71.32% and consists of one circular chromosome with a size of 9,239,851 bp hosting 8,270 predicted protein coding sequences. Phylogenetic analysis of the core genome revealed a rather distant relation to other sequenced species of the family Micromonosporaceae whereas Actinoplanes utahensis was found to be the closest species based on 16S rRNA gene sequence comparison. Besides the already published acarbose biosynthetic gene cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase- and hybrid-clusters were identified on the Actinoplanes genome. Another key feature of the genome represents the discovery of a functional actinomycete integrative and conjugative element. The complete genome sequence of Actinoplanes sp. SE50/110 marks an important step towards the rational genetic optimization of the acarbose production. In this regard, the identified actinomycete integrative and conjugative element could play a central role by providing the basis for the development of a genetic transformation system for Actinoplanes sp. SE50/110 and other Actinoplanes spp. Furthermore, the identified non-ribosomal peptide synthetase- and polyketide synthase-clusters potentially encode new antibiotics and/or other bioactive compounds, which might be of pharmacologic interest.

Exploration of sequence space as the basis of viral RNA genome segmentation.

PubMed

Moreno, Elena; Ojosnegros, Samuel; García-Arriaza, Juan; Escarmís, Cristina; Domingo, Esteban; Perales, Celia

2014-05-06

The mechanisms of viral RNA genome segmentation are unknown. On extensive passage of foot-and-mouth disease virus in baby hamster kidney-21 cells, the virus accumulated multiple point mutations and underwent a transition akin to genome segmentation. The standard single RNA genome molecule was replaced by genomes harboring internal in-frame deletions affecting the L- or capsid-coding region. These genomes were infectious and killed cells by complementation. Here we show that the point mutations in the nonstructural protein-coding region (P2, P3) that accumulated in the standard genome before segmentation increased the relative fitness of the segmented version relative to the standard genome. Fitness increase was documented by intracellular expression of virus-coded proteins and infectious progeny production by RNAs with the internal deletions placed in the sequence context of the parental and evolved genome. The complementation activity involved several viral proteins, one of them being the leader proteinase L. Thus, a history of genetic drift with accumulation of point mutations was needed to allow a major variation in the structure of a viral genome. Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.

DNA-COMPACT: DNA COMpression Based on a Pattern-Aware Contextual Modeling Technique

PubMed Central

Li, Pinghao; Wang, Shuang; Kim, Jihoon; Xiong, Hongkai; Ohno-Machado, Lucila; Jiang, Xiaoqian

2013-01-01

Genome data are becoming increasingly important for modern medicine. As the rate of increase in DNA sequencing outstrips the rate of increase in disk storage capacity, the storage and data transferring of large genome data are becoming important concerns for biomedical researchers. We propose a two-pass lossless genome compression algorithm, which highlights the synthesis of complementary contextual models, to improve the compression performance. The proposed framework could handle genome compression with and without reference sequences, and demonstrated performance advantages over best existing algorithms. The method for reference-free compression led to bit rates of 1.720 and 1.838 bits per base for bacteria and yeast, which were approximately 3.7% and 2.6% better than the state-of-the-art algorithms. Regarding performance with reference, we tested on the first Korean personal genome sequence data set, and our proposed method demonstrated a 189-fold compression rate, reducing the raw file size from 2986.8 MB to 15.8 MB at a comparable decompression cost with existing algorithms. DNAcompact is freely available at https://sourceforge.net/projects/dnacompact/for research purpose. PMID:24282536

A Concise Atlas of Thyroid Cancer Next-Generation Sequencing Panel ThyroSeq v.2

PubMed Central

Alsina, Jorge; Alsina, Raul; Gulec, Seza

2017-01-01

The next-generation sequencing technology allows high out-put genomic analysis. An innovative assay in thyroid cancer, ThyroSeq® was developed for targeted mutation detection by next generation sequencing technology in fine needle aspiration and tissue samples. ThyroSeq v.2 next generation sequencing panel offers simultaneous sequencing and detection in >1000 hotspots of 14 thyroid cancer-related genes and for 42 types of gene fusions known to occur in thyroid cancer. ThyroSeq is being increasingly used to further narrow the indeterminate category defined by cytology for thyroid nodules. From a surgical perspective, genomic profiling also provides prognostic and predictive information and closely relates to determination of surgical strategy. Both the genomic analysis technology and the informatics for the cancer genome data base are rapidly developing. In this paper, we have gathered existing information on the thyroid cancer-related genes involved in the initiation and progression of thyroid cancer. Our goal is to assemble a glossary for the current ThyroSeq genomic panel that can help elucidate the role genomics play in thyroid cancer oncogenesis. PMID:28117295

Sequence-Dependent Persistence Length of Long DNA

NASA Astrophysics Data System (ADS)

Chuang, Hui-Min; Reifenberger, Jeffrey G.; Cao, Han; Dorfman, Kevin D.

2017-12-01

Using a high-throughput genome-mapping approach, we obtained circa 50 million measurements of the extension of internal human DNA segments in a 41 nm ×41 nm nanochannel. The underlying DNA sequences, obtained by mapping to the reference human genome, are 2.5-393 kilobase pairs long and contain percent GC contents between 32.5% and 60%. Using Odijk's theory for a channel-confined wormlike chain, these data reveal that the DNA persistence length increases by almost 20% as the percent GC content increases. The increased persistence length is rationalized by a model, containing no adjustable parameters, that treats the DNA as a statistical terpolymer with a sequence-dependent intrinsic persistence length and a sequence-independent electrostatic persistence length.

Long-read sequencing data analysis for yeasts.

PubMed

Yue, Jia-Xing; Liti, Gianni

2018-06-01

Long-read sequencing technologies have become increasingly popular due to their strengths in resolving complex genomic regions. As a leading model organism with small genome size and great biotechnological importance, the budding yeast Saccharomyces cerevisiae has many isolates currently being sequenced with long reads. However, analyzing long-read sequencing data to produce high-quality genome assembly and annotation remains challenging. Here, we present a modular computational framework named long-read sequencing data analysis for yeasts (LRSDAY), the first one-stop solution that streamlines this process. Starting from the raw sequencing reads, LRSDAY can produce chromosome-level genome assembly and comprehensive genome annotation in a highly automated manner with minimal manual intervention, which is not possible using any alternative tool available to date. The annotated genomic features include centromeres, protein-coding genes, tRNAs, transposable elements (TEs), and telomere-associated elements. Although tailored for S. cerevisiae, we designed LRSDAY to be highly modular and customizable, making it adaptable to virtually any eukaryotic organism. When applying LRSDAY to an S. cerevisiae strain, it takes ∼41 h to generate a complete and well-annotated genome from ∼100× Pacific Biosciences (PacBio) running the basic workflow with four threads. Basic experience working within the Linux command-line environment is recommended for carrying out the analysis using LRSDAY.

A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions.

PubMed

Conte, Matthew A; Gammerdinger, William J; Bartie, Kerry L; Penman, David J; Kocher, Thomas D

2017-05-02

Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species. A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly (contig NG50 of 3.3Mbp) was selected using principal component analysis of likelihood scores calculated from several paired-end sequencing libraries. Comparison of the new assembly to the previous O. niloticus genome assembly reveals that recently duplicated portions of the genome are now well represented. The overall number of genes in the new assembly increased by 27.3%, including a 67% increase in pseudogenes. The new tilapia genome assembly correctly represents two recent vasa gene duplication events that have been verified with BAC sequencing. At total of 146Mbp of additional transposable element sequence are now assembled, a large proportion of which are recent insertions. Large centromeric satellite repeats are assembled and annotated in cichlid fish for the first time. Finally, the new assembly identifies the long-range structure of both a ~9Mbp XY sex determination region on LG1 in O. niloticus, and a ~50Mbp WZ sex determination region on LG3 in the related species O. aureus. This study highlights the use of long read sequencing to correctly assemble recent duplications and to characterize repeat-filled regions of the genome. The study serves as an example of the need for high quality genome assemblies and provides a framework for identifying sex determining genes in tilapia and related fish species.

Reverse Genetics and High Throughput Sequencing Methodologies for Plant Functional Genomics

PubMed Central

Ben-Amar, Anis; Daldoul, Samia; Reustle, Götz M.; Krczal, Gabriele; Mliki, Ahmed

2016-01-01

In the post-genomic era, increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of genes gives rise to a complex organism. With the advent of the next generation sequencing associated with effective computational approaches, wide variety of plant species have been fully sequenced giving a wealth of data sequence information on structure and organization of plant genomes. Since thousands of gene sequences are already known, recently developed functional genomics approaches provide powerful tools to analyze plant gene functions through various gene manipulation technologies. Integration of different omics platforms along with gene annotation and computational analysis may elucidate a complete view in a system biology level. Extensive investigations on reverse genetics methodologies were deployed for assigning biological function to a specific gene or gene product. We provide here an updated overview of these high throughout strategies highlighting recent advances in the knowledge of functional genomics in plants. PMID:28217003

ERGC: an efficient referential genome compression algorithm

PubMed Central

Saha, Subrata; Rajasekaran, Sanguthevar

2015-01-01

Motivation: Genome sequencing has become faster and more affordable. Consequently, the number of available complete genomic sequences is increasing rapidly. As a result, the cost to store, process, analyze and transmit the data is becoming a bottleneck for research and future medical applications. So, the need for devising efficient data compression and data reduction techniques for biological sequencing data is growing by the day. Although there exists a number of standard data compression algorithms, they are not efficient in compressing biological data. These generic algorithms do not exploit some inherent properties of the sequencing data while compressing. To exploit statistical and information-theoretic properties of genomic sequences, we need specialized compression algorithms. Five different next-generation sequencing data compression problems have been identified and studied in the literature. We propose a novel algorithm for one of these problems known as reference-based genome compression. Results: We have done extensive experiments using five real sequencing datasets. The results on real genomes show that our proposed algorithm is indeed competitive and performs better than the best known algorithms for this problem. It achieves compression ratios that are better than those of the currently best performing algorithms. The time to compress and decompress the whole genome is also very promising. Availability and implementation: The implementations are freely available for non-commercial purposes. They can be downloaded from http://engr.uconn.edu/∼rajasek/ERGC.zip. Contact: rajasek@engr.uconn.edu PMID:26139636

Human Genome Program

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

Not Available

1993-01-01

The DOE Human Genome program has grown tremendously, as shown by the marked increase in the number of genome-funded projects since the last workshop held in 1991. The abstracts in this book describe the genome research of DOE-funded grantees and contractors and invited guests, and all projects are represented at the workshop by posters. The 3-day meeting includes plenary sessions on ethical, legal, and social issues pertaining to the availability of genetic data; sequencing techniques, informatics support; and chromosome and cDNA mapping and sequencing.

Apollo: a sequence annotation editor

PubMed Central

Lewis, SE; Searle, SMJ; Harris, N; Gibson, M; Iyer, V; Richter, J; Wiel, C; Bayraktaroglu, L; Birney, E; Crosby, MA; Kaminker, JS; Matthews, BB; Prochnik, SE; Smith, CD; Tupy, JL; Rubin, GM; Misra, S; Mungall, CJ; Clamp, ME

2002-01-01

The well-established inaccuracy of purely computational methods for annotating genome sequences necessitates an interactive tool to allow biological experts to refine these approximations by viewing and independently evaluating the data supporting each annotation. Apollo was developed to meet this need, enabling curators to inspect genome annotations closely and edit them. FlyBase biologists successfully used Apollo to annotate the Drosophila melanogaster genome and it is increasingly being used as a starting point for the development of customized annotation editing tools for other genome projects. PMID:12537571

A high-throughput Sanger strategy for human mitochondrial genome sequencing

PubMed Central

2013-01-01

Background A population reference database of complete human mitochondrial genome (mtGenome) sequences is needed to enable the use of mitochondrial DNA (mtDNA) coding region data in forensic casework applications. However, the development of entire mtGenome haplotypes to forensic data quality standards is difficult and laborious. A Sanger-based amplification and sequencing strategy that is designed for automated processing, yet routinely produces high quality sequences, is needed to facilitate high-volume production of these mtGenome data sets. Results We developed a robust 8-amplicon Sanger sequencing strategy that regularly produces complete, forensic-quality mtGenome haplotypes in the first pass of data generation. The protocol works equally well on samples representing diverse mtDNA haplogroups and DNA input quantities ranging from 50 pg to 1 ng, and can be applied to specimens of varying DNA quality. The complete workflow was specifically designed for implementation on robotic instrumentation, which increases throughput and reduces both the opportunities for error inherent to manual processing and the cost of generating full mtGenome sequences. Conclusions The described strategy will assist efforts to generate complete mtGenome haplotypes which meet the highest data quality expectations for forensic genetic and other applications. Additionally, high-quality data produced using this protocol can be used to assess mtDNA data developed using newer technologies and chemistries. Further, the amplification strategy can be used to enrich for mtDNA as a first step in sample preparation for targeted next-generation sequencing. PMID:24341507

Complete Genome Sequences of Two Plant-Associated Pseudomonas putida Isolates with Increased Heavy-Metal Tolerance

PubMed Central

Cania, Barbara; Zadel, Urška; Schöler, Anne; Płaza, Grażyna A.; Schloter, Michael

2017-01-01

ABSTRACT We report here the complete genome sequences of two Pseudomonas putida isolates recovered from surface-sterilized roots of Sida hermaphrodita. The two isolates were characterized by an increased tolerance to zinc, cadmium, and lead. Furthermore, the strains showed typical plant growth-promoting properties, such as the production of indole acetic acid, cellulolytic enzymes, and siderophores. PMID:29167255

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes.

PubMed

Fredlake, Christopher P; Hert, Daniel G; Kan, Cheuk-Wai; Chiesl, Thomas N; Root, Brian E; Forster, Ryan E; Barron, Annelise E

2008-01-15

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require approximately 70 min to deliver approximately 650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered "hybrid" mechanism of DNA electromigration, in which DNA molecules alternate rapidly between repeating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs.

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes

PubMed Central

Fredlake, Christopher P.; Hert, Daniel G.; Kan, Cheuk-Wai; Chiesl, Thomas N.; Root, Brian E.; Forster, Ryan E.; Barron, Annelise E.

2008-01-01

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require ≈70 min to deliver ≈650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered “hybrid” mechanism of DNA electromigration, in which DNA molecules alternate rapidly between reptating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs. PMID:18184818

RSAT 2015: Regulatory Sequence Analysis Tools.

PubMed

Medina-Rivera, Alejandra; Defrance, Matthieu; Sand, Olivier; Herrmann, Carl; Castro-Mondragon, Jaime A; Delerce, Jeremy; Jaeger, Sébastien; Blanchet, Christophe; Vincens, Pierre; Caron, Christophe; Staines, Daniel M; Contreras-Moreira, Bruno; Artufel, Marie; Charbonnier-Khamvongsa, Lucie; Hernandez, Céline; Thieffry, Denis; Thomas-Chollier, Morgane; van Helden, Jacques

2015-07-01

RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Towards decoding the conifer giga-genome.

PubMed

Mackay, John; Dean, Jeffrey F D; Plomion, Christophe; Peterson, Daniel G; Cánovas, Francisco M; Pavy, Nathalie; Ingvarsson, Pär K; Savolainen, Outi; Guevara, M Ángeles; Fluch, Silvia; Vinceti, Barbara; Abarca, Dolores; Díaz-Sala, Carmen; Cervera, María-Teresa

2012-12-01

Several new initiatives have been launched recently to sequence conifer genomes including pines, spruces and Douglas-fir. Owing to the very large genome sizes ranging from 18 to 35 gigabases, sequencing even a single conifer genome had been considered unattainable until the recent throughput increases and cost reductions afforded by next generation sequencers. The purpose of this review is to describe the context for these new initiatives. A knowledge foundation has been acquired in several conifers of commercial and ecological interest through large-scale cDNA analyses, construction of genetic maps and gene mapping studies aiming to link phenotype and genotype. Exploratory sequencing in pines and spruces have pointed out some of the unique properties of these giga-genomes and suggested strategies that may be needed to extract value from their sequencing. The hope is that recent and pending developments in sequencing technology will contribute to rapidly filling the knowledge vacuum surrounding their structure, contents and evolution. Researchers are also making plans to use comparative analyses that will help to turn the data into a valuable resource for enhancing and protecting the world's conifer forests.

Increased genomic prediction accuracy in wheat breeding through spatial adjustment of field trial data.

PubMed

Lado, Bettina; Matus, Ivan; Rodríguez, Alejandra; Inostroza, Luis; Poland, Jesse; Belzile, François; del Pozo, Alejandro; Quincke, Martín; Castro, Marina; von Zitzewitz, Jarislav

2013-12-09

In crop breeding, the interest of predicting the performance of candidate cultivars in the field has increased due to recent advances in molecular breeding technologies. However, the complexity of the wheat genome presents some challenges for applying new technologies in molecular marker identification with next-generation sequencing. We applied genotyping-by-sequencing, a recently developed method to identify single-nucleotide polymorphisms, in the genomes of 384 wheat (Triticum aestivum) genotypes that were field tested under three different water regimes in Mediterranean climatic conditions: rain-fed only, mild water stress, and fully irrigated. We identified 102,324 single-nucleotide polymorphisms in these genotypes, and the phenotypic data were used to train and test genomic selection models intended to predict yield, thousand-kernel weight, number of kernels per spike, and heading date. Phenotypic data showed marked spatial variation. Therefore, different models were tested to correct the trends observed in the field. A mixed-model using moving-means as a covariate was found to best fit the data. When we applied the genomic selection models, the accuracy of predicted traits increased with spatial adjustment. Multiple genomic selection models were tested, and a Gaussian kernel model was determined to give the highest accuracy. The best predictions between environments were obtained when data from different years were used to train the model. Our results confirm that genotyping-by-sequencing is an effective tool to obtain genome-wide information for crops with complex genomes, that these data are efficient for predicting traits, and that correction of spatial variation is a crucial ingredient to increase prediction accuracy in genomic selection models.

The Genomics Education Partnership: Successful Integration of Research into Laboratory Classes at a Diverse Group of Undergraduate Institutions

PubMed Central

Shaffer, Christopher D.; Alvarez, Consuelo; Bailey, Cheryl; Barnard, Daron; Bhalla, Satish; Chandrasekaran, Chitra; Chandrasekaran, Vidya; Chung, Hui-Min; Dorer, Douglas R.; Du, Chunguang; Eckdahl, Todd T.; Poet, Jeff L.; Frohlich, Donald; Goodman, Anya L.; Gosser, Yuying; Hauser, Charles; Hoopes, Laura L.M.; Johnson, Diana; Jones, Christopher J.; Kaehler, Marian; Kokan, Nighat; Kopp, Olga R.; Kuleck, Gary A.; McNeil, Gerard; Moss, Robert; Myka, Jennifer L.; Nagengast, Alexis; Morris, Robert; Overvoorde, Paul J.; Shoop, Elizabeth; Parrish, Susan; Reed, Kelynne; Regisford, E. Gloria; Revie, Dennis; Rosenwald, Anne G.; Saville, Ken; Schroeder, Stephanie; Shaw, Mary; Skuse, Gary; Smith, Christopher; Smith, Mary; Spana, Eric P.; Spratt, Mary; Stamm, Joyce; Thompson, Jeff S.; Wawersik, Matthew; Wilson, Barbara A.; Youngblom, Jim; Leung, Wilson; Buhler, Jeremy; Mardis, Elaine R.; Lopatto, David

2010-01-01

Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning approach after participation in GEP projects. They show knowledge gains on pre- and postcourse quizzes about genes and genomes and in bioinformatic analysis. Participating faculty also report professional gains, increased access to genomics-related technology, and an overall positive experience. We have found that using a genomics research project as the core of a laboratory course is rewarding for both faculty and students. PMID:20194808

Unlimited Thirst for Genome Sequencing, Data Interpretation, and Database Usage in Genomic Era: The Road towards Fast-Track Crop Plant Improvement

PubMed Central

Govindaraj, Mahalingam

2015-01-01

The number of sequenced crop genomes and associated genomic resources is growing rapidly with the advent of inexpensive next generation sequencing methods. Databases have become an integral part of all aspects of science research, including basic and applied plant and animal sciences. The importance of databases keeps increasing as the volume of datasets from direct and indirect genomics, as well as other omics approaches, keeps expanding in recent years. The databases and associated web portals provide at a minimum a uniform set of tools and automated analysis across a wide range of crop plant genomes. This paper reviews some basic terms and considerations in dealing with crop plant databases utilization in advancing genomic era. The utilization of databases for variation analysis with other comparative genomics tools, and data interpretation platforms are well described. The major focus of this review is to provide knowledge on platforms and databases for genome-based investigations of agriculturally important crop plants. The utilization of these databases in applied crop improvement program is still being achieved widely; otherwise, the end for sequencing is not far away. PMID:25874133

Genomics in Cardiovascular Disease

PubMed Central

Roberts, Robert; Marian, A.J.; Dandona, Sonny; Stewart, Alexandre F.R.

2013-01-01

A paradigm shift towards biology occurred in the 1990’s subsequently catalyzed by the sequencing of the human genome in 2000. The cost of DNA sequencing has gone from millions to thousands of dollars with sequencing of one’s entire genome costing only $1,000. Rapid DNA sequencing is being embraced for single gene disorders, particularly for sporadic cases and those from small families. Transmission of lethal genes such as associated with Huntington’s disease can, through in-vitro fertilization, avoid passing it on to one’s offspring. DNA sequencing will meet the challenge of elucidating the genetic predisposition for common polygenic diseases, especially in determining the function of the novel common genetic risk variants and identifying the rare variants, which may also partially ascertain the source of the missing heritability. The challenge for DNA sequencing remains great, despite human genome sequences being 99.5% identical, the 3 million single nucleotide polymorphisms (SNPs) responsible for most of the unique features add up to 60 new mutations per person which, for 7 billion people, is 420 billion mutations. It is claimed that DNA sequencing has increased 10,000 fold while information storage and retrieval only 16 fold. The physician and health user will be challenged by the convergence of two major trends, whole genome sequencing and the storage/retrieval and integration of the data. PMID:23524054

Simple and efficient identification of rare recessive pathologically important sequence variants from next generation exome sequence data.

PubMed

Carr, Ian M; Morgan, Joanne; Watson, Christopher; Melnik, Svitlana; Diggle, Christine P; Logan, Clare V; Harrison, Sally M; Taylor, Graham R; Pena, Sergio D J; Markham, Alexander F; Alkuraya, Fowzan S; Black, Graeme C M; Ali, Manir; Bonthron, David T

2013-07-01

Massively parallel ("next generation") DNA sequencing (NGS) has quickly become the method of choice for seeking pathogenic mutations in rare uncharacterized monogenic diseases. Typically, before DNA sequencing, protein-coding regions are enriched from patient genomic DNA, representing either the entire genome ("exome sequencing") or selected mapped candidate loci. Sequence variants, identified as differences between the patient's and the human genome reference sequences, are then filtered according to various quality parameters. Changes are screened against datasets of known polymorphisms, such as dbSNP and the 1000 Genomes Project, in the effort to narrow the list of candidate causative variants. An increasing number of commercial services now offer to both generate and align NGS data to a reference genome. This potentially allows small groups with limited computing infrastructure and informatics skills to utilize this technology. However, the capability to effectively filter and assess sequence variants is still an important bottleneck in the identification of deleterious sequence variants in both research and diagnostic settings. We have developed an approach to this problem comprising a user-friendly suite of programs that can interactively analyze, filter and screen data from enrichment-capture NGS data. These programs ("Agile Suite") are particularly suitable for small-scale gene discovery or for diagnostic analysis. © 2013 WILEY PERIODICALS, INC.

Genome and Transcriptome Sequencing of the Ostreid herpesvirus 1 From Tomales Bay, California

NASA Astrophysics Data System (ADS)

Burge, C. A.; Langevin, S.; Closek, C. J.; Roberts, S. B.; Friedman, C. S.

2016-02-01

Mass mortalities of larval and seed bivalve molluscs attributed to the Ostreid herpesvirus 1 (OsHV-1) occur globally. OsHV-1 was fully sequenced and characterized as a member of the Family Malacoherpesviridae. Multiple strains of OsHV-1 exist and may vary in virulence, i.e. OsHV-1 µvar. For most global variants of OsHV-1, sequence data is limited to PCR-based sequencing of segments, including two recent genomes. In the United States, OsHV-1 is limited to detection in adjacent embayments in California, Tomales and Drakes bays. Limited DNA sequence data of OsHV-1 infecting oysters in Tomales Bay indicates the virus detected in Tomales Bay is similar but not identical to any one global variant of OsHV-1. In order to better understand both strain variation and virulence of OsHV-1 infecting oysters in Tomales Bay, we used genomic and transcriptomic sequencing. Meta-genomic sequencing (Illumina MiSeq) was conducted from infected oysters (n=4 per year) collected in 2003, 2007, and 2014, where full OsHV-1 genome sequences and low overall microbial diversity were achieved from highly infected oysters. Increased microbial diversity was detected in three of four samples sequenced from 2003, where qPCR based genome copy numbers of OsHV-1 were lower. Expression analysis (SOLiD RNA sequencing) of OsHV-1 genes expressed in oyster larvae at 24 hours post exposure revealed a nearly complete transcriptome, with several highly expressed genes, which are similar to recent transcriptomic analyses of other OsHV-1 variants. Taken together, our results indicate that genome and transcriptome sequencing may be powerful tools in understanding both strain variation and virulence of non-culturable marine viruses.

Improving de novo sequence assembly using machine learning and comparative genomics for overlap correction.

PubMed

Palmer, Lance E; Dejori, Mathaeus; Bolanos, Randall; Fasulo, Daniel

2010-01-15

With the rapid expansion of DNA sequencing databases, it is now feasible to identify relevant information from prior sequencing projects and completed genomes and apply it to de novo sequencing of new organisms. As an example, this paper demonstrates how such extra information can be used to improve de novo assemblies by augmenting the overlapping step. Finding all pairs of overlapping reads is a key task in many genome assemblers, and to this end, highly efficient algorithms have been developed to find alignments in large collections of sequences. It is well known that due to repeated sequences, many aligned pairs of reads nevertheless do not overlap. But no overlapping algorithm to date takes a rigorous approach to separating aligned but non-overlapping read pairs from true overlaps. We present an approach that extends the Minimus assembler by a data driven step to classify overlaps as true or false prior to contig construction. We trained several different classification models within the Weka framework using various statistics derived from overlaps of reads available from prior sequencing projects. These statistics included percent mismatch and k-mer frequencies within the overlaps as well as a comparative genomics score derived from mapping reads to multiple reference genomes. We show that in real whole-genome sequencing data from the E. coli and S. aureus genomes, by providing a curated set of overlaps to the contigging phase of the assembler, we nearly doubled the median contig length (N50) without sacrificing coverage of the genome or increasing the number of mis-assemblies. Machine learning methods that use comparative and non-comparative features to classify overlaps as true or false can be used to improve the quality of a sequence assembly.

A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing

PubMed Central

Alioto, Tyler S.; Buchhalter, Ivo; Derdak, Sophia; Hutter, Barbara; Eldridge, Matthew D.; Hovig, Eivind; Heisler, Lawrence E.; Beck, Timothy A.; Simpson, Jared T.; Tonon, Laurie; Sertier, Anne-Sophie; Patch, Ann-Marie; Jäger, Natalie; Ginsbach, Philip; Drews, Ruben; Paramasivam, Nagarajan; Kabbe, Rolf; Chotewutmontri, Sasithorn; Diessl, Nicolle; Previti, Christopher; Schmidt, Sabine; Brors, Benedikt; Feuerbach, Lars; Heinold, Michael; Gröbner, Susanne; Korshunov, Andrey; Tarpey, Patrick S.; Butler, Adam P.; Hinton, Jonathan; Jones, David; Menzies, Andrew; Raine, Keiran; Shepherd, Rebecca; Stebbings, Lucy; Teague, Jon W.; Ribeca, Paolo; Giner, Francesc Castro; Beltran, Sergi; Raineri, Emanuele; Dabad, Marc; Heath, Simon C.; Gut, Marta; Denroche, Robert E.; Harding, Nicholas J.; Yamaguchi, Takafumi N.; Fujimoto, Akihiro; Nakagawa, Hidewaki; Quesada, Víctor; Valdés-Mas, Rafael; Nakken, Sigve; Vodák, Daniel; Bower, Lawrence; Lynch, Andrew G.; Anderson, Charlotte L.; Waddell, Nicola; Pearson, John V.; Grimmond, Sean M.; Peto, Myron; Spellman, Paul; He, Minghui; Kandoth, Cyriac; Lee, Semin; Zhang, John; Létourneau, Louis; Ma, Singer; Seth, Sahil; Torrents, David; Xi, Liu; Wheeler, David A.; López-Otín, Carlos; Campo, Elías; Campbell, Peter J.; Boutros, Paul C.; Puente, Xose S.; Gerhard, Daniela S.; Pfister, Stefan M.; McPherson, John D.; Hudson, Thomas J.; Schlesner, Matthias; Lichter, Peter; Eils, Roland; Jones, David T. W.; Gut, Ivo G.

2015-01-01

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy. PMID:26647970

Complete Genome Sequence of Bordetella pertussis Strain VA-190 Isolated from a Vaccinated 10-Year-Old Patient with Whooping Cough.

PubMed

Eby, Joshua C; Turner, Lauren; Nguyen, Bryan; Kang, June; Neville, Carly; Temple, Louise

2016-09-15

The number of cases of pertussis has increased in the United States despite vaccination. We present the genome of an isolate of Bordetella pertussis from a vaccinated patient from Virginia. The genome was sequenced by long-read methodology and compared to that of a clinical isolate used for laboratory studies, D420. Copyright © 2016 Eby et al.

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.

Real-time, portable genome sequencing for Ebola surveillance

PubMed Central

Bore, Joseph Akoi; Koundouno, Raymond; Dudas, Gytis; Mikhail, Amy; Ouédraogo, Nobila; Afrough, Babak; Bah, Amadou; Baum, Jonathan HJ; Becker-Ziaja, Beate; Boettcher, Jan-Peter; Cabeza-Cabrerizo, Mar; Camino-Sanchez, Alvaro; Carter, Lisa L.; Doerrbecker, Juiliane; Enkirch, Theresa; Dorival, Isabel Graciela García; Hetzelt, Nicole; Hinzmann, Julia; Holm, Tobias; Kafetzopoulou, Liana Eleni; Koropogui, Michel; Kosgey, Abigail; Kuisma, Eeva; Logue, Christopher H; Mazzarelli, Antonio; Meisel, Sarah; Mertens, Marc; Michel, Janine; Ngabo, Didier; Nitzsche, Katja; Pallash, Elisa; Patrono, Livia Victoria; Portmann, Jasmine; Repits, Johanna Gabriella; Rickett, Natasha Yasmin; Sachse, Andrea; Singethan, Katrin; Vitoriano, Inês; Yemanaberhan, Rahel L; Zekeng, Elsa G; Trina, Racine; Bello, Alexander; Sall, Amadou Alpha; Faye, Ousmane; Faye, Oumar; Magassouba, N’Faly; Williams, Cecelia V.; Amburgey, Victoria; Winona, Linda; Davis, Emily; Gerlach, Jon; Washington, Franck; Monteil, Vanessa; Jourdain, Marine; Bererd, Marion; Camara, Alimou; Somlare, Hermann; Camara, Abdoulaye; Gerard, Marianne; Bado, Guillaume; Baillet, Bernard; Delaune, Déborah; Nebie, Koumpingnin Yacouba; Diarra, Abdoulaye; Savane, Yacouba; Pallawo, Raymond Bernard; Gutierrez, Giovanna Jaramillo; Milhano, Natacha; Roger, Isabelle; Williams, Christopher J; Yattara, Facinet; Lewandowski, Kuiama; Taylor, Jamie; Rachwal, Philip; Turner, Daniel; Pollakis, Georgios; Hiscox, Julian A.; Matthews, David A.; O’Shea, Matthew K.; Johnston, Andrew McD; Wilson, Duncan; Hutley, Emma; Smit, Erasmus; Di Caro, Antonino; Woelfel, Roman; Stoecker, Kilian; Fleischmann, Erna; Gabriel, Martin; Weller, Simon A.; Koivogui, Lamine; Diallo, Boubacar; Keita, Sakoba; Rambaut, Andrew; Formenty, Pierre; Gunther, Stephan; Carroll, Miles W.

2016-01-01

The Ebola virus disease (EVD) epidemic in West Africa is the largest on record, responsible for >28,599 cases and >11,299 deaths 1. Genome sequencing in viral outbreaks is desirable in order to characterize the infectious agent to determine its evolutionary rate, signatures of host adaptation, identification and monitoring of diagnostic targets and responses to vaccines and treatments. The Ebola virus genome (EBOV) substitution rate in the Makona strain has been estimated at between 0.87 × 10−3 to 1.42 × 10−3 mutations per site per year. This is equivalent to 16 to 27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic 2-7. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought-after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions 8. Genomic surveillance during the epidemic has been sporadic due to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities 9. In order to address this problem, we devised a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. Here we present sequence data and analysis of 142 Ebola virus (EBOV) samples collected during the period March to October 2015. We were able to generate results in less than 24 hours after receiving an Ebola positive sample, with the sequencing process taking as little as 15-60 minutes. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks. PMID:26840485

Improvement of the banana "Musa acuminata" reference sequence using NGS data and semi-automated bioinformatics methods.

PubMed

Martin, Guillaume; Baurens, Franc-Christophe; Droc, Gaëtan; Rouard, Mathieu; Cenci, Alberto; Kilian, Andrzej; Hastie, Alex; Doležel, Jaroslav; Aury, Jean-Marc; Alberti, Adriana; Carreel, Françoise; D'Hont, Angélique

2016-03-16

Recent advances in genomics indicate functional significance of a majority of genome sequences and their long range interactions. As a detailed examination of genome organization and function requires very high quality genome sequence, the objective of this study was to improve reference genome assembly of banana (Musa acuminata). We have developed a modular bioinformatics pipeline to improve genome sequence assemblies, which can handle various types of data. The pipeline comprises several semi-automated tools. However, unlike classical automated tools that are based on global parameters, the semi-automated tools proposed an expert mode for a user who can decide on suggested improvements through local compromises. The pipeline was used to improve the draft genome sequence of Musa acuminata. Genotyping by sequencing (GBS) of a segregating population and paired-end sequencing were used to detect and correct scaffold misassemblies. Long insert size paired-end reads identified scaffold junctions and fusions missed by automated assembly methods. GBS markers were used to anchor scaffolds to pseudo-molecules with a new bioinformatics approach that avoids the tedious step of marker ordering during genetic map construction. Furthermore, a genome map was constructed and used to assemble scaffolds into super scaffolds. Finally, a consensus gene annotation was projected on the new assembly from two pre-existing annotations. This approach reduced the total Musa scaffold number from 7513 to 1532 (i.e. by 80%), with an N50 that increased from 1.3 Mb (65 scaffolds) to 3.0 Mb (26 scaffolds). 89.5% of the assembly was anchored to the 11 Musa chromosomes compared to the previous 70%. Unknown sites (N) were reduced from 17.3 to 10.0%. The release of the Musa acuminata reference genome version 2 provides a platform for detailed analysis of banana genome variation, function and evolution. Bioinformatics tools developed in this work can be used to improve genome sequence assemblies in other species.

Microfluidic droplet enrichment for targeted sequencing

PubMed Central

Eastburn, Dennis J.; Huang, Yong; Pellegrino, Maurizio; Sciambi, Adam; Ptáček, Louis J.; Abate, Adam R.

2015-01-01

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications. PMID:25873629

The dynamics of genome replication using deep sequencing

PubMed Central

Müller, Carolin A.; Hawkins, Michelle; Retkute, Renata; Malla, Sunir; Wilson, Ray; Blythe, Martin J.; Nakato, Ryuichiro; Komata, Makiko; Shirahige, Katsuhiko; de Moura, Alessandro P.S.; Nieduszynski, Conrad A.

2014-01-01

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology. PMID:24089142

The unique genomic landscape surrounding the EPSPS gene in glyphosate resistant Amaranthus palmeri: a repetitive path to resistance.

PubMed

Molin, William T; Wright, Alice A; Lawton-Rauh, Amy; Saski, Christopher A

2017-01-17

The expanding number and global distributions of herbicide resistant weedy species threaten food, fuel, fiber and bioproduct sustainability and agroecosystem longevity. Amongst the most competitive weeds, Amaranthus palmeri S. Wats has rapidly evolved resistance to glyphosate primarily through massive amplification and insertion of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene across the genome. Increased EPSPS gene copy numbers results in higher titers of the EPSPS enzyme, the target of glyphosate, and confers resistance to glyphosate treatment. To understand the genomic unit and mechanism of EPSPS gene copy number proliferation, we developed and used a bacterial artificial chromosome (BAC) library from a highly resistant biotype to sequence the local genomic landscape flanking the EPSPS gene. By sequencing overlapping BACs, a 297 kb sequence was generated, hereafter referred to as the "EPSPS cassette." This region included several putative genes, dense clusters of tandem and inverted repeats, putative helitron and autonomous replication sequences, and regulatory elements. Whole genome shotgun sequencing (WGS) of two biotypes exhibiting high and no resistance to glyphosate was performed to compare genomic representation across the EPSPS cassette. Mapping of sequences for both biotypes to the reference EPSPS cassette revealed significant differences in upstream and downstream sequences relative to EPSPS with regard to both repetitive units and coding content between these biotypes. The differences in sequence may have resulted from a compounded-building mechanism such as repetitive transpositional events. The association of putative helitron sequences with the cassette suggests a possible amplification and distribution mechanism. Flow cytometry revealed that the EPSPS cassette added measurable genomic content. The adoption of glyphosate resistant cropping systems in major crops such as corn, soybean, cotton and canola coupled with excessive use of glyphosate herbicide has led to evolved glyphosate resistance in several important weeds. In Amaranthus palmeri, the amplification of the EPSPS cassette, characterized by a complex array of repetitive elements and putative helitron sequences, suggests an adaptive structural genomic mechanism that drives amplification and distribution around the genome. The added genomic content not found in glyphosate sensitive plants may be driving evolution through genome expansion.

Genomic Encyclopedia of Type Strains, Phase I: The one thousand microbial genomes (KMG-I) project

DOE PAGES

Kyrpides, Nikos C.; Woyke, Tanja; Eisen, Jonathan A.; ...

2014-06-15

The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project with the objective of sequencing 250 bacterial and archaeal genomes. The two major goals of that project were (a) to test the hypothesis that there are many benefits to the use the phylogenetic diversity of organisms in the tree of life as a primary criterion for generating their genome sequence and (b) to develop the necessary framework, technology and organization for large-scale sequencing of microbial isolate genomes. While the GEBA pilot project has not yet been entirely completed, both ofmore » the original goals have already been successfully accomplished, leading the way for the next phase of the project. Here we propose taking the GEBA project to the next level, by generating high quality draft genomes for 1,000 bacterial and archaeal strains. This represents a combined 16-fold increase in both scale and speed as compared to the GEBA pilot project (250 isolate genomes in 4+ years). We will follow a similar approach for organism selection and sequencing prioritization as was done for the GEBA pilot project (i.e. phylogenetic novelty, availability and growth of cultures of type strains and DNA extraction capability), focusing on type strains as this ensures reproducibility of our results and provides the strongest linkage between genome sequences and other knowledge about each strain. In turn, this project will constitute a pilot phase of a larger effort that will target the genome sequences of all available type strains of the Bacteria and Archaea.« less

Genomic Encyclopedia of Type Strains, Phase I: The one thousand microbial genomes (KMG-I) project

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

Kyrpides, Nikos C.; Woyke, Tanja; Eisen, Jonathan A.

The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project with the objective of sequencing 250 bacterial and archaeal genomes. The two major goals of that project were (a) to test the hypothesis that there are many benefits to the use the phylogenetic diversity of organisms in the tree of life as a primary criterion for generating their genome sequence and (b) to develop the necessary framework, technology and organization for large-scale sequencing of microbial isolate genomes. While the GEBA pilot project has not yet been entirely completed, both ofmore » the original goals have already been successfully accomplished, leading the way for the next phase of the project. Here we propose taking the GEBA project to the next level, by generating high quality draft genomes for 1,000 bacterial and archaeal strains. This represents a combined 16-fold increase in both scale and speed as compared to the GEBA pilot project (250 isolate genomes in 4+ years). We will follow a similar approach for organism selection and sequencing prioritization as was done for the GEBA pilot project (i.e. phylogenetic novelty, availability and growth of cultures of type strains and DNA extraction capability), focusing on type strains as this ensures reproducibility of our results and provides the strongest linkage between genome sequences and other knowledge about each strain. In turn, this project will constitute a pilot phase of a larger effort that will target the genome sequences of all available type strains of the Bacteria and Archaea.« less

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.

Clinical Actionability of Comprehensive Genomic Profiling for Management of Rare or Refractory Cancers

PubMed Central

Hirshfield, Kim M.; Tolkunov, Denis; Zhong, Hua; Ali, Siraj M.; Stein, Mark N.; Murphy, Susan; Vig, Hetal; Vazquez, Alexei; Glod, John; Moss, Rebecca A.; Belyi, Vladimir; Chan, Chang S.; Chen, Suzie; Goodell, Lauri; Foran, David; Yelensky, Roman; Palma, Norma A.; Sun, James X.; Miller, Vincent A.; Stephens, Philip J.; Ross, Jeffrey S.; Kaufman, Howard; Poplin, Elizabeth; Mehnert, Janice; Tan, Antoinette R.; Bertino, Joseph R.; Aisner, Joseph; DiPaola, Robert S.

2016-01-01

Background. The frequency with which targeted tumor sequencing results will lead to implemented change in care is unclear. Prospective assessment of the feasibility and limitations of using genomic sequencing is critically important. Methods. A prospective clinical study was conducted on 100 patients with diverse-histology, rare, or poor-prognosis cancers to evaluate the clinical actionability of a Clinical Laboratory Improvement Amendments (CLIA)-certified, comprehensive genomic profiling assay (FoundationOne), using formalin-fixed, paraffin-embedded tumors. The primary objectives were to assess utility, feasibility, and limitations of genomic sequencing for genomically guided therapy or other clinical purpose in the setting of a multidisciplinary molecular tumor board. Results. Of the tumors from the 92 patients with sufficient tissue, 88 (96%) had at least one genomic alteration (average 3.6, range 0–10). Commonly altered pathways included p53 (46%), RAS/RAF/MAPK (rat sarcoma; rapidly accelerated fibrosarcoma; mitogen-activated protein kinase) (45%), receptor tyrosine kinases/ligand (44%), PI3K/AKT/mTOR (phosphatidylinositol-4,5-bisphosphate 3-kinase; protein kinase B; mammalian target of rapamycin) (35%), transcription factors/regulators (31%), and cell cycle regulators (30%). Many low frequency but potentially actionable alterations were identified in diverse histologies. Use of comprehensive profiling led to implementable clinical action in 35% of tumors with genomic alterations, including genomically guided therapy, diagnostic modification, and trigger for germline genetic testing. Conclusion. Use of targeted next-generation sequencing in the setting of an institutional molecular tumor board led to implementable clinical action in more than one third of patients with rare and poor-prognosis cancers. Major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access. Early and serial sequencing in the clinical course and expanded access to genomically guided early-phase clinical trials and targeted agents may increase actionability. Implications for Practice: Identification of key factors that facilitate use of genomic tumor testing results and implementation of genomically guided therapy may lead to enhanced benefit for patients with rare or difficult to treat cancers. Clinical use of a targeted next-generation sequencing assay in the setting of an institutional molecular tumor board led to implementable clinical action in over one third of patients with rare and poor prognosis cancers. The major barriers to implementation of genomically guided therapy were clinical status of the patient and drug access both on trial and off label. Approaches to increase actionability include early and serial sequencing in the clinical course and expanded access to genomically guided early phase clinical trials and targeted agents. PMID:27566247

Ensembl Genomes 2016: more genomes, more complexity.

PubMed

Kersey, Paul Julian; Allen, James E; Armean, Irina; Boddu, Sanjay; Bolt, Bruce J; Carvalho-Silva, Denise; Christensen, Mikkel; Davis, Paul; Falin, Lee J; Grabmueller, Christoph; Humphrey, Jay; Kerhornou, Arnaud; Khobova, Julia; Aranganathan, Naveen K; Langridge, Nicholas; Lowy, Ernesto; McDowall, Mark D; Maheswari, Uma; Nuhn, Michael; Ong, Chuang Kee; Overduin, Bert; Paulini, Michael; Pedro, Helder; Perry, Emily; Spudich, Giulietta; Tapanari, Electra; Walts, Brandon; Williams, Gareth; Tello-Ruiz, Marcela; Stein, Joshua; Wei, Sharon; Ware, Doreen; Bolser, Daniel M; Howe, Kevin L; Kulesha, Eugene; Lawson, Daniel; Maslen, Gareth; Staines, Daniel M

2016-01-04

Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species, complementing the resources for vertebrate genomics developed in the context of the Ensembl project (http://www.ensembl.org). Together, the two resources provide a consistent set of programmatic and interactive interfaces to a rich range of data including reference sequence, gene models, transcriptional data, genetic variation and comparative analysis. This paper provides an update to the previous publications about the resource, with a focus on recent developments. These include the development of new analyses and views to represent polyploid genomes (of which bread wheat is the primary exemplar); and the continued up-scaling of the resource, which now includes over 23 000 bacterial genomes, 400 fungal genomes and 100 protist genomes, in addition to 55 genomes from invertebrate metazoa and 39 genomes from plants. This dramatic increase in the number of included genomes is one part of a broader effort to automate the integration of archival data (genome sequence, but also associated RNA sequence data and variant calls) within the context of reference genomes and make it available through the Ensembl user interfaces. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Ensembl Genomes 2016: more genomes, more complexity

PubMed Central

Kersey, Paul Julian; Allen, James E.; Armean, Irina; Boddu, Sanjay; Bolt, Bruce J.; Carvalho-Silva, Denise; Christensen, Mikkel; Davis, Paul; Falin, Lee J.; Grabmueller, Christoph; Humphrey, Jay; Kerhornou, Arnaud; Khobova, Julia; Aranganathan, Naveen K.; Langridge, Nicholas; Lowy, Ernesto; McDowall, Mark D.; Maheswari, Uma; Nuhn, Michael; Ong, Chuang Kee; Overduin, Bert; Paulini, Michael; Pedro, Helder; Perry, Emily; Spudich, Giulietta; Tapanari, Electra; Walts, Brandon; Williams, Gareth; Tello–Ruiz, Marcela; Stein, Joshua; Wei, Sharon; Ware, Doreen; Bolser, Daniel M.; Howe, Kevin L.; Kulesha, Eugene; Lawson, Daniel; Maslen, Gareth; Staines, Daniel M.

2016-01-01

Ensembl Genomes (http://www.ensemblgenomes.org) is an integrating resource for genome-scale data from non-vertebrate species, complementing the resources for vertebrate genomics developed in the context of the Ensembl project (http://www.ensembl.org). Together, the two resources provide a consistent set of programmatic and interactive interfaces to a rich range of data including reference sequence, gene models, transcriptional data, genetic variation and comparative analysis. This paper provides an update to the previous publications about the resource, with a focus on recent developments. These include the development of new analyses and views to represent polyploid genomes (of which bread wheat is the primary exemplar); and the continued up-scaling of the resource, which now includes over 23 000 bacterial genomes, 400 fungal genomes and 100 protist genomes, in addition to 55 genomes from invertebrate metazoa and 39 genomes from plants. This dramatic increase in the number of included genomes is one part of a broader effort to automate the integration of archival data (genome sequence, but also associated RNA sequence data and variant calls) within the context of reference genomes and make it available through the Ensembl user interfaces. PMID:26578574

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer.

PubMed

Wang, Ji; Ke, Yue Hua; Zhang, Yong; Huang, Ke Qiang; Wang, Lei; Shen, Xin Xin; Dong, Xiao Ping; Xu, Wen Bo; Ma, Xue Jun

2017-10-01

Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run. MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Identifiability, genomics and U.K. data protection law.

PubMed

Curren, Liam; Boddington, Paula; Gowans, Heather; Hawkins, Naomi; Kanellopoulou, Nadja; Kaye, Jane; Melham, Karen

2010-09-01

Analyses of individuals' genomes--their entire DNA sequence--have increased knowledge about the links between genetics and disease. Anticipated advances in 'next generation' DNA-sequencing techniques will see the routine research use of whole genomes, rather than distinct parts, within the next few years. The scientific benefits of genomic research are, however, accompanied by legal and ethical concerns. Despite the assumption that genetic research data can and will be rendered anonymous, participants' identities can sometimes be elucidated, which could cause data protection legislation to apply. We undertake a timely reappraisal of these laws--particularly new penalties--and identifiability in genomic research.

Aligner optimization increases accuracy and decreases compute times in multi-species sequence data.

PubMed

Robinson, Kelly M; Hawkins, Aziah S; Santana-Cruz, Ivette; Adkins, Ricky S; Shetty, Amol C; Nagaraj, Sushma; Sadzewicz, Lisa; Tallon, Luke J; Rasko, David A; Fraser, Claire M; Mahurkar, Anup; Silva, Joana C; Dunning Hotopp, Julie C

2017-09-01

As sequencing technologies have evolved, the tools to analyze these sequences have made similar advances. However, for multi-species samples, we observed important and adverse differences in alignment specificity and computation time for bwa- mem (Burrows-Wheeler aligner-maximum exact matches) relative to bwa-aln. Therefore, we sought to optimize bwa-mem for alignment of data from multi-species samples in order to reduce alignment time and increase the specificity of alignments. In the multi-species cases examined, there was one majority member (i.e. Plasmodium falciparum or Brugia malayi ) and one minority member (i.e. human or the Wolbachia endosymbiont w Bm) of the sequence data. Increasing bwa-mem seed length from the default value reduced the number of read pairs from the majority sequence member that incorrectly aligned to the reference genome of the minority sequence member. Combining both source genomes into a single reference genome increased the specificity of mapping, while also reducing the central processing unit (CPU) time. In Plasmodium , at a seed length of 18 nt, 24.1 % of reads mapped to the human genome using 1.7±0.1 CPU hours, while 83.6 % of reads mapped to the Plasmodium genome using 0.2±0.0 CPU hours (total: 107.7 % reads mapping; in 1.9±0.1 CPU hours). In contrast, 97.1 % of the reads mapped to a combined Plasmodium- human reference in only 0.7±0.0 CPU hours. Overall, the results suggest that combining all references into a single reference database and using a 23 nt seed length reduces the computational time, while maximizing specificity. Similar results were found for simulated sequence reads from a mock metagenomic data set. We found similar improvements to computation time in a publicly available human-only data set.

The Importance of Biological Databases in Biological Discovery.

PubMed

Baxevanis, Andreas D; Bateman, Alex

2015-06-19

Biological databases play a central role in bioinformatics. They offer scientists the opportunity to access a wide variety of biologically relevant data, including the genomic sequences of an increasingly broad range of organisms. This unit provides a brief overview of major sequence databases and portals, such as GenBank, the UCSC Genome Browser, and Ensembl. Model organism databases, including WormBase, The Arabidopsis Information Resource (TAIR), and those made available through the Mouse Genome Informatics (MGI) resource, are also covered. Non-sequence-centric databases, such as Online Mendelian Inheritance in Man (OMIM), the Protein Data Bank (PDB), MetaCyc, and the Kyoto Encyclopedia of Genes and Genomes (KEGG), are also discussed. Copyright © 2015 John Wiley & Sons, Inc.

Defining the Estimated Core Genome of Bacterial Populations Using a Bayesian Decision Model

PubMed Central

van Tonder, Andries J.; Mistry, Shilan; Bray, James E.; Hill, Dorothea M. C.; Cody, Alison J.; Farmer, Chris L.; Klugman, Keith P.; von Gottberg, Anne; Bentley, Stephen D.; Parkhill, Julian; Jolley, Keith A.; Maiden, Martin C. J.; Brueggemann, Angela B.

2014-01-01

The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domain available to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterial core genome from next-generation whole genome sequencing data and use this model to identify novel genes associated with important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developed a Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances) based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genome location. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studies demonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequence diversity in the Campylobacter jejuni and Neisseria meningitidis genomes encoded hypothetical proteins. The core gene with the highest p-distance value in C. jejuni was annotated in the reference genome as a putative hydrolase, but further work revealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistance to a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essential for DNA replication) in other C. jejuni genomes. Our Bayesian model of estimating the core genome is principled, easy to use and can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for many core genes in bacterial genomes of significant global public health importance. PMID:25144616

Complete Genome Sequences of Two Plant-Associated Pseudomonas putida Isolates with Increased Heavy-Metal Tolerance.

PubMed

Nesme, Joseph; Cania, Barbara; Zadel, Urška; Schöler, Anne; Płaza, Grażyna A; Schloter, Michael

2017-11-22

We report here the complete genome sequences of two Pseudomonas putida isolates recovered from surface-sterilized roots of Sida hermaphrodita The two isolates were characterized by an increased tolerance to zinc, cadmium, and lead. Furthermore, the strains showed typical plant growth-promoting properties, such as the production of indole acetic acid, cellulolytic enzymes, and siderophores. Copyright © 2017 Nesme et al.

Sequence analysis of the PIP5K locus in Eimeria maxima provides further evidence for eimerian genome plasticity and segmental organization.

PubMed

Song, B K; Pan, M Z; Lau, Y L; Wan, K L

2014-07-29

Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.

The draft genome of MD-2 pineapple using hybrid error correction of long reads

PubMed Central

Redwan, Raimi M.; Saidin, Akzam; Kumar, S. Vijay

2016-01-01

The introduction of the elite pineapple variety, MD-2, has caused a significant market shift in the pineapple industry. Better productivity, overall increased in fruit quality and taste, resilience to chilled storage and resistance to internal browning are among the key advantages of the MD-2 as compared with its previous predecessor, the Smooth Cayenne. Here, we present the genome sequence of the MD-2 pineapple (Ananas comosus (L.) Merr.) by using the hybrid sequencing technology from two highly reputable platforms, i.e. the PacBio long sequencing reads and the accurate Illumina short reads. Our draft genome achieved 99.6% genome coverage with 27,017 predicted protein-coding genes while 45.21% of the genome was identified as repetitive elements. Furthermore, differential expression of ripening RNASeq library of pineapple fruits revealed ethylene-related transcripts, believed to be involved in regulating the process of non-climacteric pineapple fruit ripening. The MD-2 pineapple draft genome serves as an example of how a complex heterozygous genome is amenable to whole genome sequencing by using a hybrid technology that is both economical and accurate. The genome will make genomic applications more feasible as a medium to understand complex biological processes specific to pineapple. PMID:27374615

Comparative genome analysis and characterization of the Salmonella Typhimurium strain CCRJ_26 isolated from swine carcasses using whole-genome sequencing approach.

PubMed

Panzenhagen, P H N; Cabral, C C; Suffys, P N; Franco, R M; Rodrigues, D P; Conte-Junior, C A

2018-04-01

Salmonella pathogenicity relies on virulence factors many of which are clustered within the Salmonella pathogenicity islands. Salmonella also harbours mobile genetic elements such as virulence plasmids, prophage-like elements and antimicrobial resistance genes which can contribute to increase its pathogenicity. Here, we have genetically characterized a selected S. Typhimurium strain (CCRJ_26) from our previous study with Multiple Drugs Resistant profile and high-frequency PFGE clonal profile which apparently persists in the pork production centre of Rio de Janeiro State, Brazil. By whole-genome sequencing, we described the strain's genome virulent content and characterized the repertoire of bacterial plasmids, antibiotic resistance genes and prophage-like elements. Here, we have shown evidence that strain CCRJ_26 genome possible represent a virulence-associated phenotype which may be potentially virulent in human infection. Whole-genome sequencing technologies are still costly and remain underexplored for applied microbiology in Brazil. Hence, this genomic description of S. Typhimurium strain CCRJ_26 will provide help in future molecular epidemiological studies. The analysis described here reveals a quick and useful pipeline for bacterial virulence characterization using whole-genome sequencing approach. © 2018 The Society for Applied Microbiology.

Large-scale whole-genome sequencing of the Icelandic population.

PubMed

Gudbjartsson, Daniel F; Helgason, Hannes; Gudjonsson, Sigurjon A; Zink, Florian; Oddson, Asmundur; Gylfason, Arnaldur; Besenbacher, Soren; Magnusson, Gisli; Halldorsson, Bjarni V; Hjartarson, Eirikur; Sigurdsson, Gunnar Th; Stacey, Simon N; Frigge, Michael L; Holm, Hilma; Saemundsdottir, Jona; Helgadottir, Hafdis Th; Johannsdottir, Hrefna; Sigfusson, Gunnlaugur; Thorgeirsson, Gudmundur; Sverrisson, Jon Th; Gretarsdottir, Solveig; Walters, G Bragi; Rafnar, Thorunn; Thjodleifsson, Bjarni; Bjornsson, Einar S; Olafsson, Sigurdur; Thorarinsdottir, Hildur; Steingrimsdottir, Thora; Gudmundsdottir, Thora S; Theodors, Asgeir; Jonasson, Jon G; Sigurdsson, Asgeir; Bjornsdottir, Gyda; Jonsson, Jon J; Thorarensen, Olafur; Ludvigsson, Petur; Gudbjartsson, Hakon; Eyjolfsson, Gudmundur I; Sigurdardottir, Olof; Olafsson, Isleifur; Arnar, David O; Magnusson, Olafur Th; Kong, Augustine; Masson, Gisli; Thorsteinsdottir, Unnur; Helgason, Agnar; Sulem, Patrick; Stefansson, Kari

2015-05-01

Here we describe the insights gained from sequencing the whole genomes of 2,636 Icelanders to a median depth of 20×. We found 20 million SNPs and 1.5 million insertions-deletions (indels). We describe the density and frequency spectra of sequence variants in relation to their functional annotation, gene position, pathway and conservation score. We demonstrate an excess of homozygosity and rare protein-coding variants in Iceland. We imputed these variants into 104,220 individuals down to a minor allele frequency of 0.1% and found a recessive frameshift mutation in MYL4 that causes early-onset atrial fibrillation, several mutations in ABCB4 that increase risk of liver diseases and an intronic variant in GNAS associating with increased thyroid-stimulating hormone levels when maternally inherited. These data provide a study design that can be used to determine how variation in the sequence of the human genome gives rise to human diversity.

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

Labbe, Jessy L; Uehling, Jessie K; Payen, Thibaut

The last 10 years have seen the cost of sequencing complete genomes decrease at an incredible speed. This has led to an increase in the number of genomes sequenced in all the fungal tree of life as well as a wide variety of plant genomes. The increase in sequencing has permitted us to study the evolution of organisms on a genomic scale. A number of talks during the conference discussed the importance of transposable elements (TEs) that are present in almost all species of fungi. These TEs represent an especially large percentage of genomic space in fungi that interact withmore » plants. Thierry Rouxel (INRA, Nancy, France) showed the link between speciation in the Leptosphaeria complex and the expansion of TE families. For example in the Leptosphaeria complex, one species associated with oilseed rape has experienced a recent and massive burst of movement by a few TE families. The alterations caused by these TEs took place in discrete regions of the genome leading to shuffling of the genomic landscape and the appearance of genes specific to the species, such as effectors useful for the interactions with a particular plant (Rouxel et al., 2011). Other presentations showed the importance of TEs in affecting genome organization. For example, in Amanita different species appear to have been invaded by different TE families (Veneault-Fourrey & Martin, 2011).« less

Similar Ratios of Introns to Intergenic Sequence across Animal Genomes.

PubMed

Francis, Warren R; Wörheide, Gert

2017-06-01

One central goal of genome biology is to understand how the usage of the genome differs between organisms. Our knowledge of genome composition, needed for downstream inferences, is critically dependent on gene annotations, yet problems associated with gene annotation and assembly errors are usually ignored in comparative genomics. Here, we analyze the genomes of 68 species across 12 animal phyla and some single-cell eukaryotes for general trends in genome composition and transcription, taking into account problems of gene annotation. We show that, regardless of genome size, the ratio of introns to intergenic sequence is comparable across essentially all animals, with nearly all deviations dominated by increased intergenic sequence. Genomes of model organisms have ratios much closer to 1:1, suggesting that the majority of published genomes of nonmodel organisms are underannotated and consequently omit substantial numbers of genes, with likely negative impact on evolutionary interpretations. Finally, our results also indicate that most animals transcribe half or more of their genomes arguing against differences in genome usage between animal groups, and also suggesting that the transcribed portion is more dependent on genome size than previously thought. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genome Sequence of Staphylococcus aureus VC40, a Vancomycin- and Daptomycin-Resistant Strain, To Study the Genetics of Development of Resistance to Currently Applied Last-Resort Antibiotics

PubMed Central

Berscheid, Anne; Jansen, Andrea; Oedenkoven, Marion; Szekat, Christiane; Strittmatter, Axel; Gottschalk, Gerhard; Bierbaum, Gabriele

2012-01-01

The increasing emergence of multidrug-resistant Staphylococcus aureus is a problem of global importance. Here, we report the genome of S. aureus VC40, which is resistant to the last-resort antibiotics vancomycin and daptomycin. Its genome sequence will allow insights into the mechanisms that convey full resistance to these compounds. PMID:22461548

ERGC: an efficient referential genome compression algorithm.

PubMed

Saha, Subrata; Rajasekaran, Sanguthevar

2015-11-01

Genome sequencing has become faster and more affordable. Consequently, the number of available complete genomic sequences is increasing rapidly. As a result, the cost to store, process, analyze and transmit the data is becoming a bottleneck for research and future medical applications. So, the need for devising efficient data compression and data reduction techniques for biological sequencing data is growing by the day. Although there exists a number of standard data compression algorithms, they are not efficient in compressing biological data. These generic algorithms do not exploit some inherent properties of the sequencing data while compressing. To exploit statistical and information-theoretic properties of genomic sequences, we need specialized compression algorithms. Five different next-generation sequencing data compression problems have been identified and studied in the literature. We propose a novel algorithm for one of these problems known as reference-based genome compression. We have done extensive experiments using five real sequencing datasets. The results on real genomes show that our proposed algorithm is indeed competitive and performs better than the best known algorithms for this problem. It achieves compression ratios that are better than those of the currently best performing algorithms. The time to compress and decompress the whole genome is also very promising. The implementations are freely available for non-commercial purposes. They can be downloaded from http://engr.uconn.edu/∼rajasek/ERGC.zip. rajasek@engr.uconn.edu. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The current use and attitudes towards tumor genome sequencing in breast cancer

PubMed Central

Gingras, I.; Sonnenblick, A.; de Azambuja, E.; Paesmans, M.; Delaloge, S.; Aftimos, Philippe; Piccart, M. J.; Sotiriou, C.; Ignatiadis, M.; Azim, H. A.

2016-01-01

There is increasing availability of technologies that can interrogate the genomic landscape of an individual tumor; however, their impact on daily practice remains uncertain. We conducted a 28-item survey to investigate the current attitudes towards the integration of tumor genome sequencing in breast cancer management. A link to the survey was communicated via newsletters of several oncological societies, and dedicated mailing by academic research groups. Multivariable logistic regression modeling was carried out to determine the relationship between predictors and outcomes. 215 physicians participated to the survey. The majority were medical oncologists (88%), practicing in Europe (70%) and working in academic institutions (66%). Tumor genome sequencing was requested by 82 participants (38%), of whom 21% reported low confidence in their genomic knowledge, and 56% considered tumor genome sequencing to be poorly accessible. In multivariable analysis, having time allocated to research (OR 3.37, 95% CI 1.84–6.15, p < 0.0001), working in Asia (OR 5.76, 95% CI 1.57 – 21.15, p = 0.01) and having institutional guidelines for molecular sequencing (OR 2.09, 95% 0.99–4.42, p = 0.05) were associated with a higher probability of use. In conclusion, our survey indicates that tumor genome sequencing is sometimes used, albeit not widely, in guiding management of breast cancer patients. PMID:26931736

DNA methylation at hepatitis B viral integrants is associated with methylation at flanking human genomic sequences

PubMed Central

Watanabe, Yoshiyuki; Yamamoto, Hiroyuki; Oikawa, Ritsuko; Toyota, Minoru; Yamamoto, Masakazu; Kokudo, Norihiro; Tanaka, Shinji; Arii, Shigeki; Yotsuyanagi, Hiroshi; Koike, Kazuhiko; Itoh, Fumio

2015-01-01

Integration of DNA viruses into the human genome plays an important role in various types of tumors, including hepatitis B virus (HBV)–related hepatocellular carcinoma. However, the molecular details and clinical impact of HBV integration on either human or HBV epigenomes are unknown. Here, we show that methylation of the integrated HBV DNA is related to the methylation status of the flanking human genome. We developed a next-generation sequencing-based method for structural methylation analysis of integrated viral genomes (denoted G-NaVI). This method is a novel approach that enables enrichment of viral fragments for sequencing using unique baits based on the sequence of the HBV genome. We detected integrated HBV sequences in the genome of the PLC/PRF/5 cell line and found variable levels of methylation within the integrated HBV genomes. Allele-specific methylation analysis revealed that the HBV genome often became significantly methylated when integrated into highly methylated host sites. After integration into unmethylated human genome regions such as promoters, however, the HBV DNA remains unmethylated and may eventually play an important role in tumorigenesis. The observed dynamic changes in DNA methylation of the host and viral genomes may functionally affect the biological behavior of HBV. These findings may impact public health given that millions of people worldwide are carriers of HBV. We also believe our assay will be a powerful tool to increase our understanding of the various types of DNA virus-associated tumorigenesis. PMID:25653310

Toward the 1,000 dollars human genome.

PubMed

Bennett, Simon T; Barnes, Colin; Cox, Anthony; Davies, Lisa; Brown, Clive

2005-06-01

Revolutionary new technologies, capable of transforming the economics of sequencing, are providing an unparalleled opportunity to analyze human genetic variation comprehensively at the whole-genome level within a realistic timeframe and at affordable costs. Current estimates suggest that it would cost somewhere in the region of 30 million US dollars to sequence an entire human genome using Sanger-based sequencing, and on one machine it would take about 60 years. Solexa is widely regarded as a company with the necessary disruptive technology to be the first to achieve the ultimate goal of the so-called 1,000 dollars human genome - the conceptual cost-point needed for routine analysis of individual genomes. Solexa's technology is based on completely novel sequencing chemistry capable of sequencing billions of individual DNA molecules simultaneously, a base at a time, to enable highly accurate, low cost analysis of an entire human genome in a single experiment. When applied over a large enough genomic region, these new approaches to resequencing will enable the simultaneous detection and typing of known, as well as unknown, polymorphisms, and will also offer information about patterns of linkage disequilibrium in the population being studied. Technological progress, leading to the advent of single-molecule-based approaches, is beginning to dramatically drive down costs and increase throughput to unprecedented levels, each being several orders of magnitude better than that which is currently available. A new sequencing paradigm based on single molecules will be faster, cheaper and more sensitive, and will permit routine analysis at the whole-genome level.

Genome-wide-analyses of Listeria monocytogenes from food-processing plants reveal clonal diversity and date the emergence of persisting sequence types.

PubMed

Knudsen, Gitte M; Nielsen, Jesper Boye; Marvig, Rasmus L; Ng, Yin; Worning, Peder; Westh, Henrik; Gram, Lone

2017-08-01

Whole genome sequencing is increasing used in epidemiology, e.g. for tracing outbreaks of food-borne diseases. This requires in-depth understanding of pathogen emergence, persistence and genomic diversity along the food production chain including in food processing plants. We sequenced the genomes of 80 isolates of Listeria monocytogenes sampled from Danish food processing plants over a time-period of 20 years, and analysed the sequences together with 10 public available reference genomes to advance our understanding of interplant and intraplant genomic diversity of L. monocytogenes. Except for three persisting sequence types (ST) based on Multi Locus Sequence Typing being ST7, ST8 and ST121, long-term persistence of clonal groups was limited, and new clones were introduced continuously, potentially from raw materials. No particular gene could be linked to the persistence phenotype. Using time-based phylogenetic analyses of the persistent STs, we estimate the L. monocytogenes evolutionary rate to be 0.18-0.35 single nucleotide polymorphisms/year, suggesting that the persistent STs emerged approximately 100 years ago, which correlates with the onset of industrialization and globalization of the food market. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Extraction of High Molecular Weight DNA from Fungal Rust Spores for Long Read Sequencing.

PubMed

Schwessinger, Benjamin; Rathjen, John P

2017-01-01

Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.

BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons

PubMed Central

2011-01-01

Background Visualisation of genome comparisons is invaluable for helping to determine genotypic differences between closely related prokaryotes. New visualisation and abstraction methods are required in order to improve the validation, interpretation and communication of genome sequence information; especially with the increasing amount of data arising from next-generation sequencing projects. Visualising a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Several programs, imaging libraries and internet resources already exist for this purpose, however, most are either limited in the number of comparisons they can show, are unable to adequately utilise draft genome sequence data, or require a knowledge of command-line scripting for implementation. Currently, there is no freely available desktop application that enables users to rapidly visualise comparisons between hundreds of draft or complete genomes in a single image. Results BLAST Ring Image Generator (BRIG) can generate images that show multiple prokaryote genome comparisons, without an arbitrary limit on the number of genomes compared. The output image shows similarity between a central reference sequence and other sequences as a set of concentric rings, where BLAST matches are coloured on a sliding scale indicating a defined percentage identity. Images can also include draft genome assembly information to show read coverage, assembly breakpoints and collapsed repeats. In addition, BRIG supports the mapping of unassembled sequencing reads against one or more central reference sequences. Many types of custom data and annotations can be shown using BRIG, making it a versatile approach for visualising a range of genomic comparison data. BRIG is readily accessible to any user, as it assumes no specialist computational knowledge and will perform all required file parsing and BLAST comparisons automatically. Conclusions There is a clear need for a user-friendly program that can produce genome comparisons for a large number of prokaryote genomes with an emphasis on rapidly utilising unfinished or unassembled genome data. Here we present BRIG, a cross-platform application that enables the interactive generation of comparative genomic images via a simple graphical-user interface. BRIG is freely available for all operating systems at http://sourceforge.net/projects/brig/. PMID:21824423

BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons.

PubMed

Alikhan, Nabil-Fareed; Petty, Nicola K; Ben Zakour, Nouri L; Beatson, Scott A

2011-08-08

Visualisation of genome comparisons is invaluable for helping to determine genotypic differences between closely related prokaryotes. New visualisation and abstraction methods are required in order to improve the validation, interpretation and communication of genome sequence information; especially with the increasing amount of data arising from next-generation sequencing projects. Visualising a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Several programs, imaging libraries and internet resources already exist for this purpose, however, most are either limited in the number of comparisons they can show, are unable to adequately utilise draft genome sequence data, or require a knowledge of command-line scripting for implementation. Currently, there is no freely available desktop application that enables users to rapidly visualise comparisons between hundreds of draft or complete genomes in a single image. BLAST Ring Image Generator (BRIG) can generate images that show multiple prokaryote genome comparisons, without an arbitrary limit on the number of genomes compared. The output image shows similarity between a central reference sequence and other sequences as a set of concentric rings, where BLAST matches are coloured on a sliding scale indicating a defined percentage identity. Images can also include draft genome assembly information to show read coverage, assembly breakpoints and collapsed repeats. In addition, BRIG supports the mapping of unassembled sequencing reads against one or more central reference sequences. Many types of custom data and annotations can be shown using BRIG, making it a versatile approach for visualising a range of genomic comparison data. BRIG is readily accessible to any user, as it assumes no specialist computational knowledge and will perform all required file parsing and BLAST comparisons automatically. There is a clear need for a user-friendly program that can produce genome comparisons for a large number of prokaryote genomes with an emphasis on rapidly utilising unfinished or unassembled genome data. Here we present BRIG, a cross-platform application that enables the interactive generation of comparative genomic images via a simple graphical-user interface. BRIG is freely available for all operating systems at http://sourceforge.net/projects/brig/.

Minke whale genome and aquatic adaptation in cetaceans

PubMed Central

Yim, Hyung-Soon; Cho, Yun Sung; Guang, Xuanmin; Kang, Sung Gyun; Jeong, Jae-Yeon; Cha, Sun-Shin; Oh, Hyun-Myung; Lee, Jae-Hak; Yang, Eun Chan; Kwon, Kae Kyoung; Kim, Yun Jae; Kim, Tae Wan; Kim, Wonduck; Jeon, Jeong Ho; Kim, Sang-Jin; Choi, Dong Han; Jho, Sungwoong; Kim, Hak-Min; Ko, Junsu; Kim, Hyunmin; Shin, Young-Ah; Jung, Hyun-Ju; Zheng, Yuan; Wang, Zhuo; Chen, Yan

2014-01-01

The shift from terrestrial to aquatic life by whales was a substantial evolutionary event. Here we report the whole-genome sequencing and de novo assembly of the minke whale genome, as well as the whole-genome sequences of three minke whales, a fin whale, a bottlenose dolphin and a finless porpoise. Our comparative genomic analysis identified an expansion in the whale lineage of gene families associated with stress-responsive proteins and anaerobic metabolism, whereas gene families related to body hair and sensory receptors were contracted. Our analysis also identified whale-specific mutations in genes encoding antioxidants and enzymes controlling blood pressure and salt concentration. Overall the whale-genome sequences exhibited distinct features that are associated with the physiological and morphological changes needed for life in an aquatic environment, marked by resistance to physiological stresses caused by a lack of oxygen, increased amounts of reactive oxygen species and high salt levels. PMID:24270359

Minke whale genome and aquatic adaptation in cetaceans.

PubMed

Yim, Hyung-Soon; Cho, Yun Sung; Guang, Xuanmin; Kang, Sung Gyun; Jeong, Jae-Yeon; Cha, Sun-Shin; Oh, Hyun-Myung; Lee, Jae-Hak; Yang, Eun Chan; Kwon, Kae Kyoung; Kim, Yun Jae; Kim, Tae Wan; Kim, Wonduck; Jeon, Jeong Ho; Kim, Sang-Jin; Choi, Dong Han; Jho, Sungwoong; Kim, Hak-Min; Ko, Junsu; Kim, Hyunmin; Shin, Young-Ah; Jung, Hyun-Ju; Zheng, Yuan; Wang, Zhuo; Chen, Yan; Chen, Ming; Jiang, Awei; Li, Erli; Zhang, Shu; Hou, Haolong; Kim, Tae Hyung; Yu, Lili; Liu, Sha; Ahn, Kung; Cooper, Jesse; Park, Sin-Gi; Hong, Chang Pyo; Jin, Wook; Kim, Heui-Soo; Park, Chankyu; Lee, Kyooyeol; Chun, Sung; Morin, Phillip A; O'Brien, Stephen J; Lee, Hang; Kimura, Jumpei; Moon, Dae Yeon; Manica, Andrea; Edwards, Jeremy; Kim, Byung Chul; Kim, Sangsoo; Wang, Jun; Bhak, Jong; Lee, Hyun Sook; Lee, Jung-Hyun

2014-01-01

The shift from terrestrial to aquatic life by whales was a substantial evolutionary event. Here we report the whole-genome sequencing and de novo assembly of the minke whale genome, as well as the whole-genome sequences of three minke whales, a fin whale, a bottlenose dolphin and a finless porpoise. Our comparative genomic analysis identified an expansion in the whale lineage of gene families associated with stress-responsive proteins and anaerobic metabolism, whereas gene families related to body hair and sensory receptors were contracted. Our analysis also identified whale-specific mutations in genes encoding antioxidants and enzymes controlling blood pressure and salt concentration. Overall the whale-genome sequences exhibited distinct features that are associated with the physiological and morphological changes needed for life in an aquatic environment, marked by resistance to physiological stresses caused by a lack of oxygen, increased amounts of reactive oxygen species and high salt levels.

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

Community standards for genomic resources, genetic conservation, and data integration

Treesearch

Jill Wegrzyn; Meg Staton; Emily Grau; Richard Cronn; C. Dana Nelson

2017-01-01

Genetics and genomics are increasingly important in forestry management and conservation. Next generation sequencing can increase analytical power, but still relies on building on the structure of previously acquired data. Data standards and data sharing allow the community to maximize the analytical power of high throughput genomics data. The landscape of incomplete...

Increased Genomic Prediction Accuracy in Wheat Breeding Through Spatial Adjustment of Field Trial Data

PubMed Central

Lado, Bettina; Matus, Ivan; Rodríguez, Alejandra; Inostroza, Luis; Poland, Jesse; Belzile, François; del Pozo, Alejandro; Quincke, Martín; Castro, Marina; von Zitzewitz, Jarislav

2013-01-01

In crop breeding, the interest of predicting the performance of candidate cultivars in the field has increased due to recent advances in molecular breeding technologies. However, the complexity of the wheat genome presents some challenges for applying new technologies in molecular marker identification with next-generation sequencing. We applied genotyping-by-sequencing, a recently developed method to identify single-nucleotide polymorphisms, in the genomes of 384 wheat (Triticum aestivum) genotypes that were field tested under three different water regimes in Mediterranean climatic conditions: rain-fed only, mild water stress, and fully irrigated. We identified 102,324 single-nucleotide polymorphisms in these genotypes, and the phenotypic data were used to train and test genomic selection models intended to predict yield, thousand-kernel weight, number of kernels per spike, and heading date. Phenotypic data showed marked spatial variation. Therefore, different models were tested to correct the trends observed in the field. A mixed-model using moving-means as a covariate was found to best fit the data. When we applied the genomic selection models, the accuracy of predicted traits increased with spatial adjustment. Multiple genomic selection models were tested, and a Gaussian kernel model was determined to give the highest accuracy. The best predictions between environments were obtained when data from different years were used to train the model. Our results confirm that genotyping-by-sequencing is an effective tool to obtain genome-wide information for crops with complex genomes, that these data are efficient for predicting traits, and that correction of spatial variation is a crucial ingredient to increase prediction accuracy in genomic selection models. PMID:24082033

Anonymization of electronic medical records for validating genome-wide association studies

PubMed Central

Loukides, Grigorios; Gkoulalas-Divanis, Aris; Malin, Bradley

2010-01-01

Genome-wide association studies (GWAS) facilitate the discovery of genotype–phenotype relations from population-based sequence databases, which is an integral facet of personalized medicine. The increasing adoption of electronic medical records allows large amounts of patients’ standardized clinical features to be combined with the genomic sequences of these patients and shared to support validation of GWAS findings and to enable novel discoveries. However, disseminating these data “as is” may lead to patient reidentification when genomic sequences are linked to resources that contain the corresponding patients’ identity information based on standardized clinical features. This work proposes an approach that provably prevents this type of data linkage and furnishes a result that helps support GWAS. Our approach automatically extracts potentially linkable clinical features and modifies them in a way that they can no longer be used to link a genomic sequence to a small number of patients, while preserving the associations between genomic sequences and specific sets of clinical features corresponding to GWAS-related diseases. Extensive experiments with real patient data derived from the Vanderbilt's University Medical Center verify that our approach generates data that eliminate the threat of individual reidentification, while supporting GWAS validation and clinical case analysis tasks. PMID:20385806

Comparative genomics of Lupinus angustifolius gene-rich regions: BAC library exploration, genetic mapping and cytogenetics

PubMed Central

2013-01-01

Background The narrow-leafed lupin, Lupinus angustifolius L., is a grain legume species with a relatively compact genome. The species has 2n = 40 chromosomes and its genome size is 960 Mbp/1C. During the last decade, L. angustifolius genomic studies have achieved several milestones, such as molecular-marker development, linkage maps, and bacterial artificial chromosome (BAC) libraries. Here, these resources were integratively used to identify and sequence two gene-rich regions (GRRs) of the genome. Results The genome was screened with a probe representing the sequence of a microsatellite fragment length polymorphism (MFLP) marker linked to Phomopsis stem blight resistance. BAC clones selected by hybridization were subjected to restriction fingerprinting and contig assembly, and 232 BAC-ends were sequenced and annotated. BAC fluorescence in situ hybridization (BAC-FISH) identified eight single-locus clones. Based on physical mapping, cytogenetic localization, and BAC-end annotation, five clones were chosen for sequencing. Within the sequences of clones that hybridized in FISH to a single-locus, two large GRRs were identified. The GRRs showed strong and conserved synteny to Glycine max duplicated genome regions, illustrated by both identical gene order and parallel orientation. In contrast, in the clones with dispersed FISH signals, more than one-third of sequences were transposable elements. Sequenced, single-locus clones were used to develop 12 genetic markers, increasing the number of L. angustifolius chromosomes linked to appropriate linkage groups by five pairs. Conclusions In general, probes originating from MFLP sequences can assist genome screening and gene discovery. However, such probes are not useful for positional cloning, because they tend to hybridize to numerous loci. GRRs identified in L. angustifolius contained a low number of interspersed repeats and had a high level of synteny to the genome of the model legume G. max. Our results showed that not only was the gene nucleotide sequence conserved between soybean and lupin GRRs, but the order and orientation of particular genes in syntenic blocks was homologous, as well. These findings will be valuable to the forthcoming sequencing of the lupin genome. PMID:23379841

From genomics to functional markers in the era of next-generation sequencing.

PubMed

Salgotra, R K; Gupta, B B; Stewart, C N

2014-03-01

The availability of complete genome sequences, along with other genomic resources for Arabidopsis, rice, pigeon pea, soybean and other crops, has revolutionized our understanding of the genetic make-up of plants. Next-generation DNA sequencing (NGS) has facilitated single nucleotide polymorphism discovery in plants. Functionally-characterized sequences can be identified and functional markers (FMs) for important traits can be developed at an ever-increasing ease. FMs are derived from sequence polymorphisms found in allelic variants of a functional gene. Linkage disequilibrium-based association mapping and homologous recombinants have been developed for identification of "perfect" markers for their use in crop improvement practices. Compared with many other molecular markers, FMs derived from the functionally characterized sequence genes using NGS techniques and their use provide opportunities to develop high-yielding plant genotypes resistant to various stresses at a fast pace.

Preliminary Classification of Novel Hemorrhagic Fever-Causing Viruses Using Sequence-Based PAirwise Sequence Comparison (PASC) Analysis.

PubMed

Bào, Yīmíng; Kuhn, Jens H

2018-01-01

During the last decade, genome sequence-based classification of viruses has become increasingly prominent. Viruses can be even classified based on coding-complete genome sequence data alone. Nevertheless, classification remains arduous as experts are required to establish phylogenetic trees to depict the evolutionary relationships of such sequences for preliminary taxonomic placement. Pairwise sequence comparison (PASC) of genomes is one of several novel methods for establishing relationships among viruses. This method, provided by the US National Center for Biotechnology Information as an open-access tool, circumvents phylogenetics, and yet PASC results are often in agreement with those of phylogenetic analyses. Computationally inexpensive, PASC can be easily performed by non-taxonomists. Here we describe how to use the PASC tool for the preliminary classification of novel viral hemorrhagic fever-causing viruses.

BAC sequencing using pooled methods.

PubMed

Saski, Christopher A; Feltus, F Alex; Parida, Laxmi; Haiminen, Niina

2015-01-01

Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly.

The Solanum commersonii Genome Sequence Provides Insights into Adaptation to Stress Conditions and Genome Evolution of Wild Potato Relatives

PubMed Central

Aversano, Riccardo; Contaldi, Felice; Ercolano, Maria Raffaella; Grosso, Valentina; Iorizzo, Massimo; Tatino, Filippo; Xumerle, Luciano; Dal Molin, Alessandra; Avanzato, Carla; Ferrarini, Alberto; Delledonne, Massimo; Sanseverino, Walter; Cigliano, Riccardo Aiese; Capella-Gutierrez, Salvador; Gabaldón, Toni; Frusciante, Luigi; Bradeen, James M.; Carputo, Domenico

2015-01-01

Here, we report the draft genome sequence of Solanum commersonii, which consists of ∼830 megabases with an N50 of 44,303 bp anchored to 12 chromosomes, using the potato (Solanum tuberosum) genome sequence as a reference. Compared with potato, S. commersonii shows a striking reduction in heterozygosity (1.5% versus 53 to 59%), and differences in genome sizes were mainly due to variations in intergenic sequence length. Gene annotation by ab initio prediction supported by RNA-seq data produced a catalog of 1703 predicted microRNAs, 18,882 long noncoding RNAs of which 20% are shown to target cold-responsive genes, and 39,290 protein-coding genes with a significant repertoire of nonredundant nucleotide binding site-encoding genes and 126 cold-related genes that are lacking in S. tuberosum. Phylogenetic analyses indicate that domesticated potato and S. commersonii lineages diverged ∼2.3 million years ago. Three duplication periods corresponding to genome enrichment for particular gene families related to response to salt stress, water transport, growth, and defense response were discovered. The draft genome sequence of S. commersonii substantially increases our understanding of the domesticated germplasm, facilitating translation of acquired knowledge into advances in crop stability in light of global climate and environmental changes. PMID:25873387

Applications and challenges of next-generation sequencing in Brassica species.

PubMed

Wei, Lijuan; Xiao, Meili; Hayward, Alice; Fu, Donghui

2013-12-01

Next-generation sequencing (NGS) produces numerous (often millions) short DNA sequence reads, typically varying between 25 and 400 bp in length, at a relatively low cost and in a short time. This revolutionary technology is being increasingly applied in whole-genome, transcriptome, epigenome and small RNA sequencing, molecular marker and gene discovery, comparative and evolutionary genomics, and association studies. The Brassica genus comprises some of the most agro-economically important crops, providing abundant vegetables, condiments, fodder, oil and medicinal products. Many Brassica species have undergone the process of polyploidization, which makes their genomes exceptionally complex and can create difficulties in genomics research. NGS injects new vigor into Brassica research, yet also faces specific challenges in the analysis of complex crop genomes and traits. In this article, we review the advantages and limitations of different NGS technologies and their applications and challenges, using Brassica as an advanced model system for agronomically important, polyploid crops. Specifically, we focus on the use of NGS for genome resequencing, transcriptome sequencing, development of single-nucleotide polymorphism markers, and identification of novel microRNAs and their targets. We present trends and advances in NGS technology in relation to Brassica crop improvement, with wide application for sophisticated genomics research into agronomically important polyploid crops.

Adaptive efficient compression of genomes

PubMed Central

2012-01-01

Modern high-throughput sequencing technologies are able to generate DNA sequences at an ever increasing rate. In parallel to the decreasing experimental time and cost necessary to produce DNA sequences, computational requirements for analysis and storage of the sequences are steeply increasing. Compression is a key technology to deal with this challenge. Recently, referential compression schemes, storing only the differences between a to-be-compressed input and a known reference sequence, gained a lot of interest in this field. However, memory requirements of the current algorithms are high and run times often are slow. In this paper, we propose an adaptive, parallel and highly efficient referential sequence compression method which allows fine-tuning of the trade-off between required memory and compression speed. When using 12 MB of memory, our method is for human genomes on-par with the best previous algorithms in terms of compression ratio (400:1) and compression speed. In contrast, it compresses a complete human genome in just 11 seconds when provided with 9 GB of main memory, which is almost three times faster than the best competitor while using less main memory. PMID:23146997

Genomics of crop wild relatives: expanding the gene pool for crop improvement.

PubMed

Brozynska, Marta; Furtado, Agnelo; Henry, Robert J

2016-04-01

Plant breeders require access to new genetic diversity to satisfy the demands of a growing human population for more food that can be produced in a variable or changing climate and to deliver the high-quality food with nutritional and health benefits demanded by consumers. The close relatives of domesticated plants, crop wild relatives (CWRs), represent a practical gene pool for use by plant breeders. Genomics of CWR generates data that support the use of CWR to expand the genetic diversity of crop plants. Advances in DNA sequencing technology are enabling the efficient sequencing of CWR and their increased use in crop improvement. As the sequencing of genomes of major crop species is completed, attention has shifted to analysis of the wider gene pool of major crops including CWR. A combination of de novo sequencing and resequencing is required to efficiently explore useful genetic variation in CWR. Analysis of the nuclear genome, transcriptome and maternal (chloroplast and mitochondrial) genome of CWR is facilitating their use in crop improvement. Genome analysis results in discovery of useful alleles in CWR and identification of regions of the genome in which diversity has been lost in domestication bottlenecks. Targeting of high priority CWR for sequencing will maximize the contribution of genome sequencing of CWR. Coordination of global efforts to apply genomics has the potential to accelerate access to and conservation of the biodiversity essential to the sustainability of agriculture and food production. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Genomic Enzymology: Web Tools for Leveraging Protein Family Sequence-Function Space and Genome Context to Discover Novel Functions.

PubMed

Gerlt, John A

2017-08-22

The exponentially increasing number of protein and nucleic acid sequences provides opportunities to discover novel enzymes, metabolic pathways, and metabolites/natural products, thereby adding to our knowledge of biochemistry and biology. The challenge has evolved from generating sequence information to mining the databases to integrating and leveraging the available information, i.e., the availability of "genomic enzymology" web tools. Web tools that allow identification of biosynthetic gene clusters are widely used by the natural products/synthetic biology community, thereby facilitating the discovery of novel natural products and the enzymes responsible for their biosynthesis. However, many novel enzymes with interesting mechanisms participate in uncharacterized small-molecule metabolic pathways; their discovery and functional characterization also can be accomplished by leveraging information in protein and nucleic acid databases. This Perspective focuses on two genomic enzymology web tools that assist the discovery novel metabolic pathways: (1) Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) for generating sequence similarity networks to visualize and analyze sequence-function space in protein families and (2) Enzyme Function Initiative-Genome Neighborhood Tool (EFI-GNT) for generating genome neighborhood networks to visualize and analyze the genome context in microbial and fungal genomes. Both tools have been adapted to other applications to facilitate target selection for enzyme discovery and functional characterization. As the natural products community has demonstrated, the enzymology community needs to embrace the essential role of web tools that allow the protein and genome sequence databases to be leveraged for novel insights into enzymological problems.

Increasing genomic diversity and evidence of constrained lifestyle evolution due to insertion sequences in Aeromonas salmonicida.

PubMed

Vincent, Antony T; Trudel, Mélanie V; Freschi, Luca; Nagar, Vandan; Gagné-Thivierge, Cynthia; Levesque, Roger C; Charette, Steve J

2016-01-12

Aeromonads make up a group of Gram-negative bacteria that includes human and fish pathogens. The Aeromonas salmonicida species has the peculiarity of including five known subspecies. However, few studies of the genomes of A. salmonicida subspecies have been reported to date. We sequenced the genomes of additional A. salmonicida isolates, including three from India, using next-generation sequencing in order to gain a better understanding of the genomic and phylogenetic links between A. salmonicida subspecies. Their relative phylogenetic positions were confirmed by a core genome phylogeny based on 1645 gene sequences. The Indian isolates, which formed a sub-group together with A. salmonicida subsp. pectinolytica, were able to grow at either at 18 °C and 37 °C, unlike the A. salmonicida psychrophilic isolates that did not grow at 37 °C. Amino acid frequencies, GC content, tRNA composition, loss and gain of genes during evolution, pseudogenes as well as genes under positive selection and the mobilome were studied to explain this intraspecies dichotomy. Insertion sequences appeared to be an important driving force that locked the psychrophilic strains into their particular lifestyle in order to conserve their genomic integrity. This observation, based on comparative genomics, is in agreement with previous results showing that insertion sequence mobility induced by heat in A. salmonicida subspecies causes genomic plasticity, resulting in a deleterious effect on the virulence of the bacterium. We provide a proof-of-concept that selfish DNAs play a major role in the evolution of bacterial species by modeling genomes.

RNA-Seq Alignment to Individualized Genomes Improves Transcript Abundance Estimates in Multiparent Populations

PubMed Central

Munger, Steven C.; Raghupathy, Narayanan; Choi, Kwangbom; Simons, Allen K.; Gatti, Daniel M.; Hinerfeld, Douglas A.; Svenson, Karen L.; Keller, Mark P.; Attie, Alan D.; Hibbs, Matthew A.; Graber, Joel H.; Chesler, Elissa J.; Churchill, Gary A.

2014-01-01

Massively parallel RNA sequencing (RNA-seq) has yielded a wealth of new insights into transcriptional regulation. A first step in the analysis of RNA-seq data is the alignment of short sequence reads to a common reference genome or transcriptome. Genetic variants that distinguish individual genomes from the reference sequence can cause reads to be misaligned, resulting in biased estimates of transcript abundance. Fine-tuning of read alignment algorithms does not correct this problem. We have developed Seqnature software to construct individualized diploid genomes and transcriptomes for multiparent populations and have implemented a complete analysis pipeline that incorporates other existing software tools. We demonstrate in simulated and real data sets that alignment to individualized transcriptomes increases read mapping accuracy, improves estimation of transcript abundance, and enables the direct estimation of allele-specific expression. Moreover, when applied to expression QTL mapping we find that our individualized alignment strategy corrects false-positive linkage signals and unmasks hidden associations. We recommend the use of individualized diploid genomes over reference sequence alignment for all applications of high-throughput sequencing technology in genetically diverse populations. PMID:25236449

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

Insights into the Evolution of Mitochondrial Genome Size from Complete Sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae)

PubMed Central

Alverson, Andrew J.; Wei, XiaoXin; Rice, Danny W.; Stern, David B.; Barry, Kerrie; Palmer, Jeffrey D.

2010-01-01

The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)—the two smallest characterized cucurbit mitochondrial genomes—and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors. PMID:20118192

MendeLIMS: a web-based laboratory information management system for clinical genome sequencing.

PubMed

Grimes, Susan M; Ji, Hanlee P

2014-08-27

Large clinical genomics studies using next generation DNA sequencing require the ability to select and track samples from a large population of patients through many experimental steps. With the number of clinical genome sequencing studies increasing, it is critical to maintain adequate laboratory information management systems to manage the thousands of patient samples that are subject to this type of genetic analysis. To meet the needs of clinical population studies using genome sequencing, we developed a web-based laboratory information management system (LIMS) with a flexible configuration that is adaptable to continuously evolving experimental protocols of next generation DNA sequencing technologies. Our system is referred to as MendeLIMS, is easily implemented with open source tools and is also highly configurable and extensible. MendeLIMS has been invaluable in the management of our clinical genome sequencing studies. We maintain a publicly available demonstration version of the application for evaluation purposes at http://mendelims.stanford.edu. MendeLIMS is programmed in Ruby on Rails (RoR) and accesses data stored in SQL-compliant relational databases. Software is freely available for non-commercial use at http://dna-discovery.stanford.edu/software/mendelims/.

Next Generation Sequencing of Actinobacteria for the Discovery of Novel Natural Products

PubMed Central

Gomez-Escribano, Juan Pablo; Alt, Silke; Bibb, Mervyn J.

2016-01-01

Like many fields of the biosciences, actinomycete natural products research has been revolutionised by next-generation DNA sequencing (NGS). Hundreds of new genome sequences from actinobacteria are made public every year, many of them as a result of projects aimed at identifying new natural products and their biosynthetic pathways through genome mining. Advances in these technologies in the last five years have meant not only a reduction in the cost of whole genome sequencing, but also a substantial increase in the quality of the data, having moved from obtaining a draft genome sequence comprised of several hundred short contigs, sometimes of doubtful reliability, to the possibility of obtaining an almost complete and accurate chromosome sequence in a single contig, allowing a detailed study of gene clusters and the design of strategies for refactoring and full gene cluster synthesis. The impact that these technologies are having in the discovery and study of natural products from actinobacteria, including those from the marine environment, is only starting to be realised. In this review we provide a historical perspective of the field, analyse the strengths and limitations of the most relevant technologies, and share the insights acquired during our genome mining projects. PMID:27089350

Genomic signal processing methods for computation of alignment-free distances from DNA sequences.

PubMed

Borrayo, Ernesto; Mendizabal-Ruiz, E Gerardo; Vélez-Pérez, Hugo; Romo-Vázquez, Rebeca; Mendizabal, Adriana P; Morales, J Alejandro

2014-01-01

Genomic signal processing (GSP) refers to the use of digital signal processing (DSP) tools for analyzing genomic data such as DNA sequences. A possible application of GSP that has not been fully explored is the computation of the distance between a pair of sequences. In this work we present GAFD, a novel GSP alignment-free distance computation method. We introduce a DNA sequence-to-signal mapping function based on the employment of doublet values, which increases the number of possible amplitude values for the generated signal. Additionally, we explore the use of three DSP distance metrics as descriptors for categorizing DNA signal fragments. Our results indicate the feasibility of employing GAFD for computing sequence distances and the use of descriptors for characterizing DNA fragments.

Genomic Signal Processing Methods for Computation of Alignment-Free Distances from DNA Sequences

PubMed Central

Borrayo, Ernesto; Mendizabal-Ruiz, E. Gerardo; Vélez-Pérez, Hugo; Romo-Vázquez, Rebeca; Mendizabal, Adriana P.; Morales, J. Alejandro

2014-01-01

Genomic signal processing (GSP) refers to the use of digital signal processing (DSP) tools for analyzing genomic data such as DNA sequences. A possible application of GSP that has not been fully explored is the computation of the distance between a pair of sequences. In this work we present GAFD, a novel GSP alignment-free distance computation method. We introduce a DNA sequence-to-signal mapping function based on the employment of doublet values, which increases the number of possible amplitude values for the generated signal. Additionally, we explore the use of three DSP distance metrics as descriptors for categorizing DNA signal fragments. Our results indicate the feasibility of employing GAFD for computing sequence distances and the use of descriptors for characterizing DNA fragments. PMID:25393409

Comparative Genomics in Drosophila.

PubMed

Oti, Martin; Pane, Attilio; Sammeth, Michael

2018-01-01

Since the pioneering studies of Thomas Hunt Morgan and coworkers at the dawn of the twentieth century, Drosophila melanogaster and its sister species have tremendously contributed to unveil the rules underlying animal genetics, development, behavior, evolution, and human disease. Recent advances in DNA sequencing technologies launched Drosophila into the post-genomic era and paved the way for unprecedented comparative genomics investigations. The complete sequencing and systematic comparison of the genomes from 12 Drosophila species represents a milestone achievement in modern biology, which allowed a plethora of different studies ranging from the annotation of known and novel genomic features to the evolution of chromosomes and, ultimately, of entire genomes. Despite the efforts of countless laboratories worldwide, the vast amount of data that were produced over the past 15 years is far from being fully explored.In this chapter, we will review some of the bioinformatic approaches that were developed to interrogate the genomes of the 12 Drosophila species. Setting off from alignments of the entire genomic sequences, the degree of conservation can be separately evaluated for every region of the genome, providing already first hints about elements that are under purifying selection and therefore likely functional. Furthermore, the careful analysis of repeated sequences sheds light on the evolutionary dynamics of transposons, an enigmatic and fascinating class of mobile elements housed in the genomes of animals and plants. Comparative genomics also aids in the computational identification of the transcriptionally active part of the genome, first and foremost of protein-coding loci, but also of transcribed nevertheless apparently noncoding regions, which were once considered "junk" DNA. Eventually, the synergy between functional and comparative genomics also facilitates in silico and in vivo studies on cis-acting regulatory elements, like transcription factor binding sites, that due to the high degree of sequence variability usually impose increased challenges for bioinformatics approaches.

Paediatric genomics: diagnosing rare disease in children.

PubMed

Wright, Caroline F; FitzPatrick, David R; Firth, Helen V

2018-05-01

The majority of rare diseases affect children, most of whom have an underlying genetic cause for their condition. However, making a molecular diagnosis with current technologies and knowledge is often still a challenge. Paediatric genomics is an immature but rapidly evolving field that tackles this issue by incorporating next-generation sequencing technologies, especially whole-exome sequencing and whole-genome sequencing, into research and clinical workflows. This complex multidisciplinary approach, coupled with the increasing availability of population genetic variation data, has already resulted in an increased discovery rate of causative genes and in improved diagnosis of rare paediatric disease. Importantly, for affected families, a better understanding of the genetic basis of rare disease translates to more accurate prognosis, management, surveillance and genetic advice; stimulates research into new therapies; and enables provision of better support.

Genomic prediction using preselected DNA variants from a GWAS with whole-genome sequence data in Holstein-Friesian cattle.

PubMed

Veerkamp, Roel F; Bouwman, Aniek C; Schrooten, Chris; Calus, Mario P L

2016-12-01

Whole-genome sequence data is expected to capture genetic variation more completely than common genotyping panels. Our objective was to compare the proportion of variance explained and the accuracy of genomic prediction by using imputed sequence data or preselected SNPs from a genome-wide association study (GWAS) with imputed whole-genome sequence data. Phenotypes were available for 5503 Holstein-Friesian bulls. Genotypes were imputed up to whole-genome sequence (13,789,029 segregating DNA variants) by using run 4 of the 1000 bull genomes project. The program GCTA was used to perform GWAS for protein yield (PY), somatic cell score (SCS) and interval from first to last insemination (IFL). From the GWAS, subsets of variants were selected and genomic relationship matrices (GRM) were used to estimate the variance explained in 2087 validation animals and to evaluate the genomic prediction ability. Finally, two GRM were fitted together in several models to evaluate the effect of selected variants that were in competition with all the other variants. The GRM based on full sequence data explained only marginally more genetic variation than that based on common SNP panels: for PY, SCS and IFL, genomic heritability improved from 0.81 to 0.83, 0.83 to 0.87 and 0.69 to 0.72, respectively. Sequence data also helped to identify more variants linked to quantitative trait loci and resulted in clearer GWAS peaks across the genome. The proportion of total variance explained by the selected variants combined in a GRM was considerably smaller than that explained by all variants (less than 0.31 for all traits). When selected variants were used, accuracy of genomic predictions decreased and bias increased. Although 35 to 42 variants were detected that together explained 13 to 19% of the total variance (18 to 23% of the genetic variance) when fitted alone, there was no advantage in using dense sequence information for genomic prediction in the Holstein data used in our study. Detection and selection of variants within a single breed are difficult due to long-range linkage disequilibrium. Stringent selection of variants resulted in more biased genomic predictions, although this might be due to the training population being the same dataset from which the selected variants were identified.

Paul Spellman, Ph.D., Talks about TCGA at AACR 2011 - TCGA

Cancer.gov

Dr. Paul Spellman talks about The Cancer Genome Atlas (TCGA) and how this could help further the treatment of cancer. TCGA is a project working to catalog genetic mutations responsible for cancer. Clinicians are sequencing the genomes of patients with any of 20 different cancers and hope that this could target clinical trials at the specific patient sub-groups that would benefit most. Dr. Spellman explains how an increasing number of laboratories are becoming able to conduct genome sequencing and contribute to the TCGA project, discusses how clinicians could apply the findings in practice to decide on treatment and effect patient outlook and suggests that in future patients may start to request for their genome to be sequenced in order to aid their treatment.

Computational complexity of algorithms for sequence comparison, short-read assembly and genome alignment.

PubMed

Baichoo, Shakuntala; Ouzounis, Christos A

A multitude of algorithms for sequence comparison, short-read assembly and whole-genome alignment have been developed in the general context of molecular biology, to support technology development for high-throughput sequencing, numerous applications in genome biology and fundamental research on comparative genomics. The computational complexity of these algorithms has been previously reported in original research papers, yet this often neglected property has not been reviewed previously in a systematic manner and for a wider audience. We provide a review of space and time complexity of key sequence analysis algorithms and highlight their properties in a comprehensive manner, in order to identify potential opportunities for further research in algorithm or data structure optimization. The complexity aspect is poised to become pivotal as we will be facing challenges related to the continuous increase of genomic data on unprecedented scales and complexity in the foreseeable future, when robust biological simulation at the cell level and above becomes a reality. Copyright © 2017 Elsevier B.V. All rights reserved.

CHOgenome.org 2.0: Genome resources and website updates.

PubMed

Kremkow, Benjamin G; Baik, Jong Youn; MacDonald, Madolyn L; Lee, Kelvin H

2015-07-01

Chinese hamster ovary (CHO) cells are a major host cell line for the production of therapeutic proteins, and CHO cell and Chinese hamster (CH) genomes have recently been sequenced using next-generation sequencing methods. CHOgenome.org was launched in 2011 (version 1.0) to serve as a database repository and to provide bioinformatics tools for the CHO community. CHOgenome.org (version 1.0) maintained GenBank CHO-K1 genome data, identified CHO-omics literature, and provided a CHO-specific BLAST service. Recent major updates to CHOgenome.org (version 2.0) include new sequence and annotation databases for both CHO and CH genomes, a more user-friendly website, and new research tools, including a proteome browser and a genome viewer. CHO cell-line specific sequences and annotations facilitate cell line development opportunities, several of which are discussed. Moving forward, CHOgenome.org will host the increasing amount of CHO-omics data and continue to make useful bioinformatics tools available to the CHO community. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

STORMSeq: an open-source, user-friendly pipeline for processing personal genomics data in the cloud.

PubMed

Karczewski, Konrad J; Fernald, Guy Haskin; Martin, Alicia R; Snyder, Michael; Tatonetti, Nicholas P; Dudley, Joel T

2014-01-01

The increasing public availability of personal complete genome sequencing data has ushered in an era of democratized genomics. However, read mapping and variant calling software is constantly improving and individuals with personal genomic data may prefer to customize and update their variant calls. Here, we describe STORMSeq (Scalable Tools for Open-Source Read Mapping), a graphical interface cloud computing solution that does not require a parallel computing environment or extensive technical experience. This customizable and modular system performs read mapping, read cleaning, and variant calling and annotation. At present, STORMSeq costs approximately $2 and 5-10 hours to process a full exome sequence and $30 and 3-8 days to process a whole genome sequence. We provide this open-access and open-source resource as a user-friendly interface in Amazon EC2.

Draft genome sequence of the rubber tree Hevea brasiliensis.

PubMed

Rahman, Ahmad Yamin Abdul; Usharraj, Abhilash O; Misra, Biswapriya B; Thottathil, Gincy P; Jayasekaran, Kandakumar; Feng, Yun; Hou, Shaobin; Ong, Su Yean; Ng, Fui Ling; Lee, Ling Sze; Tan, Hock Siew; Sakaff, Muhd Khairul Luqman Muhd; Teh, Beng Soon; Khoo, Bee Feong; Badai, Siti Suriawati; Aziz, Nurohaida Ab; Yuryev, Anton; Knudsen, Bjarne; Dionne-Laporte, Alexandre; Mchunu, Nokuthula P; Yu, Qingyi; Langston, Brennick J; Freitas, Tracey Allen K; Young, Aaron G; Chen, Rui; Wang, Lei; Najimudin, Nazalan; Saito, Jennifer A; Alam, Maqsudul

2013-02-02

Hevea brasiliensis, a member of the Euphorbiaceae family, is the major commercial source of natural rubber (NR). NR is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since 1876. Here, we report the draft genome sequence of H. brasiliensis. The assembly spans ~1.1 Gb of the estimated 2.15 Gb haploid genome. Overall, ~78% of the genome was identified as repetitive DNA. Gene prediction shows 68,955 gene models, of which 12.7% are unique to Hevea. Most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified. The knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber.

Draft genome sequence of the rubber tree Hevea brasiliensis

PubMed Central

2013-01-01

Background Hevea brasiliensis, a member of the Euphorbiaceae family, is the major commercial source of natural rubber (NR). NR is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since 1876. Results Here, we report the draft genome sequence of H. brasiliensis. The assembly spans ~1.1 Gb of the estimated 2.15 Gb haploid genome. Overall, ~78% of the genome was identified as repetitive DNA. Gene prediction shows 68,955 gene models, of which 12.7% are unique to Hevea. Most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified. Conclusions The knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber. PMID:23375136

1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life

DOE PAGES

Mukherjee, Supratim; Seshadri, Rekha; Varghese, Neha J.; ...

2017-06-12

We present 1,003 reference genomes that were sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) initiative, selected to maximize sequence coverage of phylogenetic space. These genomes double the number of existing type strains and expand their overall phylogenetic diversity by 25%. Comparative analyses with previously available finished and draft genomes reveal a 10.5% increase in novel protein families as a function of phylogenetic diversity. The GEBA genomes recruit 25 million previously unassigned metagenomic proteins from 4,650 samples, improving their phylogenetic and functional interpretation. We identify numerous biosynthetic clusters and experimentally validate a divergent phenazine cluster withmore » potential new chemical structure and antimicrobial activity. This Resource is the largest single release of reference genomes to date. Bacterial and archaeal isolate sequence space is still far from saturated, and future endeavors in this direction will continue to be a valuable resource for scientific discovery.« less

1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life

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

Mukherjee, Supratim; Seshadri, Rekha; Varghese, Neha J.

We present 1,003 reference genomes that were sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) initiative, selected to maximize sequence coverage of phylogenetic space. These genomes double the number of existing type strains and expand their overall phylogenetic diversity by 25%. Comparative analyses with previously available finished and draft genomes reveal a 10.5% increase in novel protein families as a function of phylogenetic diversity. The GEBA genomes recruit 25 million previously unassigned metagenomic proteins from 4,650 samples, improving their phylogenetic and functional interpretation. We identify numerous biosynthetic clusters and experimentally validate a divergent phenazine cluster withmore » potential new chemical structure and antimicrobial activity. This Resource is the largest single release of reference genomes to date. Bacterial and archaeal isolate sequence space is still far from saturated, and future endeavors in this direction will continue to be a valuable resource for scientific discovery.« less

P2RP: a Web-based framework for the identification and analysis of regulatory proteins in prokaryotic genomes.

PubMed

Barakat, Mohamed; Ortet, Philippe; Whitworth, David E

2013-04-20

Regulatory proteins (RPs) such as transcription factors (TFs) and two-component system (TCS) proteins control how prokaryotic cells respond to changes in their external and/or internal state. Identification and annotation of TFs and TCSs is non-trivial, and between-genome comparisons are often confounded by different standards in annotation. There is a need for user-friendly, fast and convenient tools to allow researchers to overcome the inherent variability in annotation between genome sequences. We have developed the web-server P2RP (Predicted Prokaryotic Regulatory Proteins), which enables users to identify and annotate TFs and TCS proteins within their sequences of interest. Users can input amino acid or genomic DNA sequences, and predicted proteins therein are scanned for the possession of DNA-binding domains and/or TCS domains. RPs identified in this manner are categorised into families, unambiguously annotated, and a detailed description of their features generated, using an integrated software pipeline. P2RP results can then be outputted in user-specified formats. Biologists have an increasing need for fast and intuitively usable tools, which is why P2RP has been developed as an interactive system. As well as assisting experimental biologists to interrogate novel sequence data, it is hoped that P2RP will be built into genome annotation pipelines and re-annotation processes, to increase the consistency of RP annotation in public genomic sequences. P2RP is the first publicly available tool for predicting and analysing RP proteins in users' sequences. The server is freely available and can be accessed along with documentation at http://www.p2rp.org.

Draft genome sequence of carbapenem-resistant Shewanella algae strain AC isolated from small abalone (Haliotis diversicolor).

PubMed

Huang, Yao-Ting; Cheng, Jan-Fang; Chen, Shi-Yu; Hong, Yu-Kai; Wu, Zong-Yen; Liu, Po-Yu

2018-06-19

Shewanella algae is an environmental marine bacteria and an emerging opportunistic human pathogen. Moreover, there are increasing reports of strains showing multi-drug resistance, particularly carbapenem-resistant isolates. Although S. algae have been found in bivalve shellfish aquaculture, there is very little genome-wide data on resistant determinants in S. algae from shellfish. In the study, we aimed to determine the whole genome sequence of carbapenem-resistant S. algae strain AC isolated from small abalone in Taiwan. Genome DNA was sequenced using an Illumina MiSeq platform using 250bp paired-end reads. De novo genome assembly was performed using Velvet v1.2.07. The whole genome was annotated and several candidate genes for antimicrobial resistance were identified. The genome size was calculated at 4,751,156bp, with a mean G＋C content of 53.09%. A total of 4,164 protein-coding sequences, 7 rRNAs, 85 tRNAs, and 5 non-coding RNAs were identified. The genome contains genes associated with resistance to β-lactams, trimethoprim, tetracycline, colistin, and quinolone resistance. Multiple efflux pump genes were also detected. Small abalone is a potential source of foodborne drug resistant S. algae. The genome sequence of a carbapenem-resistant S. algae strain AC isolated from small abalone will provide valuable information for further study of the dissemination of resistance genes at the human-animal interface. Copyright © 2018. Published by Elsevier Ltd.

A nine-scaffold genome assembly of the nine chromosome sugar beet

USDA-ARS?s Scientific Manuscript database

A sugar beet genome sequence is required to take full advantage of the increasingly powerful approaches directed a single nucleotide resolution across the whole genome. A high quality reference genome serves as a benchmark from which other genotypes might be compared and exploited for sugar beet imp...

Genome skimming: A rapid approach to gaining diverse biological insights into multicellular pathogens

USDA-ARS?s Scientific Manuscript database

New genome sequence information can now be generated very quickly and cheaply for virtually any organism. The dive into genomics is increasingly tempting to scientists studying plant pathogens and other eukaryotic species without reference genomes. The ease of data collection, however, is tempered ...

West Nile virus (WNV) genome RNAs with up to three adjacent mutations that disrupt long distance 5'-3' cyclization sequence basepairs are viable

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

Basu, Mausumi; Brinton, Margo A., E-mail: mbrinton@gsu.ed

2011-03-30

Mosquito-borne flavivirus genomes contain conserved 5' and 3' cyclization sequences (CYC) that facilitate long distance RNA-RNA interactions. In previous studies, flavivirus replicon RNA replication was completely inhibited by single or multiple mismatching CYC nt substitutions. In the present study, full-length WNV genomes with one, two or three mismatching CYC substitutions showed reduced replication efficiencies but were viable and generated revertants with increased replication efficiency. Several different three adjacent mismatching CYC substitution mutant RNAs were rescued by a second site mutation that created an additional basepair (nts 147-10913) on the internal genomic side of the 5'-3' CYC. The finding that full-lengthmore » genomes with up to three mismatching CYC mutations are viable and can be rescued by a single nt spontaneous mutation indicates that more than three adjacent CYC basepair substitutions would be required to increase the safety of vaccine genomes by creating mismatches in inter-genomic recombinants.« less

A De-Novo Genome Analysis Pipeline (DeNoGAP) for large-scale comparative prokaryotic genomics studies.

PubMed

Thakur, Shalabh; Guttman, David S

2016-06-30

Comparative analysis of whole genome sequence data from closely related prokaryotic species or strains is becoming an increasingly important and accessible approach for addressing both fundamental and applied biological questions. While there are number of excellent tools developed for performing this task, most scale poorly when faced with hundreds of genome sequences, and many require extensive manual curation. We have developed a de-novo genome analysis pipeline (DeNoGAP) for the automated, iterative and high-throughput analysis of data from comparative genomics projects involving hundreds of whole genome sequences. The pipeline is designed to perform reference-assisted and de novo gene prediction, homolog protein family assignment, ortholog prediction, functional annotation, and pan-genome analysis using a range of proven tools and databases. While most existing methods scale quadratically with the number of genomes since they rely on pairwise comparisons among predicted protein sequences, DeNoGAP scales linearly since the homology assignment is based on iteratively refined hidden Markov models. This iterative clustering strategy enables DeNoGAP to handle a very large number of genomes using minimal computational resources. Moreover, the modular structure of the pipeline permits easy updates as new analysis programs become available. DeNoGAP integrates bioinformatics tools and databases for comparative analysis of a large number of genomes. The pipeline offers tools and algorithms for annotation and analysis of completed and draft genome sequences. The pipeline is developed using Perl, BioPerl and SQLite on Ubuntu Linux version 12.04 LTS. Currently, the software package accompanies script for automated installation of necessary external programs on Ubuntu Linux; however, the pipeline should be also compatible with other Linux and Unix systems after necessary external programs are installed. DeNoGAP is freely available at https://sourceforge.net/projects/denogap/ .

Impact of Genomic Counseling on Informed Decision-Making among ostensibly Healthy Individuals Seeking Personal Genome Sequencing: the HealthSeq Project.

PubMed

Suckiel, Sabrina A; Linderman, Michael D; Sanderson, Saskia C; Diaz, George A; Wasserstein, Melissa; Kasarskis, Andrew; Schadt, Eric E; Zinberg, Randi E

2016-10-01

Personal genome sequencing is increasingly utilized by healthy individuals for predispositional screening and other applications. However, little is known about the impact of 'genomic counseling' on informed decision-making in this context. Our primary aim was to compare measures of participants' informed decision-making before and after genomic counseling in the HealthSeq project, a longitudinal cohort study of individuals receiving personal results from whole genome sequencing (WGS). Our secondary aims were to assess the impact of the counseling on WGS knowledge and concerns, and to explore participants' satisfaction with the counseling. Questionnaires were administered to participants (n = 35) before and after their pre-test genomic counseling appointment. Informed decision-making was measured using the Decisional Conflict Scale (DCS) and the Satisfaction with Decision Scale (SDS). DCS scores decreased after genomic counseling (mean: 11.34 before vs. 5.94 after; z = -4.34, p < 0.001, r = 0.52), and SDS scores increased (mean: 27.91 vs. 29.06 respectively; z = 2.91, p = 0.004, r = 0.35). Satisfaction with counseling was high (mean (SD) = 26.91 (2.68), on a scale where 6 = low and 30 = high satisfaction). HealthSeq participants felt that their decision regarding receiving personal results from WGS was more informed after genomic counseling. Further research comparing the impact of different genomic counseling models is needed.

Whole-genome sequencing for comparative genomics and de novo genome assembly.

PubMed

Benjak, Andrej; Sala, Claudia; Hartkoorn, Ruben C

2015-01-01

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

Draft Genome Sequences of Two Species of "Difficult-to-Identify" Human-Pathogenic Corynebacteria: Implications for Better Identification Tests.

PubMed

Pacheco, Luis G C; Mattos-Guaraldi, Ana L; Santos, Carolina S; Veras, Adonney A O; Guimarães, Luis C; Abreu, Vinícius; Pereira, Felipe L; Soares, Siomar C; Dorella, Fernanda A; Carvalho, Alex F; Leal, Carlos G; Figueiredo, Henrique C P; Ramos, Juliana N; Vieira, Veronica V; Farfour, Eric; Guiso, Nicole; Hirata, Raphael; Azevedo, Vasco; Silva, Artur; Ramos, Rommel T J

2015-01-01

Non-diphtheriae Corynebacterium species have been increasingly recognized as the causative agents of infections in humans. Differential identification of these bacteria in the clinical microbiology laboratory by the most commonly used biochemical tests is challenging, and normally requires additional molecular methods. Herein, we present the annotated draft genome sequences of two isolates of "difficult-to-identify" human-pathogenic corynebacterial species: C. xerosis and C. minutissimum. The genome sequences of ca. 2.7 Mbp, with a mean number of 2,580 protein encoding genes, were also compared with the publicly available genome sequences of strains of C. amycolatum and C. striatum. These results will aid the exploration of novel biochemical reactions to improve existing identification tests as well as the development of more accurate molecular identification methods through detection of species-specific target genes for isolate's identification or drug susceptibility profiling.

Control of transcriptional pausing by biased thermal fluctuations on repetitive genomic sequences

PubMed Central

Imashimizu, Masahiko; Afek, Ariel; Takahashi, Hiroki; Lubkowska, Lucyna; Lukatsky, David B.

2016-01-01

In the process of transcription elongation, RNA polymerase (RNAP) pauses at highly nonrandom positions across genomic DNA, broadly regulating transcription; however, molecular mechanisms responsible for the recognition of such pausing positions remain poorly understood. Here, using a combination of statistical mechanical modeling and high-throughput sequencing and biochemical data, we evaluate the effect of thermal fluctuations on the regulation of RNAP pausing. We demonstrate that diffusive backtracking of RNAP, which is biased by repetitive DNA sequence elements, causes transcriptional pausing. This effect stems from the increased microscopic heterogeneity of an elongation complex, and thus is entropy-dominated. This report shows a linkage between repetitive sequence elements encoded in the genome and regulation of RNAP pausing driven by thermal fluctuations. PMID:27830653

Read clouds uncover variation in complex regions of the human genome

PubMed Central

Bishara, Alex; Liu, Yuling; Weng, Ziming; Kashef-Haghighi, Dorna; Newburger, Daniel E.; West, Robert; Sidow, Arend; Batzoglou, Serafim

2015-01-01

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies. PMID:26286554

Legume Genome Initiative at the University of Oklahoma

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

Bruce A. Roe

2004-02-27

Consolidated Appropriations Resolution, 2003 Conference Report for the Department of Energy's Biological and Environmental Research (BER) program provided $481,000 for the Legume Genome Initiative at the University of Oklahoma. These funds were used to support our research that is aimed at determining the entire sequence of the gene rich regions of the genome of the legume, Medicago truncatula, by allowing us to obtain a greater degree of finished BAC sequences from the draft sequences we have already obtained through research funded by the Noble Foundation. During the funding period we increased the number of Medicago truncatula BACs with finished (Bermudamore » standard) sequences from 109 to 359, and the total number of BACs for which we collected sequence data from 584 to 842, 359 of which reached phase 2 (ordered and oriented contigs). We also sequenced a series of pooled BAC clones that cover additional euchromatic (gene rich) genomic regions. This work resulted in 6 refereed publications, see below. Genes whose sequence was determined during this study included multiple members of the plant disease resistance (R-gene) family as well as several genes involved in flavinoid biosynthesis, nitrogen fixation and plant-microbial symbosis. This work also served as a prelude to obtaining NSF funding for the international collaborative effort to complete the entire sequence of the Medicago truncatula genomic euchromatic regions using a BAC based approach.« less

Whole-exome/genome sequencing and genomics.

PubMed

Grody, Wayne W; Thompson, Barry H; Hudgins, Louanne

2013-12-01

As medical genetics has progressed from a descriptive entity to one focused on the functional relationship between genes and clinical disorders, emphasis has been placed on genomics. Genomics, a subelement of genetics, is the study of the genome, the sum total of all the genes of an organism. The human genome, which is contained in the 23 pairs of nuclear chromosomes and in the mitochondrial DNA of each cell, comprises >6 billion nucleotides of genetic code. There are some 23,000 protein-coding genes, a surprisingly small fraction of the total genetic material, with the remainder composed of noncoding DNA, regulatory sequences, and introns. The Human Genome Project, launched in 1990, produced a draft of the genome in 2001 and then a finished sequence in 2003, on the 50th anniversary of the initial publication of Watson and Crick's paper on the double-helical structure of DNA. Since then, this mass of genetic information has been translated at an ever-increasing pace into useable knowledge applicable to clinical medicine. The recent advent of massively parallel DNA sequencing (also known as shotgun, high-throughput, and next-generation sequencing) has brought whole-genome analysis into the clinic for the first time, and most of the current applications are directed at children with congenital conditions that are undiagnosable by using standard genetic tests for single-gene disorders. Thus, pediatricians must become familiar with this technology, what it can and cannot offer, and its technical and ethical challenges. Here, we address the concepts of human genomic analysis and its clinical applicability for primary care providers.

Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest

PubMed Central

2013-01-01

Background The mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak destroyed more than 15 million hectares of pine forests, with major environmental effects on forest health, and economic effects on the forest industry. The outbreak has in part been driven by climate change, and will contribute to increased carbon emissions through decaying forests. Results We developed a genome sequence resource for the mountain pine beetle to better understand the unique aspects of this insect's biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa, and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole-transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione S-transferase, and plant cell wall-degrading enzyme gene families important to the survival of the mountain pine beetle in its harsh and nutrient-poor host environment, and examine genome-wide single-nucleotide polymorphism variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome, and its tissue-specific transcription suggests a functional role for this beetle. Conclusions Despite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome sequence reported in Coleoptera, and will provide an important resource for the Curculionoidea and other insects. PMID:23537049

CoVaCS: a consensus variant calling system.

PubMed

Chiara, Matteo; Gioiosa, Silvia; Chillemi, Giovanni; D'Antonio, Mattia; Flati, Tiziano; Picardi, Ernesto; Zambelli, Federico; Horner, David Stephen; Pesole, Graziano; Castrignanò, Tiziana

2018-02-05

The advent and ongoing development of next generation sequencing technologies (NGS) has led to a rapid increase in the rate of human genome re-sequencing data, paving the way for personalized genomics and precision medicine. The body of genome resequencing data is progressively increasing underlining the need for accurate and time-effective bioinformatics systems for genotyping - a crucial prerequisite for identification of candidate causal mutations in diagnostic screens. Here we present CoVaCS, a fully automated, highly accurate system with a web based graphical interface for genotyping and variant annotation. Extensive tests on a gold standard benchmark data-set -the NA12878 Illumina platinum genome- confirm that call-sets based on our consensus strategy are completely in line with those attained by similar command line based approaches, and far more accurate than call-sets from any individual tool. Importantly our system exhibits better sensitivity and higher specificity than equivalent commercial software. CoVaCS offers optimized pipelines integrating state of the art tools for variant calling and annotation for whole genome sequencing (WGS), whole-exome sequencing (WES) and target-gene sequencing (TGS) data. The system is currently hosted at Cineca, and offers the speed of a HPC computing facility, a crucial consideration when large numbers of samples must be analysed. Importantly, all the analyses are performed automatically allowing high reproducibility of the results. As such, we believe that CoVaCS can be a valuable tool for the analysis of human genome resequencing studies. CoVaCS is available at: https://bioinformatics.cineca.it/covacs .

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 Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

PubMed

Christen, Matthias; Deutsch, Samuel; Christen, Beat

2015-08-21

Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

Draft Genome of the Pearl Oyster Pinctada fucata: A Platform for Understanding Bivalve Biology

PubMed Central

Takeuchi, Takeshi; Kawashima, Takeshi; Koyanagi, Ryo; Gyoja, Fuki; Tanaka, Makiko; Ikuta, Tetsuro; Shoguchi, Eiichi; Fujiwara, Mayuki; Shinzato, Chuya; Hisata, Kanako; Fujie, Manabu; Usami, Takeshi; Nagai, Kiyohito; Maeyama, Kaoru; Okamoto, Kikuhiko; Aoki, Hideo; Ishikawa, Takashi; Masaoka, Tetsuji; Fujiwara, Atushi; Endo, Kazuyoshi; Endo, Hirotoshi; Nagasawa, Hiromichi; Kinoshita, Shigeharu; Asakawa, Shuichi; Watabe, Shugo; Satoh, Nori

2012-01-01

The study of the pearl oyster Pinctada fucata is key to increasing our understanding of the molecular mechanisms involved in pearl biosynthesis and biology of bivalve molluscs. We sequenced ∼1150-Mb genome at ∼40-fold coverage using the Roche 454 GS-FLX and Illumina GAIIx sequencers. The sequences were assembled into contigs with N50 = 1.6 kb (total contig assembly reached to 1024 Mb) and scaffolds with N50 = 14.5 kb. The pearl oyster genome is AT-rich, with a GC content of 34%. DNA transposons, retrotransposons, and tandem repeat elements occupied 0.4, 1.5, and 7.9% of the genome, respectively (a total of 9.8%). Version 1.0 of the P. fucata draft genome contains 23 257 complete gene models, 70% of which are supported by the corresponding expressed sequence tags. The genes include those reported to have an association with bio-mineralization. Genes encoding transcription factors and signal transduction molecules are present in numbers comparable with genomes of other metazoans. Genome-wide molecular phylogeny suggests that the lophotrochozoan represents a distinct clade from ecdysozoans. Our draft genome of the pearl oyster thus provides a platform for the identification of selection markers and genes for calcification, knowledge of which will be important in the pearl industry. PMID:22315334

Application of industrial scale genomics to discovery of therapeutic targets in heart failure.

PubMed

Mehraban, F; Tomlinson, J E

2001-12-01

In recent years intense activity in both academic and industrial sectors has provided a wealth of information on the human genome with an associated impressive increase in the number of novel gene sequences deposited in sequence data repositories and patent applications. This genomic industrial revolution has transformed the way in which drug target discovery is now approached. In this article we discuss how various differential gene expression (DGE) technologies are being utilized for cardiovascular disease (CVD) drug target discovery. Other approaches such as sequencing cDNA from cardiovascular derived tissues and cells coupled with bioinformatic sequence analysis are used with the aim of identifying novel gene sequences that may be exploited towards target discovery. Additional leverage from gene sequence information is obtained through identification of polymorphisms that may confer disease susceptibility and/or affect drug responsiveness. Pharmacogenomic studies are described wherein gene expression-based techniques are used to evaluate drug response and/or efficacy. Industrial-scale genomics supports and addresses not only novel target gene discovery but also the burgeoning issues in pharmaceutical and clinical cardiovascular medicine relative to polymorphic gene responses.

Potential Uses and Inherent Challenges of Using Genome-Scale Sequencing to Augment Current Newborn Screening.

PubMed

Berg, Jonathan S; Powell, Cynthia M

2015-10-05

Since newborn screening (NBS) began in the 1960s, technological advances have enabled its expansion to include an increasing number of disorders. Recent developments now make it possible to sequence an infant's genome relatively quickly and economically. Clinical application of whole-exome and whole-genome sequencing is expanding at a rapid pace but presents many challenges. Its utility in NBS has yet to be demonstrated and its application in the pediatric population requires examination, not only for potential clinical benefits, but also for the unique ethical challenges it presents. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

Genomic Diversity in the Endosymbiotic Bacterium Rhizobium leguminosarum.

PubMed

Sánchez-Cañizares, Carmen; Jorrín, Beatriz; Durán, David; Nadendla, Suvarna; Albareda, Marta; Rubio-Sanz, Laura; Lanza, Mónica; González-Guerrero, Manuel; Prieto, Rosa Isabel; Brito, Belén; Giglio, Michelle G; Rey, Luis; Ruiz-Argüeso, Tomás; Palacios, José M; Imperial, Juan

2018-01-24

Rhizobium leguminosarum bv. viciae is a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae , 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

AbsIDconvert: An absolute approach for converting genetic identifiers at different granularities

PubMed Central

2012-01-01

Background High-throughput molecular biology techniques yield vast amounts of data, often by detecting small portions of ribonucleotides corresponding to specific identifiers. Existing bioinformatic methodologies categorize and compare these elements using inferred descriptive annotation given this sequence information irrespective of the fact that it may not be representative of the identifier as a whole. Results All annotations, no matter the granularity, can be aligned to genomic sequences and therefore annotated by genomic intervals. We have developed AbsIDconvert, a methodology for converting between genomic identifiers by first mapping them onto a common universal coordinate system using an interval tree which is subsequently queried for overlapping identifiers. AbsIDconvert has many potential uses, including gene identifier conversion, identification of features within a genomic region, and cross-species comparisons. The utility is demonstrated in three case studies: 1) comparative genomic study mapping plasmodium gene sequences to corresponding human and mosquito transcriptional regions; 2) cross-species study of Incyte clone sequences; and 3) analysis of human Ensembl transcripts mapped by Affymetrix®; and Agilent microarray probes. AbsIDconvert currently supports ID conversion of 53 species for a given list of input identifiers, genomic sequence, or genome intervals. Conclusion AbsIDconvert provides an efficient and reliable mechanism for conversion between identifier domains of interest. The flexibility of this tool allows for custom definition identifier domains contingent upon the availability and determination of a genomic mapping interval. As the genomes and the sequences for genetic elements are further refined, this tool will become increasingly useful and accurate. AbsIDconvert is freely available as a web application or downloadable as a virtual machine at: http://bioinformatics.louisville.edu/abid/. PMID:22967011

Draft Genome Sequences of Acinetobacter and Bacillus Strains Isolated from Spacecraft-Associated Surfaces

PubMed Central

Seuylemezian, Arman; Vaishampayan, Parag; Cooper, Kerry

2018-01-01

ABSTRACT We report here the draft genome sequences of four strains isolated from spacecraft-associated surfaces exhibiting increased resistance to stressors such as UV radiation and exposure to H2O2. The draft genomes of strains 1P01SCT, FO-92T, 50v1, and 2P01AA had sizes of 5,500,894 bp, 4,699,376 bp, 3,174,402 bp, and 4,328,804 bp, respectively. PMID:29439046

Transposable Element Dynamics among Asymbiotic and Ectomycorrhizal Amanita Fungi

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

Hess, Jaqueline; Skrede, Inger; Wolfe, Benjamin E.

Transposable elements (TEs) are ubiquitous inhabitants of eukaryotic genomes and their proliferation and dispersal shape genome architectures and diversity. Nevertheless, TE dynamics are often explored for one species at a time and are rarely considered in ecological contexts. Recent work with plant pathogens suggests a link between symbiosis and TE abundance. The genomes of pathogenic fungi appear to house an increased abundance of TEs, and TEs are frequently associated with the genes involved in symbiosis. To investigate whether this pattern is general, and relevant to mutualistic plant-fungal symbioses, we sequenced the genomes of related asymbiotic (AS) and ectomycorrhizal (ECM) Amanitamore » fungi. We used methods developed to interrogate both assembled and unassembled sequences, and characterized and quantified TEs across three AS and three ECM species, including the AS outgroup Volvariella volvacea. The ECM genomes are characterized by abundant numbers of TEs, an especially prominent feature of unassembled sequencing libraries. Increased TE activity in ECM species is also supported by phylogenetic analysis of the three most abundant TE superfamilies; phylogenies revealed many radiations within contemporary ECM species. However, the AS species Amanita thiersii also houses extensive amplifications of elements, highlighting the influence of additional evolutionary parameters on TE abundance. Our analyses provide further evidence for a link between symbiotic associations among plants and fungi, and increased TE activity, while highlighting the importance individual species’ natural histories may have in shaping genome architecture.« less

Transposable Element Dynamics among Asymbiotic and Ectomycorrhizal Amanita Fungi

DOE PAGES

Hess, Jaqueline; Skrede, Inger; Wolfe, Benjamin E.; ...

2014-06-12

Transposable elements (TEs) are ubiquitous inhabitants of eukaryotic genomes and their proliferation and dispersal shape genome architectures and diversity. Nevertheless, TE dynamics are often explored for one species at a time and are rarely considered in ecological contexts. Recent work with plant pathogens suggests a link between symbiosis and TE abundance. The genomes of pathogenic fungi appear to house an increased abundance of TEs, and TEs are frequently associated with the genes involved in symbiosis. To investigate whether this pattern is general, and relevant to mutualistic plant-fungal symbioses, we sequenced the genomes of related asymbiotic (AS) and ectomycorrhizal (ECM) Amanitamore » fungi. We used methods developed to interrogate both assembled and unassembled sequences, and characterized and quantified TEs across three AS and three ECM species, including the AS outgroup Volvariella volvacea. The ECM genomes are characterized by abundant numbers of TEs, an especially prominent feature of unassembled sequencing libraries. Increased TE activity in ECM species is also supported by phylogenetic analysis of the three most abundant TE superfamilies; phylogenies revealed many radiations within contemporary ECM species. However, the AS species Amanita thiersii also houses extensive amplifications of elements, highlighting the influence of additional evolutionary parameters on TE abundance. Our analyses provide further evidence for a link between symbiotic associations among plants and fungi, and increased TE activity, while highlighting the importance individual species’ natural histories may have in shaping genome architecture.« less

Influenza Virus Database (IVDB): an integrated information resource and analysis platform for influenza virus research.

PubMed

Chang, Suhua; Zhang, Jiajie; Liao, Xiaoyun; Zhu, Xinxing; Wang, Dahai; Zhu, Jiang; Feng, Tao; Zhu, Baoli; Gao, George F; Wang, Jian; Yang, Huanming; Yu, Jun; Wang, Jing

2007-01-01

Frequent outbreaks of highly pathogenic avian influenza and the increasing data available for comparative analysis require a central database specialized in influenza viruses (IVs). We have established the Influenza Virus Database (IVDB) to integrate information and create an analysis platform for genetic, genomic, and phylogenetic studies of the virus. IVDB hosts complete genome sequences of influenza A virus generated by Beijing Institute of Genomics (BIG) and curates all other published IV sequences after expert annotation. Our Q-Filter system classifies and ranks all nucleotide sequences into seven categories according to sequence content and integrity. IVDB provides a series of tools and viewers for comparative analysis of the viral genomes, genes, genetic polymorphisms and phylogenetic relationships. A search system has been developed for users to retrieve a combination of different data types by setting search options. To facilitate analysis of global viral transmission and evolution, the IV Sequence Distribution Tool (IVDT) has been developed to display the worldwide geographic distribution of chosen viral genotypes and to couple genomic data with epidemiological data. The BLAST, multiple sequence alignment and phylogenetic analysis tools were integrated for online data analysis. Furthermore, IVDB offers instant access to pre-computed alignments and polymorphisms of IV genes and proteins, and presents the results as SNP distribution plots and minor allele distributions. IVDB is publicly available at http://influenza.genomics.org.cn.

FMLRC: Hybrid long read error correction using an FM-index.

PubMed

Wang, Jeremy R; Holt, James; McMillan, Leonard; Jones, Corbin D

2018-02-09

Long read sequencing is changing the landscape of genomic research, especially de novo assembly. Despite the high error rate inherent to long read technologies, increased read lengths dramatically improve the continuity and accuracy of genome assemblies. However, the cost and throughput of these technologies limits their application to complex genomes. One solution is to decrease the cost and time to assemble novel genomes by leveraging "hybrid" assemblies that use long reads for scaffolding and short reads for accuracy. We describe a novel method leveraging a multi-string Burrows-Wheeler Transform with auxiliary FM-index to correct errors in long read sequences using a set of complementary short reads. We demonstrate that our method efficiently produces significantly more high quality corrected sequence than existing hybrid error-correction methods. We also show that our method produces more contiguous assemblies, in many cases, than existing state-of-the-art hybrid and long-read only de novo assembly methods. Our method accurately corrects long read sequence data using complementary short reads. We demonstrate higher total throughput of corrected long reads and a corresponding increase in contiguity of the resulting de novo assemblies. Improved throughput and computational efficiency than existing methods will help better economically utilize emerging long read sequencing technologies.

FRESCO: Referential compression of highly similar sequences.

PubMed

Wandelt, Sebastian; Leser, Ulf

2013-01-01

In many applications, sets of similar texts or sequences are of high importance. Prominent examples are revision histories of documents or genomic sequences. Modern high-throughput sequencing technologies are able to generate DNA sequences at an ever-increasing rate. In parallel to the decreasing experimental time and cost necessary to produce DNA sequences, computational requirements for analysis and storage of the sequences are steeply increasing. Compression is a key technology to deal with this challenge. Recently, referential compression schemes, storing only the differences between a to-be-compressed input and a known reference sequence, gained a lot of interest in this field. In this paper, we propose a general open-source framework to compress large amounts of biological sequence data called Framework for REferential Sequence COmpression (FRESCO). Our basic compression algorithm is shown to be one to two orders of magnitudes faster than comparable related work, while achieving similar compression ratios. We also propose several techniques to further increase compression ratios, while still retaining the advantage in speed: 1) selecting a good reference sequence; and 2) rewriting a reference sequence to allow for better compression. In addition,we propose a new way of further boosting the compression ratios by applying referential compression to already referentially compressed files (second-order compression). This technique allows for compression ratios way beyond state of the art, for instance,4,000:1 and higher for human genomes. We evaluate our algorithms on a large data set from three different species (more than 1,000 genomes, more than 3 TB) and on a collection of versions of Wikipedia pages. Our results show that real-time compression of highly similar sequences at high compression ratios is possible on modern hardware.

The minimum information about a genome sequence (MIGS) specification

PubMed Central

Field, Dawn; Garrity, George; Gray, Tanya; Morrison, Norman; Selengut, Jeremy; Sterk, Peter; Tatusova, Tatiana; Thomson, Nicholas; Allen, Michael J; Angiuoli, Samuel V; Ashburner, Michael; Axelrod, Nelson; Baldauf, Sandra; Ballard, Stuart; Boore, Jeffrey; Cochrane, Guy; Cole, James; Dawyndt, Peter; De Vos, Paul; dePamphilis, Claude; Edwards, Robert; Faruque, Nadeem; Feldman, Robert; Gilbert, Jack; Gilna, Paul; Glöckner, Frank Oliver; Goldstein, Philip; Guralnick, Robert; Haft, Dan; Hancock, David; Hermjakob, Henning; Hertz-Fowler, Christiane; Hugenholtz, Phil; Joint, Ian; Kagan, Leonid; Kane, Matthew; Kennedy, Jessie; Kowalchuk, George; Kottmann, Renzo; Kolker, Eugene; Kravitz, Saul; Kyrpides, Nikos; Leebens-Mack, Jim; Lewis, Suzanna E; Li, Kelvin; Lister, Allyson L; Lord, Phillip; Maltsev, Natalia; Markowitz, Victor; Martiny, Jennifer; Methe, Barbara; Mizrachi, Ilene; Moxon, Richard; Nelson, Karen; Parkhill, Julian; Proctor, Lita; White, Owen; Sansone, Susanna-Assunta; Spiers, Andrew; Stevens, Robert; Swift, Paul; Taylor, Chris; Tateno, Yoshio; Tett, Adrian; Turner, Sarah; Ussery, David; Vaughan, Bob; Ward, Naomi; Whetzel, Trish; Gil, Ingio San; Wilson, Gareth; Wipat, Anil

2008-01-01

With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the ‘transparency’ of the information contained in existing genomic databases. PMID:18464787

INDIGO – INtegrated Data Warehouse of MIcrobial GenOmes with Examples from the Red Sea Extremophiles

PubMed Central

Alam, Intikhab; Antunes, André; Kamau, Allan Anthony; Ba alawi, Wail; Kalkatawi, Manal; Stingl, Ulrich; Bajic, Vladimir B.

2013-01-01

Background The next generation sequencing technologies substantially increased the throughput of microbial genome sequencing. To functionally annotate newly sequenced microbial genomes, a variety of experimental and computational methods are used. Integration of information from different sources is a powerful approach to enhance such annotation. Functional analysis of microbial genomes, necessary for downstream experiments, crucially depends on this annotation but it is hampered by the current lack of suitable information integration and exploration systems for microbial genomes. Results We developed a data warehouse system (INDIGO) that enables the integration of annotations for exploration and analysis of newly sequenced microbial genomes. INDIGO offers an opportunity to construct complex queries and combine annotations from multiple sources starting from genomic sequence to protein domain, gene ontology and pathway levels. This data warehouse is aimed at being populated with information from genomes of pure cultures and uncultured single cells of Red Sea bacteria and Archaea. Currently, INDIGO contains information from Salinisphaera shabanensis, Haloplasma contractile, and Halorhabdus tiamatea - extremophiles isolated from deep-sea anoxic brine lakes of the Red Sea. We provide examples of utilizing the system to gain new insights into specific aspects on the unique lifestyle and adaptations of these organisms to extreme environments. Conclusions We developed a data warehouse system, INDIGO, which enables comprehensive integration of information from various resources to be used for annotation, exploration and analysis of microbial genomes. It will be regularly updated and extended with new genomes. It is aimed to serve as a resource dedicated to the Red Sea microbes. In addition, through INDIGO, we provide our Automatic Annotation of Microbial Genomes (AAMG) pipeline. The INDIGO web server is freely available at http://www.cbrc.kaust.edu.sa/indigo. PMID:24324765

INDIGO - INtegrated data warehouse of microbial genomes with examples from the red sea extremophiles.

PubMed

Alam, Intikhab; Antunes, André; Kamau, Allan Anthony; Ba Alawi, Wail; Kalkatawi, Manal; Stingl, Ulrich; Bajic, Vladimir B

2013-01-01

The next generation sequencing technologies substantially increased the throughput of microbial genome sequencing. To functionally annotate newly sequenced microbial genomes, a variety of experimental and computational methods are used. Integration of information from different sources is a powerful approach to enhance such annotation. Functional analysis of microbial genomes, necessary for downstream experiments, crucially depends on this annotation but it is hampered by the current lack of suitable information integration and exploration systems for microbial genomes. We developed a data warehouse system (INDIGO) that enables the integration of annotations for exploration and analysis of newly sequenced microbial genomes. INDIGO offers an opportunity to construct complex queries and combine annotations from multiple sources starting from genomic sequence to protein domain, gene ontology and pathway levels. This data warehouse is aimed at being populated with information from genomes of pure cultures and uncultured single cells of Red Sea bacteria and Archaea. Currently, INDIGO contains information from Salinisphaera shabanensis, Haloplasma contractile, and Halorhabdus tiamatea - extremophiles isolated from deep-sea anoxic brine lakes of the Red Sea. We provide examples of utilizing the system to gain new insights into specific aspects on the unique lifestyle and adaptations of these organisms to extreme environments. We developed a data warehouse system, INDIGO, which enables comprehensive integration of information from various resources to be used for annotation, exploration and analysis of microbial genomes. It will be regularly updated and extended with new genomes. It is aimed to serve as a resource dedicated to the Red Sea microbes. In addition, through INDIGO, we provide our Automatic Annotation of Microbial Genomes (AAMG) pipeline. The INDIGO web server is freely available at http://www.cbrc.kaust.edu.sa/indigo.

Unexpected effects of different genetic backgrounds on identification of genomic rearrangements via whole-genome next generation sequencing.

PubMed

Chen, Zhangguo; Gowan, Katherine; Leach, Sonia M; Viboolsittiseri, Sawanee S; Mishra, Ameet K; Kadoishi, Tanya; Diener, Katrina; Gao, Bifeng; Jones, Kenneth; Wang, Jing H

2016-10-21

Whole genome next generation sequencing (NGS) is increasingly employed to detect genomic rearrangements in cancer genomes, especially in lymphoid malignancies. We recently established a unique mouse model by specifically deleting a key non-homologous end-joining DNA repair gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in germinal center B cells. This mouse model spontaneously develops mature B cell lymphomas (termed G1XP lymphomas). Here, we attempt to employ whole genome NGS to identify novel structural rearrangements, in particular inter-chromosomal translocations (CTXs), in these G1XP lymphomas. We sequenced six lymphoma samples, aligned our NGS data with mouse reference genome (in C57BL/6J (B6) background) and identified CTXs using CREST algorithm. Surprisingly, we detected widespread CTXs in both lymphomas and wildtype control samples, majority of which were false positive and attributable to different genetic backgrounds. In addition, we validated our NGS pipeline by sequencing multiple control samples from distinct tissues of different genetic backgrounds of mouse (B6 vs non-B6). Lastly, our studies showed that widespread false positive CTXs can be generated by simply aligning sequences from different genetic backgrounds of mouse. We conclude that mapping and alignment with reference genome might not be a preferred method for analyzing whole-genome NGS data obtained from a genetic background different from reference genome. Given the complex genetic background of different mouse strains or the heterogeneity of cancer genomes in human patients, in order to minimize such systematic artifacts and uncover novel CTXs, a preferred method might be de novo assembly of personalized normal control genome and cancer cell genome, instead of mapping and aligning NGS data to mouse or human reference genome. Thus, our studies have critical impact on the manner of data analysis for cancer genomics.

Draft genome of the red harvester ant Pogonomyrmex barbatus.

PubMed

Smith, Chris R; Smith, Christopher D; Robertson, Hugh M; Helmkampf, Martin; Zimin, Aleksey; Yandell, Mark; Holt, Carson; Hu, Hao; Abouheif, Ehab; Benton, Richard; Cash, Elizabeth; Croset, Vincent; Currie, Cameron R; Elhaik, Eran; Elsik, Christine G; Favé, Marie-Julie; Fernandes, Vilaiwan; Gibson, Joshua D; Graur, Dan; Gronenberg, Wulfila; Grubbs, Kirk J; Hagen, Darren E; Viniegra, Ana Sofia Ibarraran; Johnson, Brian R; Johnson, Reed M; Khila, Abderrahman; Kim, Jay W; Mathis, Kaitlyn A; Munoz-Torres, Monica C; Murphy, Marguerite C; Mustard, Julie A; Nakamura, Rin; Niehuis, Oliver; Nigam, Surabhi; Overson, Rick P; Placek, Jennifer E; Rajakumar, Rajendhran; Reese, Justin T; Suen, Garret; Tao, Shu; Torres, Candice W; Tsutsui, Neil D; Viljakainen, Lumi; Wolschin, Florian; Gadau, Jürgen

2011-04-05

We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.

Thermophiles in the genomic era: Biodiversity, science, and applications.

PubMed

Urbieta, M Sofía; Donati, Edgardo R; Chan, Kok-Gan; Shahar, Saleha; Sin, Lee Li; Goh, Kian Mau

2015-11-01

Thermophiles and hyperthermophiles are present in various regions of the Earth, including volcanic environments, hot springs, mud pots, fumaroles, geysers, coastal thermal springs, and even deep-sea hydrothermal vents. They are also found in man-made environments, such as heated compost facilities, reactors, and spray dryers. Thermophiles, hyperthermophiles, and their bioproducts facilitate various industrial, agricultural, and medicinal applications and offer potential solutions to environmental damages and the demand for biofuels. Intensified efforts to sequence the entire genome of hyperthermophiles and thermophiles are increasing rapidly, as evidenced by the fact that over 120 complete genome sequences of the hyperthermophiles Aquificae, Thermotogae, Crenarchaeota, and Euryarchaeota are now available. In this review, we summarise the major current applications of thermophiles and thermozymes. In addition, emphasis is placed on recent progress in understanding the biodiversity, genomes, transcriptomes, metagenomes, and single-cell sequencing of thermophiles in the genomic era. Copyright © 2015 Elsevier Inc. All rights reserved.

STORMSeq: An Open-Source, User-Friendly Pipeline for Processing Personal Genomics Data in the Cloud

PubMed Central

Karczewski, Konrad J.; Fernald, Guy Haskin; Martin, Alicia R.; Snyder, Michael; Tatonetti, Nicholas P.; Dudley, Joel T.

2014-01-01

The increasing public availability of personal complete genome sequencing data has ushered in an era of democratized genomics. However, read mapping and variant calling software is constantly improving and individuals with personal genomic data may prefer to customize and update their variant calls. Here, we describe STORMSeq (Scalable Tools for Open-Source Read Mapping), a graphical interface cloud computing solution that does not require a parallel computing environment or extensive technical experience. This customizable and modular system performs read mapping, read cleaning, and variant calling and annotation. At present, STORMSeq costs approximately $2 and 5–10 hours to process a full exome sequence and $30 and 3–8 days to process a whole genome sequence. We provide this open-access and open-source resource as a user-friendly interface in Amazon EC2. PMID:24454756

Interpreting Microbial Biosynthesis in the Genomic Age: Biological and Practical Considerations

PubMed Central

Miller, Ian J.; Chevrette, Marc G.; Kwan, Jason C.

2017-01-01

Genome mining has become an increasingly powerful, scalable, and economically accessible tool for the study of natural product biosynthesis and drug discovery. However, there remain important biological and practical problems that can complicate or obscure biosynthetic analysis in genomic and metagenomic sequencing projects. Here, we focus on limitations of available technology as well as computational and experimental strategies to overcome them. We review the unique challenges and approaches in the study of symbiotic and uncultured systems, as well as those associated with biosynthetic gene cluster (BGC) assembly and product prediction. Finally, to explore sequencing parameters that affect the recovery and contiguity of large and repetitive BGCs assembled de novo, we simulate Illumina and PacBio sequencing of the Salinispora tropica genome focusing on assembly of the salinilactam (slm) BGC. PMID:28587290

The past, present and future of mitochondrial genomics: have we sequenced enough mtDNAs?

PubMed

Smith, David Roy

2016-01-01

The year 2014 saw more than a thousand new mitochondrial genome sequences deposited in GenBank-an almost 15% increase from the previous year. Hundreds of peer-reviewed articles accompanied these genomes, making mitochondrial DNAs (mtDNAs) the most sequenced and reported type of eukaryotic chromosome. These mtDNA data have advanced a wide range of scientific fields, from forensics to anthropology to medicine to molecular evolution. But for many biological lineages, mtDNAs are so well sampled that newly published genomes are arguably no longer contributing significantly to the progression of science, and in some cases they are tying up valuable resources, particularly journal editors and referees. Is it time to acknowledge that as a research community we have published enough mitochondrial genome papers? Here, I address this question, exploring the history, milestones and impacts of mitochondrial genomics, the benefits and drawbacks of continuing to publish mtDNAs at a high rate and what the future may hold for such an important and popular genetic marker. I highlight groups for which mtDNAs are still poorly sampled, thus meriting further investigation, and recommend that more energy be spent characterizing aspects of mitochondrial genomes apart from the DNA sequence, such as their chromosomal and transcriptional architectures. Ultimately, one should be mindful before writing a mitochondrial genome paper. Consider perhaps sending the sequence directly to GenBank instead, and be sure to annotate it correctly before submission. © The Author 2015. Published by Oxford University Press.

Library construction for next-generation sequencing: Overviews and challenges

PubMed Central

Head, Steven R.; Komori, H. Kiyomi; LaMere, Sarah A.; Whisenant, Thomas; Van Nieuwerburgh, Filip; Salomon, Daniel R.; Ordoukhanian, Phillip

2014-01-01

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed. PMID:24502796

Comparative Analysis of the First Complete Enterococcus faecium Genome

PubMed Central

Lam, Margaret M. C.; Seemann, Torsten; Bulach, Dieter M.; Gladman, Simon L.; Chen, Honglei; Haring, Volker; Moore, Robert J.; Ballard, Susan; Grayson, M. Lindsay; Johnson, Paul D. R.; Howden, Benjamin P.

2012-01-01

Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infections in health care facilities around the globe. In particular, infections caused by vancomycin-resistant Enterococcus faecium are becoming increasingly common. Comparative and functional genomic studies of E. faecium isolates have so far been limited owing to the lack of a fully assembled E. faecium genome sequence. Here we address this issue and report the complete 3.0-Mb genome sequence of the multilocus sequence type 17 vancomycin-resistant Enterococcus faecium strain Aus0004, isolated from the bloodstream of a patient in Melbourne, Australia, in 1998. The genome comprises a 2.9-Mb circular chromosome and three circular plasmids. The chromosome harbors putative E. faecium virulence factors such as enterococcal surface protein, hemolysin, and collagen-binding adhesin. Aus0004 has a very large accessory genome (38%) that includes three prophage and two genomic islands absent among 22 other E. faecium genomes. One of the prophage was present as inverted 50-kb repeats that appear to have facilitated a 683-kb chromosomal inversion across the replication terminus, resulting in a striking replichore imbalance. Other distinctive features include 76 insertion sequence elements and a single chromosomal copy of Tn1549 containing the vanB vancomycin resistance element. A complete E. faecium genome will be a useful resource to assist our understanding of this emerging nosocomial pathogen. PMID:22366422

Emerging Genomic Tools for Legume Breeding: Current Status and Future Prospects

PubMed Central

Pandey, Manish K.; Roorkiwal, Manish; Singh, Vikas K.; Ramalingam, Abirami; Kudapa, Himabindu; Thudi, Mahendar; Chitikineni, Anu; Rathore, Abhishek; Varshney, Rajeev K.

2016-01-01

Legumes play a vital role in ensuring global nutritional food security and improving soil quality through nitrogen fixation. Accelerated higher genetic gains is required to meet the demand of ever increasing global population. In recent years, speedy developments have been witnessed in legume genomics due to advancements in next-generation sequencing (NGS) and high-throughput genotyping technologies. Reference genome sequences for many legume crops have been reported in the last 5 years. The availability of the draft genome sequences and re-sequencing of elite genotypes for several important legume crops have made it possible to identify structural variations at large scale. Availability of large-scale genomic resources and low-cost and high-throughput genotyping technologies are enhancing the efficiency and resolution of genetic mapping and marker-trait association studies. Most importantly, deployment of molecular breeding approaches has resulted in development of improved lines in some legume crops such as chickpea and groundnut. In order to support genomics-driven crop improvement at a fast pace, the deployment of breeder-friendly genomics and decision support tools seems appear to be critical in breeding programs in developing countries. This review provides an overview of emerging genomics and informatics tools/approaches that will be the key driving force for accelerating genomics-assisted breeding and ultimately ensuring nutritional and food security in developing countries. PMID:27199998

Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery

PubMed Central

Karas, Vlad O; Sinnott-Armstrong, Nicholas A; Varghese, Vici; Shafer, Robert W; Greenleaf, William J; Sherlock, Gavin

2018-01-01

Abstract Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation. PMID:29361139

Quantitative trait loci markers derived from whole genome sequence data increases the reliability of genomic prediction.

PubMed

Brøndum, R F; Su, G; Janss, L; Sahana, G; Guldbrandtsen, B; Boichard, D; Lund, M S

2015-06-01

This study investigated the effect on the reliability of genomic prediction when a small number of significant variants from single marker analysis based on whole genome sequence data were added to the regular 54k single nucleotide polymorphism (SNP) array data. The extra markers were selected with the aim of augmenting the custom low-density Illumina BovineLD SNP chip (San Diego, CA) used in the Nordic countries. The single-marker analysis was done breed-wise on all 16 index traits included in the breeding goals for Nordic Holstein, Danish Jersey, and Nordic Red cattle plus the total merit index itself. Depending on the trait's economic weight, 15, 10, or 5 quantitative trait loci (QTL) were selected per trait per breed and 3 to 5 markers were selected to tag each QTL. After removing duplicate markers (same marker selected for more than one trait or breed) and filtering for high pairwise linkage disequilibrium and assaying performance on the array, a total of 1,623 QTL markers were selected for inclusion on the custom chip. Genomic prediction analyses were performed for Nordic and French Holstein and Nordic Red animals using either a genomic BLUP or a Bayesian variable selection model. When using the genomic BLUP model including the QTL markers in the analysis, reliability was increased by up to 4 percentage points for production traits in Nordic Holstein animals, up to 3 percentage points for Nordic Reds, and up to 5 percentage points for French Holstein. Smaller gains of up to 1 percentage point was observed for mastitis, but only a 0.5 percentage point increase was seen for fertility. When using a Bayesian model accuracies were generally higher with only 54k data compared with the genomic BLUP approach, but increases in reliability were relatively smaller when QTL markers were included. Results from this study indicate that the reliability of genomic prediction can be increased by including markers significant in genome-wide association studies on whole genome sequence data alongside the 54k SNP set. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Genomics-assisted breeding in four major pulse crops of developing countries: present status and prospects.

PubMed

Bohra, Abhishek; Pandey, Manish K; Jha, Uday C; Singh, Balwant; Singh, Indra P; Datta, Dibendu; Chaturvedi, Sushil K; Nadarajan, N; Varshney, Rajeev K

2014-06-01

Given recent advances in pulse molecular biology, genomics-driven breeding has emerged as a promising approach to address the issues of limited genetic gain and low productivity in various pulse crops. The global population is continuously increasing and is expected to reach nine billion by 2050. This huge population pressure will lead to severe shortage of food, natural resources and arable land. Such an alarming situation is most likely to arise in developing countries due to increase in the proportion of people suffering from protein and micronutrient malnutrition. Pulses being a primary and affordable source of proteins and minerals play a key role in alleviating the protein calorie malnutrition, micronutrient deficiencies and other undernourishment-related issues. Additionally, pulses are a vital source of livelihood generation for millions of resource-poor farmers practising agriculture in the semi-arid and sub-tropical regions. Limited success achieved through conventional breeding so far in most of the pulse crops will not be enough to feed the ever increasing population. In this context, genomics-assisted breeding (GAB) holds promise in enhancing the genetic gains. Though pulses have long been considered as orphan crops, recent advances in the area of pulse genomics are noteworthy, e.g. discovery of genome-wide genetic markers, high-throughput genotyping and sequencing platforms, high-density genetic linkage/QTL maps and, more importantly, the availability of whole-genome sequence. With genome sequence in hand, there is a great scope to apply genome-wide methods for trait mapping using association studies and to choose desirable genotypes via genomic selection. It is anticipated that GAB will speed up the progress of genetic improvement of pulses, leading to the rapid development of cultivars with higher yield, enhanced stress tolerance and wider adaptability.

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

PubMed

Goya, Stephanie; Valinotto, Laura E; Tittarelli, Estefania; Rojo, Gabriel L; Nabaes Jodar, Mercedes S; Greninger, Alexander L; Zaiat, Jonathan J; Marti, Marcelo A; Mistchenko, Alicia S; Viegas, Mariana

2018-01-01

Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load ≥ E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1-3 (HPIV1-3) were also obtained with the selected optimal methodology.

Genomic Sequence Variation Markup Language (GSVML).

PubMed

Nakaya, Jun; Kimura, Michio; Hiroi, Kaei; Ido, Keisuke; Yang, Woosung; Tanaka, Hiroshi

2010-02-01

With the aim of making good use of internationally accumulated genomic sequence variation data, which is increasing rapidly due to the explosive amount of genomic research at present, the development of an interoperable data exchange format and its international standardization are necessary. Genomic Sequence Variation Markup Language (GSVML) will focus on genomic sequence variation data and human health applications, such as gene based medicine or pharmacogenomics. We developed GSVML through eight steps, based on case analysis and domain investigations. By focusing on the design scope to human health applications and genomic sequence variation, we attempted to eliminate ambiguity and to ensure practicability. We intended to satisfy the requirements derived from the use case analysis of human-based clinical genomic applications. Based on database investigations, we attempted to minimize the redundancy of the data format, while maximizing the data covering range. We also attempted to ensure communication and interface ability with other Markup Languages, for exchange of omics data among various omics researchers or facilities. The interface ability with developing clinical standards, such as the Health Level Seven Genotype Information model, was analyzed. We developed the human health-oriented GSVML comprising variation data, direct annotation, and indirect annotation categories; the variation data category is required, while the direct and indirect annotation categories are optional. The annotation categories contain omics and clinical information, and have internal relationships. For designing, we examined 6 cases for three criteria as human health application and 15 data elements for three criteria as data formats for genomic sequence variation data exchange. The data format of five international SNP databases and six Markup Languages and the interface ability to the Health Level Seven Genotype Model in terms of 317 items were investigated. GSVML was developed as a potential data exchanging format for genomic sequence variation data exchange focusing on human health applications. The international standardization of GSVML is necessary, and is currently underway. GSVML can be applied to enhance the utilization of genomic sequence variation data worldwide by providing a communicable platform between clinical and research applications. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Human genetics and genomics a decade after the release of the draft sequence of the human genome.

PubMed

Naidoo, Nasheen; Pawitan, Yudi; Soong, Richie; Cooper, David N; Ku, Chee-Seng

2011-10-01

Substantial progress has been made in human genetics and genomics research over the past ten years since the publication of the draft sequence of the human genome in 2001. Findings emanating directly from the Human Genome Project, together with those from follow-on studies, have had an enormous impact on our understanding of the architecture and function of the human genome. Major developments have been made in cataloguing genetic variation, the International HapMap Project, and with respect to advances in genotyping technologies. These developments are vital for the emergence of genome-wide association studies in the investigation of complex diseases and traits. In parallel, the advent of high-throughput sequencing technologies has ushered in the 'personal genome sequencing' era for both normal and cancer genomes, and made possible large-scale genome sequencing studies such as the 1000 Genomes Project and the International Cancer Genome Consortium. The high-throughput sequencing and sequence-capture technologies are also providing new opportunities to study Mendelian disorders through exome sequencing and whole-genome sequencing. This paper reviews these major developments in human genetics and genomics over the past decade.

Human genetics and genomics a decade after the release of the draft sequence of the human genome

PubMed Central

2011-01-01

Substantial progress has been made in human genetics and genomics research over the past ten years since the publication of the draft sequence of the human genome in 2001. Findings emanating directly from the Human Genome Project, together with those from follow-on studies, have had an enormous impact on our understanding of the architecture and function of the human genome. Major developments have been made in cataloguing genetic variation, the International HapMap Project, and with respect to advances in genotyping technologies. These developments are vital for the emergence of genome-wide association studies in the investigation of complex diseases and traits. In parallel, the advent of high-throughput sequencing technologies has ushered in the 'personal genome sequencing' era for both normal and cancer genomes, and made possible large-scale genome sequencing studies such as the 1000 Genomes Project and the International Cancer Genome Consortium. The high-throughput sequencing and sequence-capture technologies are also providing new opportunities to study Mendelian disorders through exome sequencing and whole-genome sequencing. This paper reviews these major developments in human genetics and genomics over the past decade. PMID:22155605

GenomicTools: a computational platform for developing high-throughput analytics in genomics.

PubMed

Tsirigos, Aristotelis; Haiminen, Niina; Bilal, Erhan; Utro, Filippo

2012-01-15

Recent advances in sequencing technology have resulted in the dramatic increase of sequencing data, which, in turn, requires efficient management of computational resources, such as computing time, memory requirements as well as prototyping of computational pipelines. We present GenomicTools, a flexible computational platform, comprising both a command-line set of tools and a C++ API, for the analysis and manipulation of high-throughput sequencing data such as DNA-seq, RNA-seq, ChIP-seq and MethylC-seq. GenomicTools implements a variety of mathematical operations between sets of genomic regions thereby enabling the prototyping of computational pipelines that can address a wide spectrum of tasks ranging from pre-processing and quality control to meta-analyses. Additionally, the GenomicTools platform is designed to analyze large datasets of any size by minimizing memory requirements. In practical applications, where comparable, GenomicTools outperforms existing tools in terms of both time and memory usage. The GenomicTools platform (version 2.0.0) was implemented in C++. The source code, documentation, user manual, example datasets and scripts are available online at http://code.google.com/p/ibm-cbc-genomic-tools.

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

PubMed Central

2011-01-01

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol. PMID:21205303

Sensitive Next-Generation Sequencing Method Reveals Deep Genetic Diversity of HIV-1 in the Democratic Republic of the Congo.

PubMed

Rodgers, Mary A; Wilkinson, Eduan; Vallari, Ana; McArthur, Carole; Sthreshley, Larry; Brennan, Catherine A; Cloherty, Gavin; de Oliveira, Tulio

2017-03-15

As the epidemiological epicenter of the human immunodeficiency virus (HIV) pandemic, the Democratic Republic of the Congo (DRC) is a reservoir of circulating HIV strains exhibiting high levels of diversity and recombination. In this study, we characterized HIV specimens collected in two rural areas of the DRC between 2001 and 2003 to identify rare strains of HIV. The env gp41 region was sequenced and characterized for 172 HIV-positive specimens. The env sequences were predominantly subtype A (43.02%), but 7 other subtypes (33.14%), 20 circulating recombinant forms (CRFs; 11.63%), and 20 unclassified (11.63%) sequences were also found. Of the rare and unclassified subtypes, 18 specimens were selected for next-generation sequencing (NGS) by a modified HIV-switching mechanism at the 5' end of the RNA template (SMART) method to obtain full-genome sequences. NGS produced 14 new complete genomes, which included pure subtype C ( n = 2), D ( n = 1), F1 ( n = 1), H ( n = 3), and J ( n = 1) genomes. The two subtype C genomes and one of the subtype H genomes branched basal to their respective subtype branches but had no evidence of recombination. The remaining 6 genomes were complex recombinants of 2 or more subtypes, including subtypes A1, F, G, H, J, and K and unclassified fragments, including one subtype CRF25 isolate, which branched basal to all CRF25 references. Notably, all recombinant subtype H fragments branched basal to the H clade. Spatial-geographical analysis indicated that the diverse sequences identified here did not expand globally. The full-genome and subgenomic sequences identified in our study population significantly increase the documented diversity of the strains involved in the continually evolving HIV-1 pandemic. IMPORTANCE Very little is known about the ancestral HIV-1 strains that founded the global pandemic, and very few complete genome sequences are available from patients in the Congo Basin, where HIV-1 expanded early in the global pandemic. By sequencing a subgenomic fragment of the HIV-1 envelope from study participants in the DRC, we identified rare variants for complete genome sequencing. The basal branching of some of the complete genome sequences that we recovered suggests that these strains are more closely related to ancestral HIV-1 strains than to previously reported strains and is evidence that the local diversification of HIV in the DRC continues to outpace the diversity of global strains decades after the emergence of the pandemic. Copyright © 2017 Rodgers et al.

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies

PubMed Central

Karamitros, Timokratis; Piorkowska, Renata; Katzourakis, Aris; Magiorkinis, Gkikas; Mbisa, Jean Lutamyo

2016-01-01

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal. PMID:27309375

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies.

PubMed

Karamitros, Timokratis; Harrison, Ian; Piorkowska, Renata; Katzourakis, Aris; Magiorkinis, Gkikas; Mbisa, Jean Lutamyo

2016-01-01

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal.

Reconstruction of the ancestral plastid genome in Geraniaceae reveals a correlation between genome rearrangements, repeats, and nucleotide substitution rates.

PubMed

Weng, Mao-Lun; Blazier, John C; Govindu, Madhumita; Jansen, Robert K

2014-03-01

Geraniaceae plastid genomes are highly rearranged, and each of the four genera already sequenced in the family has a distinct genome organization. This study reports plastid genome sequences of six additional species, Francoa sonchifolia, Melianthus villosus, and Viviania marifolia from Geraniales, and Pelargonium alternans, California macrophylla, and Hypseocharis bilobata from Geraniaceae. These genome sequences, combined with previously published species, provide sufficient taxon sampling to reconstruct the ancestral plastid genome organization of Geraniaceae and the rearrangements unique to each genus. The ancestral plastid genome of Geraniaceae has a 4 kb inversion and a reduced, Pelargonium-like small single copy region. Our ancestral genome reconstruction suggests that a few minor rearrangements occurred in the stem branch of Geraniaceae followed by independent rearrangements in each genus. The genomic comparison demonstrates that a series of inverted repeat boundary shifts and inversions played a major role in shaping genome organization in the family. The distribution of repeats is strongly associated with breakpoints in the rearranged genomes, and the proportion and the number of large repeats (>20 bp and >60 bp) are significantly correlated with the degree of genome rearrangements. Increases in the degree of plastid genome rearrangements are correlated with the acceleration in nonsynonymous substitution rates (dN) but not with synonymous substitution rates (dS). Possible mechanisms that might contribute to this correlation, including DNA repair system and selection, are discussed.

Simultaneous mutation and copy number variation (CNV) detection by multiplex PCR-based GS-FLX sequencing.

PubMed

Goossens, Dirk; Moens, Lotte N; Nelis, Eva; Lenaerts, An-Sofie; Glassee, Wim; Kalbe, Andreas; Frey, Bruno; Kopal, Guido; De Jonghe, Peter; De Rijk, Peter; Del-Favero, Jurgen

2009-03-01

We evaluated multiplex PCR amplification as a front-end for high-throughput sequencing, to widen the applicability of massive parallel sequencers for the detailed analysis of complex genomes. Using multiplex PCR reactions, we sequenced the complete coding regions of seven genes implicated in peripheral neuropathies in 40 individuals on a GS-FLX genome sequencer (Roche). The resulting dataset showed highly specific and uniform amplification. Comparison of the GS-FLX sequencing data with the dataset generated by Sanger sequencing confirmed the detection of all variants present and proved the sensitivity of the method for mutation detection. In addition, we showed that we could exploit the multiplexed PCR amplicons to determine individual copy number variation (CNV), increasing the spectrum of detected variations to both genetic and genomic variants. We conclude that our straightforward procedure substantially expands the applicability of the massive parallel sequencers for sequencing projects of a moderate number of amplicons (50-500) with typical applications in resequencing exons in positional or functional candidate regions and molecular genetic diagnostics. 2008 Wiley-Liss, Inc.

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...

Genome Science: A Video Tour of the Washington University Genome Sequencing Center for High School and Undergraduate Students

ERIC Educational Resources Information Center

Flowers, Susan K.; Easter, Carla; Holmes, Andrea; Cohen, Brian; Bednarski, April E.; Mardis, Elaine R.; Wilson, Richard K.; Elgin, Sarah C. R.

2005-01-01

Sequencing of the human genome has ushered in a new era of biology. The technologies developed to facilitate the sequencing of the human genome are now being applied to the sequencing of other genomes. In 2004, a partnership was formed between Washington University School of Medicine Genome Sequencing Center's Outreach Program and Washington…

Genomics and integrated systems biology in Plasmodium falciparum: a path to malaria control and eradication.

PubMed

Le Roch, K G; Chung, D-W D; Ponts, N

2012-01-01

The first draft of the human malaria parasite's genome was released in 2002. Since then, the malaria scientific community has witnessed a steady embrace of new and powerful functional genomic studies. Over the years, these approaches have slowly revolutionized malaria research and enabled the comprehensive, unbiased investigation of various aspects of the parasite's biology. These genome-wide analyses delivered a refined annotation of the parasite's genome, delivered a better knowledge of its RNA, proteins and metabolite derivatives, and fostered the discovery of new vaccine and drug targets. Despite the positive impacts of these genomic studies, most research and investment still focus on protein targets, drugs and vaccine candidates that were known before the publication of the parasite genome sequence. However, recent access to next-generation sequencing technologies, along with an increased number of genome-wide applications, is expanding the impact of the parasite genome on biomedical research, contributing to a paradigm shift in research activities that may possibly lead to new optimized diagnosis and treatments. This review provides an update of Plasmodium falciparum genome sequences and an overview of the rapid development of genomics and system biology applications that have an immense potential of creating powerful tools for a successful malaria eradication campaign. © 2011 Blackwell Publishing Ltd.

In silico comparison of genomic regions containing genes coding for enzymes and transcription factors for the phenylpropanoid pathway in Phaseolus vulgaris L. and Glycine max L. Merr

PubMed Central

Reinprecht, Yarmilla; Yadegari, Zeinab; Perry, Gregory E.; Siddiqua, Mahbuba; Wright, Lori C.; McClean, Phillip E.; Pauls, K. Peter

2013-01-01

Legumes contain a variety of phytochemicals derived from the phenylpropanoid pathway that have important effects on human health as well as seed coat color, plant disease resistance and nodulation. However, the information about the genes involved in this important pathway is fragmentary in common bean (Phaseolus vulgaris L.). The objectives of this research were to isolate genes that function in and control the phenylpropanoid pathway in common bean, determine their genomic locations in silico in common bean and soybean, and analyze sequences of the 4CL gene family in two common bean genotypes. Sequences of phenylpropanoid pathway genes available for common bean or other plant species were aligned, and the conserved regions were used to design sequence-specific primers. The PCR products were cloned and sequenced and the gene sequences along with common bean gene-based (g) markers were BLASTed against the Glycine max v.1.0 genome and the P. vulgaris v.1.0 (Andean) early release genome. In addition, gene sequences were BLASTed against the OAC Rex (Mesoamerican) genome sequence assembly. In total, fragments of 46 structural and regulatory phenylpropanoid pathway genes were characterized in this way and placed in silico on common bean and soybean sequence maps. The maps contain over 250 common bean g and SSR (simple sequence repeat) markers and identify the positions of more than 60 additional phenylpropanoid pathway gene sequences, plus the putative locations of seed coat color genes. The majority of cloned phenylpropanoid pathway gene sequences were mapped to one location in the common bean genome but had two positions in soybean. The comparison of the genomic maps confirmed previous studies, which show that common bean and soybean share genomic regions, including those containing phenylpropanoid pathway gene sequences, with conserved synteny. Indels identified in the comparison of Andean and Mesoamerican common bean 4CL gene sequences might be used to develop inter-pool phenylpropanoid pathway gene-based markers. We anticipate that the information obtained by this study will simplify and accelerate selections of common bean with specific phenylpropanoid pathway alleles to increase the contents of beneficial phenylpropanoids in common bean and other legumes. PMID:24046770

CoryneBase: Corynebacterium Genomic Resources and Analysis Tools at Your Fingertips

PubMed Central

Tan, Mui Fern; Jakubovics, Nick S.; Wee, Wei Yee; Mutha, Naresh V. R.; Wong, Guat Jah; Ang, Mia Yang; Yazdi, Amir Hessam; Choo, Siew Woh

2014-01-01

Corynebacteria are used for a wide variety of industrial purposes but some species are associated with human diseases. With increasing number of corynebacterial genomes having been sequenced, comparative analysis of these strains may provide better understanding of their biology, phylogeny, virulence and taxonomy that may lead to the discoveries of beneficial industrial strains or contribute to better management of diseases. To facilitate the ongoing research of corynebacteria, a specialized central repository and analysis platform for the corynebacterial research community is needed to host the fast-growing amount of genomic data and facilitate the analysis of these data. Here we present CoryneBase, a genomic database for Corynebacterium with diverse functionality for the analysis of genomes aimed to provide: (1) annotated genome sequences of Corynebacterium where 165,918 coding sequences and 4,180 RNAs can be found in 27 species; (2) access to comprehensive Corynebacterium data through the use of advanced web technologies for interactive web interfaces; and (3) advanced bioinformatic analysis tools consisting of standard BLAST for homology search, VFDB BLAST for sequence homology search against the Virulence Factor Database (VFDB), Pairwise Genome Comparison (PGC) tool for comparative genomic analysis, and a newly designed Pathogenomics Profiling Tool (PathoProT) for comparative pathogenomic analysis. CoryneBase offers the access of a range of Corynebacterium genomic resources as well as analysis tools for comparative genomics and pathogenomics. It is publicly available at http://corynebacterium.um.edu.my/. PMID:24466021

Whole-Genome-Sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374.

PubMed

Wang, Xiaoli; Xie, Yingzhou; Li, Gang; Liu, Jialin; Li, Xiaobin; Tian, Lijun; Sun, Jingyong; Ou, Hong-Yu; Qu, Hongping

2018-01-01

Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 10 2 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance.

Whole-Genome-Sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374

PubMed Central

Wang, Xiaoli; Xie, Yingzhou; Li, Gang; Liu, Jialin; Li, Xiaobin; Tian, Lijun; Sun, Jingyong; Qu, Hongping

2018-01-01

ABSTRACT Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 102 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance. PMID:29338592

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

Plastome Sequence Determination and Comparative Analysis for Members of the Lolium-Festuca Grass Species Complex

PubMed Central

Hand, Melanie L.; Spangenberg, German C.; Forster, John W.; Cogan, Noel O. I.

2013-01-01

Chloroplast genome sequences are of broad significance in plant biology, due to frequent use in molecular phylogenetics, comparative genomics, population genetics, and genetic modification studies. The present study used a second-generation sequencing approach to determine and assemble the plastid genomes (plastomes) of four representatives from the agriculturally important Lolium-Festuca species complex of pasture grasses (Lolium multiflorum, Festuca pratensis, Festuca altissima, and Festuca ovina). Total cellular DNA was extracted from either roots or leaves, was sequenced, and the output was filtered for plastome-related reads. A comparison between sources revealed fewer plastome-related reads from root-derived template but an increase in incidental bacterium-derived sequences. Plastome assembly and annotation indicated high levels of sequence identity and a conserved organization and gene content between species. However, frequent deletions within the F. ovina plastome appeared to contribute to a smaller plastid genome size. Comparative analysis with complete plastome sequences from other members of the Poaceae confirmed conservation of most grass-specific features. Detailed analysis of the rbcL–psaI intergenic region, however, revealed a “hot-spot” of variation characterized by independent deletion events. The evolutionary implications of this observation are discussed. The complete plastome sequences are anticipated to provide the basis for potential organelle-specific genetic modification of pasture grasses. PMID:23550121

Development of self-compressing BLSOM for comprehensive analysis of big sequence data.

PubMed

Kikuchi, Akihito; Ikemura, Toshimichi; Abe, Takashi

2015-01-01

With the remarkable increase in genomic sequence data from various organisms, novel tools are needed for comprehensive analyses of available big sequence data. We previously developed a Batch-Learning Self-Organizing Map (BLSOM), which can cluster genomic fragment sequences according to phylotype solely dependent on oligonucleotide composition and applied to genome and metagenomic studies. BLSOM is suitable for high-performance parallel-computing and can analyze big data simultaneously, but a large-scale BLSOM needs a large computational resource. We have developed Self-Compressing BLSOM (SC-BLSOM) for reduction of computation time, which allows us to carry out comprehensive analysis of big sequence data without the use of high-performance supercomputers. The strategy of SC-BLSOM is to hierarchically construct BLSOMs according to data class, such as phylotype. The first-layer BLSOM was constructed with each of the divided input data pieces that represents the data subclass, such as phylotype division, resulting in compression of the number of data pieces. The second BLSOM was constructed with a total of weight vectors obtained in the first-layer BLSOMs. We compared SC-BLSOM with the conventional BLSOM by analyzing bacterial genome sequences. SC-BLSOM could be constructed faster than BLSOM and cluster the sequences according to phylotype with high accuracy, showing the method's suitability for efficient knowledge discovery from big sequence data.

Origin of the Y genome in Elymus and its relationship to other genomes in Triticeae based on evidence from elongation factor G (EF-G) gene sequences.

PubMed

Sun, Genlou; Komatsuda, Takao

2010-08-01

It is well known that Elymus arose through hybridization between representatives of different genera. Cytogenetic analyses show that all its members include the St genome in combination with one or more of four other genomes, the H, Y, P, and W genomes. The origins of the H, P, and W genomes are known, but not for the Y genome. We analyzed the single copy nuclear gene coding for elongation factor G (EF-G) from 28 accessions of polyploid Elymus species and 45 accessions of diploid Triticeae species in order to investigate origin of the Y genome and its relationship to other genomes in the tribe Triticeae. Sequence comparisons among the St, H, Y, P, W, and E genomes detected genome-specific polymorphisms at 66 nucleotide positions. The St and Y genomes are relatively dissimilar. The phylogeny of the Y genome sequences was investigated for the first time. They were most similar to the W genome sequences. The Y genome sequences were placed in two different groups. These two groups were included in an unresolved clade that included the W and E sequences as well as sequences from many annual species. The H genomes sequences were in a clade with the F, P, and Ns genome sequences as sister groups. These two clades were more closely related to each other and to the L and Xp genomes than they were to the St genome sequences. These data support the hypothesis that the Y genome evolved in a diploid species and has a different origin from the St genome. Copyright 2010 Elsevier Inc. All rights reserved.

Company profile: Complete Genomics Inc.

PubMed

Reid, Clifford

2011-02-01

Complete Genomics Inc. is a life sciences company that focuses on complete human genome sequencing. It is taking a completely different approach to DNA sequencing than other companies in the industry. Rather than building a general-purpose platform for sequencing all organisms and all applications, it has focused on a single application - complete human genome sequencing. The company's Complete Genomics Analysis Platform (CGA™ Platform) comprises an integrated package of biochemistry, instrumentation and software that sequences human genomes at the highest quality, lowest cost and largest scale available. Complete Genomics offers a turnkey service that enables customers to outsource their human genome sequencing to the company's genome sequencing center in Mountain View, CA, USA. Customers send in their DNA samples, the company does all the library preparation, DNA sequencing, assembly and variant analysis, and customers receive research-ready data that they can use for biological discovery.

Centromere Locations in Brassica A and C Genomes Revealed Through Half-Tetrad Analysis

PubMed Central

Mason, Annaliese S.; Rousseau-Gueutin, Mathieu; Morice, Jérôme; Bayer, Philipp E.; Besharat, Naghmeh; Cousin, Anouska; Pradhan, Aneeta; Parkin, Isobel A. P.; Chèvre, Anne-Marie; Batley, Jacqueline; Nelson, Matthew N.

2016-01-01

Locating centromeres on genome sequences can be challenging. The high density of repetitive elements in these regions makes sequence assembly problematic, especially when using short-read sequencing technologies. It can also be difficult to distinguish between active and recently extinct centromeres through sequence analysis. An effective solution is to identify genetically active centromeres (functional in meiosis) by half-tetrad analysis. This genetic approach involves detecting heterozygosity along chromosomes in segregating populations derived from gametes (half-tetrads). Unreduced gametes produced by first division restitution mechanisms comprise complete sets of nonsister chromatids. Along these chromatids, heterozygosity is maximal at the centromeres, and homologous recombination events result in homozygosity toward the telomeres. We genotyped populations of half-tetrad-derived individuals (from Brassica interspecific hybrids) using a high-density array of physically anchored SNP markers (Illumina Brassica 60K Infinium array). Mapping the distribution of heterozygosity in these half-tetrad individuals allowed the genetic mapping of all 19 centromeres of the Brassica A and C genomes to the reference Brassica napus genome. Gene and transposable element density across the B. napus genome were also assessed and corresponded well to previously reported genetic map positions. Known centromere-specific sequences were located in the reference genome, but mostly matched unanchored sequences, suggesting that the core centromeric regions may not yet be assembled into the pseudochromosomes of the reference genome. The increasing availability of genetic markers physically anchored to reference genomes greatly simplifies the genetic and physical mapping of centromeres using half-tetrad analysis. We discuss possible applications of this approach, including in species where half-tetrads are currently difficult to isolate. PMID:26614742

Centromere Locations in Brassica A and C Genomes Revealed Through Half-Tetrad Analysis.

PubMed

Mason, Annaliese S; Rousseau-Gueutin, Mathieu; Morice, Jérôme; Bayer, Philipp E; Besharat, Naghmeh; Cousin, Anouska; Pradhan, Aneeta; Parkin, Isobel A P; Chèvre, Anne-Marie; Batley, Jacqueline; Nelson, Matthew N

2016-02-01

Locating centromeres on genome sequences can be challenging. The high density of repetitive elements in these regions makes sequence assembly problematic, especially when using short-read sequencing technologies. It can also be difficult to distinguish between active and recently extinct centromeres through sequence analysis. An effective solution is to identify genetically active centromeres (functional in meiosis) by half-tetrad analysis. This genetic approach involves detecting heterozygosity along chromosomes in segregating populations derived from gametes (half-tetrads). Unreduced gametes produced by first division restitution mechanisms comprise complete sets of nonsister chromatids. Along these chromatids, heterozygosity is maximal at the centromeres, and homologous recombination events result in homozygosity toward the telomeres. We genotyped populations of half-tetrad-derived individuals (from Brassica interspecific hybrids) using a high-density array of physically anchored SNP markers (Illumina Brassica 60K Infinium array). Mapping the distribution of heterozygosity in these half-tetrad individuals allowed the genetic mapping of all 19 centromeres of the Brassica A and C genomes to the reference Brassica napus genome. Gene and transposable element density across the B. napus genome were also assessed and corresponded well to previously reported genetic map positions. Known centromere-specific sequences were located in the reference genome, but mostly matched unanchored sequences, suggesting that the core centromeric regions may not yet be assembled into the pseudochromosomes of the reference genome. The increasing availability of genetic markers physically anchored to reference genomes greatly simplifies the genetic and physical mapping of centromeres using half-tetrad analysis. We discuss possible applications of this approach, including in species where half-tetrads are currently difficult to isolate. Copyright © 2016 by the Genetics Society of America.

How do students react to analyzing their own genomes in a whole-genome sequencing course?: outcomes of a longitudinal cohort study.

PubMed

Sanderson, Saskia C; Linderman, Michael D; Zinberg, Randi; Bashir, Ali; Kasarskis, Andrew; Zweig, Micol; Suckiel, Sabrina; Shah, Hardik; Mahajan, Milind; Diaz, George A; Schadt, Eric E

2015-11-01

Health-care professionals need to be trained to work with whole-genome sequencing (WGS) in their practice. Our aim was to explore how students responded to a novel genome analysis course that included the option to analyze their own genomes. This was an observational cohort study. Questionnaires were administered before (T3) and after the genome analysis course (T4), as well as 6 months later (T5). In-depth interviews were conducted at T5. All students (n = 19) opted to analyze their own genomes. At T5, 12 of 15 students stated that analyzing their own genomes had been useful. Ten reported they had applied their knowledge in the workplace. Technical WGS knowledge increased (mean of 63.8% at T3, mean of 72.5% at T4; P = 0.005). In-depth interviews suggested that analyzing their own genomes may increase students' motivation to learn and their understanding of the patient experience. Most (but not all) of the students reported low levels of WGS results-related distress and low levels of regret about their decision to analyze their own genomes. Giving students the option of analyzing their own genomes may increase motivation to learn, but some students may experience personal WGS results-related distress and regret. Additional evidence is required before considering incorporating optional personal genome analysis into medical education on a large scale.

Read clouds uncover variation in complex regions of the human genome.

PubMed

Bishara, Alex; Liu, Yuling; Weng, Ziming; Kashef-Haghighi, Dorna; Newburger, Daniel E; West, Robert; Sidow, Arend; Batzoglou, Serafim

2015-10-01

Although an increasing amount of human genetic variation is being identified and recorded, determining variants within repeated sequences of the human genome remains a challenge. Most population and genome-wide association studies have therefore been unable to consider variation in these regions. Core to the problem is the lack of a sequencing technology that produces reads with sufficient length and accuracy to enable unique mapping. Here, we present a novel methodology of using read clouds, obtained by accurate short-read sequencing of DNA derived from long fragment libraries, to confidently align short reads within repeat regions and enable accurate variant discovery. Our novel algorithm, Random Field Aligner (RFA), captures the relationships among the short reads governed by the long read process via a Markov Random Field. We utilized a modified version of the Illumina TruSeq synthetic long-read protocol, which yielded shallow-sequenced read clouds. We test RFA through extensive simulations and apply it to discover variants on the NA12878 human sample, for which shallow TruSeq read cloud sequencing data are available, and on an invasive breast carcinoma genome that we sequenced using the same method. We demonstrate that RFA facilitates accurate recovery of variation in 155 Mb of the human genome, including 94% of 67 Mb of segmental duplication sequence and 96% of 11 Mb of transcribed sequence, that are currently hidden from short-read technologies. © 2015 Bishara et al.; Published by Cold Spring Harbor Laboratory Press.

Curated eutherian third party data gene data sets.

PubMed

Premzl, Marko

2016-03-01

The free available eutherian genomic sequence data sets advanced scientific field of genomics. Of note, future revisions of gene data sets were expected, due to incompleteness of public eutherian genomic sequence assemblies and potential genomic sequence errors. The eutherian comparative genomic analysis protocol was proposed as guidance in protection against potential genomic sequence errors in public eutherian genomic sequences. The protocol was applicable in updates of 7 major eutherian gene data sets, including 812 complete coding sequences deposited in European Nucleotide Archive as curated third party data gene data sets.

Comparative analysis of syntenic genes in grass genomes reveals accelerated rates of gene structure and coding sequence evolution in polyploid wheat

USDA-ARS?s Scientific Manuscript database

Cycles of whole genome duplication (WGD) and diploidization are hallmarks of eukaryotic genome evolution and speciation. Polyploid wheat (Triticum aestivum) has had a massive increase in genome size largely due to recent WGDs. How these processes may impact the dynamics of gene evolution was studied...

Complete genome sequence of the European sheatfish virus.

PubMed

Mavian, Carla; López-Bueno, Alberto; Fernández Somalo, María Pilar; Alcamí, Antonio; Alejo, Alí

2012-06-01

Viral diseases are an increasing threat to the thriving aquaculture industry worldwide. An emerging group of fish pathogens is formed by several ranaviruses, which have been isolated at different locations from freshwater and seawater fish species since 1985. We report the complete genome sequence of European sheatfish ranavirus (ESV), the first ranavirus isolated in Europe, which causes high mortality rates in infected sheatfish (Silurus glanis) and in other species. Analysis of the genome sequence shows that ESV belongs to the amphibian-like ranaviruses and is closely related to the epizootic hematopoietic necrosis virus (EHNV), a disease agent geographically confined to the Australian continent and notifiable to the World Organization for Animal Health.

Complete Genome Sequence of the European Sheatfish Virus

PubMed Central

Mavian, Carla; López-Bueno, Alberto; Fernández Somalo, María Pilar; Alcamí, Antonio

2012-01-01

Viral diseases are an increasing threat to the thriving aquaculture industry worldwide. An emerging group of fish pathogens is formed by several ranaviruses, which have been isolated at different locations from freshwater and seawater fish species since 1985. We report the complete genome sequence of European sheatfish ranavirus (ESV), the first ranavirus isolated in Europe, which causes high mortality rates in infected sheatfish (Silurus glanis) and in other species. Analysis of the genome sequence shows that ESV belongs to the amphibian-like ranaviruses and is closely related to the epizootic hematopoietic necrosis virus (EHNV), a disease agent geographically confined to the Australian continent and notifiable to the World Organization for Animal Health. PMID:22570241

Were protein internal repeats formed by "bricolage"?

PubMed

Lavorgna, G; Patthy, L; Boncinelli, E

2001-03-01

Is evolution an engineer, or is it a tinkerer--a "bricoleur"--building up complex molecules in organisms by increasing and adapting the materials at hand? An analysis of completely sequenced genomes suggests the latter, showing that increasing repetition of modules within the proteins encoded by these genomes is correlated with increasing complexity of the organism.

Consequences of splitting whole-genome sequencing effort over multiple breeds on imputation accuracy.

PubMed

Bouwman, Aniek C; Veerkamp, Roel F

2014-10-03

The aim of this study was to determine the consequences of splitting sequencing effort over multiple breeds for imputation accuracy from a high-density SNP chip towards whole-genome sequence. Such information would assist for instance numerical smaller cattle breeds, but also pig and chicken breeders, who have to choose wisely how to spend their sequencing efforts over all the breeds or lines they evaluate. Sequence data from cattle breeds was used, because there are currently relatively many individuals from several breeds sequenced within the 1,000 Bull Genomes project. The advantage of whole-genome sequence data is that it carries the causal mutations, but the question is whether it is possible to impute the causal variants accurately. This study therefore focussed on imputation accuracy of variants with low minor allele frequency and breed specific variants. Imputation accuracy was assessed for chromosome 1 and 29 as the correlation between observed and imputed genotypes. For chromosome 1, the average imputation accuracy was 0.70 with a reference population of 20 Holstein, and increased to 0.83 when the reference population was increased by including 3 other dairy breeds with 20 animals each. When the same amount of animals from the Holstein breed were added the accuracy improved to 0.88, while adding the 3 other breeds to the reference population of 80 Holstein improved the average imputation accuracy marginally to 0.89. For chromosome 29, the average imputation accuracy was lower. Some variants benefitted from the inclusion of other breeds in the reference population, initially determined by the MAF of the variant in each breed, but even Holstein specific variants did gain imputation accuracy from the multi-breed reference population. This study shows that splitting sequencing effort over multiple breeds and combining the reference populations is a good strategy for imputation from high-density SNP panels towards whole-genome sequence when reference populations are small and sequencing effort is limiting. When sequencing effort is limiting and interest lays in multiple breeds or lines this provides imputation of each breed.

Disclosure of Incidental Findings From Next-Generation Sequencing in Pediatric Genomic Research

PubMed Central

Abdul-Karim, Ruqayyah; Berkman, Benjamin E.; Wendler, David; Rid, Annette; Khan, Javed; Badgett, Tom

2013-01-01

Next-generation sequencing technologies will likely be used with increasing frequency in pediatric research. One consequence will be the increased identification of individual genomic research findings that are incidental to the aims of the research. Although researchers and ethicists have raised theoretical concerns about incidental findings in the context of genetic research, next-generation sequencing will make this once largely hypothetical concern an increasing reality. Most commentators have begun to accept the notion that there is some duty to disclose individual genetic research results to research subjects; however, the scope of that duty remains unclear. These issues are especially complicated in the pediatric setting, where subjects cannot currently but typically will eventually be able to make their own medical decisions at the age of adulthood. This article discusses the management of incidental findings in the context of pediatric genomic research. We provide an overview of the current literature and propose a framework to manage incidental findings in this unique context, based on what we believe is a limited responsibility to disclose. We hope this will be a useful source of guidance for investigators, institutional review boards, and bioethicists that anticipates the complicated ethical issues raised by advances in genomic technology. PMID:23400601

REFGEN and TREENAMER: Automated Sequence Data Handling for Phylogenetic Analysis in the Genomic Era

PubMed Central

Leonard, Guy; Stevens, Jamie R.; Richards, Thomas A.

2009-01-01

The phylogenetic analysis of nucleotide sequences and increasingly that of amino acid sequences is used to address a number of biological questions. Access to extensive datasets, including numerous genome projects, means that standard phylogenetic analyses can include many hundreds of sequences. Unfortunately, most phylogenetic analysis programs do not tolerate the sequence naming conventions of genome databases. Managing large numbers of sequences and standardizing sequence labels for use in phylogenetic analysis programs can be a time consuming and laborious task. Here we report the availability of an online resource for the management of gene sequences recovered from public access genome databases such as GenBank. These web utilities include the facility for renaming every sequence in a FASTA alignment file, with each sequence label derived from a user-defined combination of the species name and/or database accession number. This facility enables the user to keep track of the branching order of the sequences/taxa during multiple tree calculations and re-optimisations. Post phylogenetic analysis, these webpages can then be used to rename every label in the subsequent tree files (with a user-defined combination of species name and/or database accession number). Together these programs drastically reduce the time required for managing sequence alignments and labelling phylogenetic figures. Additional features of our platform include the automatic removal of identical accession numbers (recorded in the report file) and generation of species and accession number lists for use in supplementary materials or figure legends. PMID:19812722

CloVR-Comparative: automated, cloud-enabled comparative microbial genome sequence analysis pipeline.

PubMed

Agrawal, Sonia; Arze, Cesar; Adkins, Ricky S; Crabtree, Jonathan; Riley, David; Vangala, Mahesh; Galens, Kevin; Fraser, Claire M; Tettelin, Hervé; White, Owen; Angiuoli, Samuel V; Mahurkar, Anup; Fricke, W Florian

2017-04-27

The benefit of increasing genomic sequence data to the scientific community depends on easy-to-use, scalable bioinformatics support. CloVR-Comparative combines commonly used bioinformatics tools into an intuitive, automated, and cloud-enabled analysis pipeline for comparative microbial genomics. CloVR-Comparative runs on annotated complete or draft genome sequences that are uploaded by the user or selected via a taxonomic tree-based user interface and downloaded from NCBI. CloVR-Comparative runs reference-free multiple whole-genome alignments to determine unique, shared and core coding sequences (CDSs) and single nucleotide polymorphisms (SNPs). Output includes short summary reports and detailed text-based results files, graphical visualizations (phylogenetic trees, circular figures), and a database file linked to the Sybil comparative genome browser. Data up- and download, pipeline configuration and monitoring, and access to Sybil are managed through CloVR-Comparative web interface. CloVR-Comparative and Sybil are distributed as part of the CloVR virtual appliance, which runs on local computers or the Amazon EC2 cloud. Representative datasets (e.g. 40 draft and complete Escherichia coli genomes) are processed in <36 h on a local desktop or at a cost of <$20 on EC2. CloVR-Comparative allows anybody with Internet access to run comparative genomics projects, while eliminating the need for on-site computational resources and expertise.

Ancient genomics

PubMed Central

Der Sarkissian, Clio; Allentoft, Morten E.; Ávila-Arcos, María C.; Barnett, Ross; Campos, Paula F.; Cappellini, Enrico; Ermini, Luca; Fernández, Ruth; da Fonseca, Rute; Ginolhac, Aurélien; Hansen, Anders J.; Jónsson, Hákon; Korneliussen, Thorfinn; Margaryan, Ashot; Martin, Michael D.; Moreno-Mayar, J. Víctor; Raghavan, Maanasa; Rasmussen, Morten; Velasco, Marcela Sandoval; Schroeder, Hannes; Schubert, Mikkel; Seguin-Orlando, Andaine; Wales, Nathan; Gilbert, M. Thomas P.; Willerslev, Eske; Orlando, Ludovic

2015-01-01

The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field's focus was previously limited to mitochondrial DNA and a few nuclear markers, whole genome sequences from the deep past can now be retrieved. This breakthrough is tightly connected to the massive sequence throughput of next generation sequencing platforms and the ability to target short and degraded DNA molecules. Many ancient specimens previously unsuitable for DNA analyses because of extensive degradation can now successfully be used as source materials. Additionally, the analytical power obtained by increasing the number of sequence reads to billions effectively means that contamination issues that have haunted aDNA research for decades, particularly in human studies, can now be efficiently and confidently quantified. At present, whole genomes have been sequenced from ancient anatomically modern humans, archaic hominins, ancient pathogens and megafaunal species. Those have revealed important functional and phenotypic information, as well as unexpected adaptation, migration and admixture patterns. As such, the field of aDNA has entered the new era of genomics and has provided valuable information when testing specific hypotheses related to the past. PMID:25487338

Leptospira species molecular epidemiology in the genomic era.

PubMed

Caimi, K; Repetto, S A; Varni, V; Ruybal, P

2017-10-01

Leptospirosis is a zoonotic disease which global burden is increasing often related to climatic change. Hundreds of whole genome sequences from worldwide isolates of Leptospira spp. are available nowadays, together with online tools that permit to assign MLST sequence types (STs) directly from raw sequence data. In this work we have applied R7L-MLST to near 500 genomes and strains collection globally distributed. All 10 pathogenic species as well as intermediate were typed using this MLST scheme. The correlation observed between STs and serogroups in our previous work, is still satisfied with this higher dataset sustaining the implementation of MLST to assist serological classification as a complementary approach. Bayesian phylogenetic analysis of concatenated sequences from R7-MLST loci allowed us to resolve taxonomic inconsistencies but also showed that events such as recombination, gene conversion or lateral gene transfer played an important role in the evolution of Leptospira genus. Whole genome sequencing allows us to contribute with suitable epidemiologic information useful to apply in the design of control strategies and also in diagnostic methods for this illness. Copyright © 2017 Elsevier B.V. All rights reserved.

ngs.plot: Quick mining and visualization of next-generation sequencing data by integrating genomic databases.

PubMed

Shen, Li; Shao, Ningyi; Liu, Xiaochuan; Nestler, Eric

2014-04-15

Understanding the relationship between the millions of functional DNA elements and their protein regulators, and how they work in conjunction to manifest diverse phenotypes, is key to advancing our understanding of the mammalian genome. Next-generation sequencing technology is now used widely to probe these protein-DNA interactions and to profile gene expression at a genome-wide scale. As the cost of DNA sequencing continues to fall, the interpretation of the ever increasing amount of data generated represents a considerable challenge. We have developed ngs.plot - a standalone program to visualize enrichment patterns of DNA-interacting proteins at functionally important regions based on next-generation sequencing data. We demonstrate that ngs.plot is not only efficient but also scalable. We use a few examples to demonstrate that ngs.plot is easy to use and yet very powerful to generate figures that are publication ready. We conclude that ngs.plot is a useful tool to help fill the gap between massive datasets and genomic information in this era of big sequencing data.

ngs.plot: Quick mining and visualization of next-generation sequencing data by integrating genomic databases

PubMed Central

2014-01-01

Background Understanding the relationship between the millions of functional DNA elements and their protein regulators, and how they work in conjunction to manifest diverse phenotypes, is key to advancing our understanding of the mammalian genome. Next-generation sequencing technology is now used widely to probe these protein-DNA interactions and to profile gene expression at a genome-wide scale. As the cost of DNA sequencing continues to fall, the interpretation of the ever increasing amount of data generated represents a considerable challenge. Results We have developed ngs.plot – a standalone program to visualize enrichment patterns of DNA-interacting proteins at functionally important regions based on next-generation sequencing data. We demonstrate that ngs.plot is not only efficient but also scalable. We use a few examples to demonstrate that ngs.plot is easy to use and yet very powerful to generate figures that are publication ready. Conclusions We conclude that ngs.plot is a useful tool to help fill the gap between massive datasets and genomic information in this era of big sequencing data. PMID:24735413

Artificial Intelligence, DNA Mimicry, and Human Health.

PubMed

Stefano, George B; Kream, Richard M

2017-08-14

The molecular evolution of genomic DNA across diverse plant and animal phyla involved dynamic registrations of sequence modifications to maintain existential homeostasis to increasingly complex patterns of environmental stressors. As an essential corollary, driver effects of positive evolutionary pressure are hypothesized to effect concerted modifications of genomic DNA sequences to meet expanded platforms of regulatory controls for successful implementation of advanced physiological requirements. It is also clearly apparent that preservation of updated registries of advantageous modifications of genomic DNA sequences requires coordinate expansion of convergent cellular proofreading/error correction mechanisms that are encoded by reciprocally modified genomic DNA. Computational expansion of operationally defined DNA memory extends to coordinate modification of coding and previously under-emphasized noncoding regions that now appear to represent essential reservoirs of untapped genetic information amenable to evolutionary driven recruitment into the realm of biologically active domains. Additionally, expansion of DNA memory potential via chemical modification and activation of noncoding sequences is targeted to vertical augmentation and integration of an expanded cadre of transcriptional and epigenetic regulatory factors affecting linear coding of protein amino acid sequences within open reading frames.

MEGANTE: A Web-Based System for Integrated Plant Genome Annotation

PubMed Central

Numa, Hisataka; Itoh, Takeshi

2014-01-01

The recent advancement of high-throughput genome sequencing technologies has resulted in a considerable increase in demands for large-scale genome annotation. While annotation is a crucial step for downstream data analyses and experimental studies, this process requires substantial expertise and knowledge of bioinformatics. Here we present MEGANTE, a web-based annotation system that makes plant genome annotation easy for researchers unfamiliar with bioinformatics. Without any complicated configuration, users can perform genomic sequence annotations simply by uploading a sequence and selecting the species to query. MEGANTE automatically runs several analysis programs and integrates the results to select the appropriate consensus exon–intron structures and to predict open reading frames (ORFs) at each locus. Functional annotation, including a similarity search against known proteins and a functional domain search, are also performed for the predicted ORFs. The resultant annotation information is visualized with a widely used genome browser, GBrowse. For ease of analysis, the results can be downloaded in Microsoft Excel format. All of the query sequences and annotation results are stored on the server side so that users can access their own data from virtually anywhere on the web. The current release of MEGANTE targets 24 plant species from the Brassicaceae, Fabaceae, Musaceae, Poaceae, Salicaceae, Solanaceae, Rosaceae and Vitaceae families, and it allows users to submit a sequence up to 10 Mb in length and to save up to 100 sequences with the annotation information on the server. The MEGANTE web service is available at https://megante.dna.affrc.go.jp/. PMID:24253915

Ultra-barcoding in cacao (Theobroma spp.; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA.

PubMed

Kane, Nolan; Sveinsson, Saemundur; Dempewolf, Hannes; Yang, Ji Yong; Zhang, Dapeng; Engels, Johannes M M; Cronk, Quentin

2012-02-01

To reliably identify lineages below the species level such as subspecies or varieties, we propose an extension to DNA-barcoding using next-generation sequencing to produce whole organellar genomes and substantial nuclear ribosomal sequence. Because this method uses much longer versions of the traditional DNA-barcoding loci in the plastid and ribosomal DNA, we call our approach ultra-barcoding (UBC). We used high-throughput next-generation sequencing to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an individual of the related species T. grandiflorum, as well as an additional publicly available whole plastid genome of T. cacao. All individuals of T. cacao examined were uniquely distinguished, and evidence of reticulation and gene flow was observed. Sequence variation was observed in some of the canonical barcoding regions between species, but other regions of the chloroplast were more variable both within species and between species, as were ribosomal spacers. Furthermore, no single region provides the level of data available using the complete plastid genome and rDNA. Our data demonstrate that UBC is a viable, increasingly cost-effective approach for reliably distinguishing varieties and even individual genotypes of T. cacao. This approach shows great promise for applications where very closely related or interbreeding taxa must be distinguished.

Draft genome sequence of Inquilinus limosus strain MP06, a multidrug-resistant clinical isolate

PubMed Central

Pino, Marylú; Conza, José Di; Gutkind, Gabriel

2015-01-01

The bacterium, Inquilinus limosus, with its remarkable antimicrobial multiresistant profile, has increasingly been isolated in cystic fibrosis patients. We report draft genome sequence of a strain MP06, which is of considerable interest in elucidating the associated mechanisms of antibiotic resistance in this bacterium and for an insight about its persistence in airways of these patients. PMID:26691451

Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence

PubMed Central

2011-01-01

Background Many plants have large and complex genomes with an abundance of repeated sequences. Many plants are also polyploid. Both of these attributes typify the genome architecture in the tribe Triticeae, whose members include economically important wheat, rye and barley. Large genome sizes, an abundance of repeated sequences, and polyploidy present challenges to genome-wide SNP discovery using next-generation sequencing (NGS) of total genomic DNA by making alignment and clustering of short reads generated by the NGS platforms difficult, particularly in the absence of a reference genome sequence. Results An annotation-based, genome-wide SNP discovery pipeline is reported using NGS data for large and complex genomes without a reference genome sequence. Roche 454 shotgun reads with low genome coverage of one genotype are annotated in order to distinguish single-copy sequences and repeat junctions from repetitive sequences and sequences shared by paralogous genes. Multiple genome equivalents of shotgun reads of another genotype generated with SOLiD or Solexa are then mapped to the annotated Roche 454 reads to identify putative SNPs. A pipeline program package, AGSNP, was developed and used for genome-wide SNP discovery in Aegilops tauschii-the diploid source of the wheat D genome, and with a genome size of 4.02 Gb, of which 90% is repetitive sequences. Genomic DNA of Ae. tauschii accession AL8/78 was sequenced with the Roche 454 NGS platform. Genomic DNA and cDNA of Ae. tauschii accession AS75 was sequenced primarily with SOLiD, although some Solexa and Roche 454 genomic sequences were also generated. A total of 195,631 putative SNPs were discovered in gene sequences, 155,580 putative SNPs were discovered in uncharacterized single-copy regions, and another 145,907 putative SNPs were discovered in repeat junctions. These SNPs were dispersed across the entire Ae. tauschii genome. To assess the false positive SNP discovery rate, DNA containing putative SNPs was amplified by PCR from AL8/78 and AS75 and resequenced with the ABI 3730 xl. In a sample of 302 randomly selected putative SNPs, 84.0% in gene regions, 88.0% in repeat junctions, and 81.3% in uncharacterized regions were validated. Conclusion An annotation-based genome-wide SNP discovery pipeline for NGS platforms was developed. The pipeline is suitable for SNP discovery in genomic libraries of complex genomes and does not require a reference genome sequence. The pipeline is applicable to all current NGS platforms, provided that at least one such platform generates relatively long reads. The pipeline package, AGSNP, and the discovered 497,118 Ae. tauschii SNPs can be accessed at (http://avena.pw.usda.gov/wheatD/agsnp.shtml). PMID:21266061

The contribution of alu elements to mutagenic DNA double-strand break repair.

PubMed

Morales, Maria E; White, Travis B; Streva, Vincent A; DeFreece, Cecily B; Hedges, Dale J; Deininger, Prescott L

2015-03-01

Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both the rate and nature of DNA repair events.

Alignment-free genome tree inference by learning group-specific distance metrics.

PubMed

Patil, Kaustubh R; McHardy, Alice C

2013-01-01

Understanding the evolutionary relationships between organisms is vital for their in-depth study. Gene-based methods are often used to infer such relationships, which are not without drawbacks. One can now attempt to use genome-scale information, because of the ever increasing number of genomes available. This opportunity also presents a challenge in terms of computational efficiency. Two fundamentally different methods are often employed for sequence comparisons, namely alignment-based and alignment-free methods. Alignment-free methods rely on the genome signature concept and provide a computationally efficient way that is also applicable to nonhomologous sequences. The genome signature contains evolutionary signal as it is more similar for closely related organisms than for distantly related ones. We used genome-scale sequence information to infer taxonomic distances between organisms without additional information such as gene annotations. We propose a method to improve genome tree inference by learning specific distance metrics over the genome signature for groups of organisms with similar phylogenetic, genomic, or ecological properties. Specifically, our method learns a Mahalanobis metric for a set of genomes and a reference taxonomy to guide the learning process. By applying this method to more than a thousand prokaryotic genomes, we showed that, indeed, better distance metrics could be learned for most of the 18 groups of organisms tested here. Once a group-specific metric is available, it can be used to estimate the taxonomic distances for other sequenced organisms from the group. This study also presents a large scale comparison between 10 methods--9 alignment-free and 1 alignment-based.

Whole Genome Sequencing of Greater Amberjack (Seriola dumerili) for SNP Identification on Aligned Scaffolds and Genome Structural Variation Analysis Using Parallel Resequencing

PubMed Central

Aokic, Jun-ya; Kawase, Junya; Hamada, Kazuhisa; Fujimoto, Hiroshi; Yamamoto, Ikki; Usuki, Hironori

2018-01-01

Greater amberjack (Seriola dumerili) is distributed in tropical and temperate waters worldwide and is an important aquaculture fish. We carried out de novo sequencing of the greater amberjack genome to construct a reference genome sequence to identify single nucleotide polymorphisms (SNPs) for breeding amberjack by marker-assisted or gene-assisted selection as well as to identify functional genes for biological traits. We obtained 200 times coverage and constructed a high-quality genome assembly using next generation sequencing technology. The assembled sequences were aligned onto a yellowtail (Seriola quinqueradiata) radiation hybrid (RH) physical map by sequence homology. A total of 215 of the longest amberjack sequences, with a total length of 622.8 Mbp (92% of the total length of the genome scaffolds), were lined up on the yellowtail RH map. We resequenced the whole genomes of 20 greater amberjacks and mapped the resulting sequences onto the reference genome sequence. About 186,000 nonredundant SNPs were successfully ordered on the reference genome. Further, we found differences in the genome structural variations between two greater amberjack populations using BreakDancer. We also analyzed the greater amberjack transcriptome and mapped the annotated sequences onto the reference genome sequence. PMID:29785397

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease.

PubMed

Carss, Keren J; Arno, Gavin; Erwood, Marie; Stephens, Jonathan; Sanchis-Juan, Alba; Hull, Sarah; Megy, Karyn; Grozeva, Detelina; Dewhurst, Eleanor; Malka, Samantha; Plagnol, Vincent; Penkett, Christopher; Stirrups, Kathleen; Rizzo, Roberta; Wright, Genevieve; Josifova, Dragana; Bitner-Glindzicz, Maria; Scott, Richard H; Clement, Emma; Allen, Louise; Armstrong, Ruth; Brady, Angela F; Carmichael, Jenny; Chitre, Manali; Henderson, Robert H H; Hurst, Jane; MacLaren, Robert E; Murphy, Elaine; Paterson, Joan; Rosser, Elisabeth; Thompson, Dorothy A; Wakeling, Emma; Ouwehand, Willem H; Michaelides, Michel; Moore, Anthony T; Webster, Andrew R; Raymond, F Lucy

2017-01-05

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease. Copyright © 2017. Published by Elsevier Inc.

Genome Sequencing of Steroid Producing Bacteria Using Ion Torrent Technology and a Reference Genome.

PubMed

Sola-Landa, Alberto; Rodríguez-García, Antonio; Barreiro, Carlos; Pérez-Redondo, Rosario

2017-01-01

The Next-Generation Sequencing technology has enormously eased the bacterial genome sequencing and several tens of thousands of genomes have been sequenced during the last 10 years. Most of the genome projects are published as draft version, however, for certain applications the complete genome sequence is required.In this chapter, we describe the strategy that allowed the complete genome sequencing of Mycobacterium neoaurum NRRL B-3805, an industrial strain exploited for steroid production, using Ion Torrent sequencing reads and the genome of a close strain as the reference. This protocol can be applied to analyze the genetic variations between closely related strains; for example, to elucidate the point mutations between a parental strain and a random mutagenesis-derived mutant.

Whole genome sequencing and bioinformatics analysis of two Egyptian genomes.

PubMed

ElHefnawi, Mahmoud; Jeon, Sungwon; Bhak, Youngjune; ElFiky, Asmaa; Horaiz, Ahmed; Jun, JeHoon; Kim, Hyunho; Bhak, Jong

2018-05-15

We report two Egyptian male genomes (EGP1 and EGP2) sequenced at ~ 30× sequencing depths. EGP1 had 4.7 million variants, where 198,877 were novel variants while EGP2 had 209,109 novel variants out of 4.8 million variants. The mitochondrial haplogroup of the two individuals were identified to be H7b1 and L2a1c, respectively. We also identified the Y haplogroup of EGP1 (R1b) and EGP2 (J1a2a1a2 > P58 > FGC11). EGP1 had a mutation in the NADH gene of the mitochondrial genome ND4 (m.11778 G > A) that causes Leber's hereditary optic neuropathy. Some SNPs shared by the two genomes were associated with an increased level of cholesterol and triglycerides, probably related with Egyptians obesity. Comparison of these genomes with African and Western-Asian genomes can provide insights on Egyptian ancestry and genetic history. This resource can be used to further understand genomic diversity and functional classification of variants as well as human migration and evolution across Africa and Western-Asia. Copyright © 2017. Published by Elsevier B.V.

ISEScan: automated identification of insertion sequence elements in prokaryotic genomes.

PubMed

Xie, Zhiqun; Tang, Haixu

2017-11-01

The insertion sequence (IS) elements are the smallest but most abundant autonomous transposable elements in prokaryotic genomes, which play a key role in prokaryotic genome organization and evolution. With the fast growing genomic data, it is becoming increasingly critical for biology researchers to be able to accurately and automatically annotate ISs in prokaryotic genome sequences. The available automatic IS annotation systems are either providing only incomplete IS annotation or relying on the availability of existing genome annotations. Here, we present a new IS elements annotation pipeline to address these issues. ISEScan is a highly sensitive software pipeline based on profile hidden Markov models constructed from manually curated IS elements. ISEScan performs better than existing IS annotation systems when tested on prokaryotic genomes with curated annotations of IS elements. Applying it to 2784 prokaryotic genomes, we report the global distribution of IS families across taxonomic clades in Archaea and Bacteria. ISEScan is implemented in Python and released as an open source software at https://github.com/xiezhq/ISEScan. hatang@indiana.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

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

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes.

PubMed

Staňková, Helena; Hastie, Alex R; Chan, Saki; Vrána, Jan; Tulpová, Zuzana; Kubaláková, Marie; Visendi, Paul; Hayashi, Satomi; Luo, Mingcheng; Batley, Jacqueline; Edwards, David; Doležel, Jaroslav; Šimková, Hana

2016-07-01

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

A Hybrid Approach for the Automated Finishing of Bacterial Genomes

PubMed Central

Robins, William P.; Chin, Chen-Shan; Webster, Dale; Paxinos, Ellen; Hsu, David; Ashby, Meredith; Wang, Susana; Peluso, Paul; Sebra, Robert; Sorenson, Jon; Bullard, James; Yen, Jackie; Valdovino, Marie; Mollova, Emilia; Luong, Khai; Lin, Steven; LaMay, Brianna; Joshi, Amruta; Rowe, Lori; Frace, Michael; Tarr, Cheryl L.; Turnsek, Maryann; Davis, Brigid M; Kasarskis, Andrew; Mekalanos, John J.; Waldor, Matthew K.; Schadt, Eric E.

2013-01-01

Dramatic improvements in DNA sequencing technology have revolutionized our ability to characterize most genomic diversity. However, accurate resolution of large structural events has remained challenging due to the comparatively shorter read lengths of second-generation technologies. Emerging third-generation sequencing technologies, which yield markedly increased read length on rapid time scales and for low cost, have the potential to address assembly limitations. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at > 99.9% accuracy. Complex regions with clinically significant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 reference we obtain 14 and 8 scaffolds greater than 1kb, respectively, correcting several errors in the underlying source data. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly. PMID:22750883

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

Genomic sequencing and the impact of molecular diagnosis on patient care.

PubMed

Solomon, Benjamin D

2015-02-01

Evolving sequencing technologies allow more accurate, efficient and affordable genomic analysis. As a result, these technologies are increasingly available, especially to provide molecular diagnoses for patients with suspected genetic disorders. However, there are many challenges to using genomic sequencing to benefit patients, including concerns that there is insufficient evidence that identifying an underlying molecular explanation may positively impact a patient's healthcare. This concern has many repercussions, including funding and/or (in some countries and healthcare systems) insurance reimbursement for genomic sequencing. To investigate this concern, all monogenic disorders were analyzed based on the impact of achieving molecular diagnosis. Of the 2,849 individual genes in which germline mutations cause disorders (not including contiguous gene syndromes or what may be categorized as susceptibility alleles), our analyses showed a specific, available intervention related to at least one affected organ system for 1,419 (49.8%) genes. In 95.6% of these genes, the intervention(s) would be recommended during the pediatric time frame.

SeqWare Query Engine: storing and searching sequence data in the cloud.

PubMed

O'Connor, Brian D; Merriman, Barry; Nelson, Stanley F

2010-12-21

Since the introduction of next-generation DNA sequencers the rapid increase in sequencer throughput, and associated drop in costs, has resulted in more than a dozen human genomes being resequenced over the last few years. These efforts are merely a prelude for a future in which genome resequencing will be commonplace for both biomedical research and clinical applications. The dramatic increase in sequencer output strains all facets of computational infrastructure, especially databases and query interfaces. The advent of cloud computing, and a variety of powerful tools designed to process petascale datasets, provide a compelling solution to these ever increasing demands. In this work, we present the SeqWare Query Engine which has been created using modern cloud computing technologies and designed to support databasing information from thousands of genomes. Our backend implementation was built using the highly scalable, NoSQL HBase database from the Hadoop project. We also created a web-based frontend that provides both a programmatic and interactive query interface and integrates with widely used genome browsers and tools. Using the query engine, users can load and query variants (SNVs, indels, translocations, etc) with a rich level of annotations including coverage and functional consequences. As a proof of concept we loaded several whole genome datasets including the U87MG cell line. We also used a glioblastoma multiforme tumor/normal pair to both profile performance and provide an example of using the Hadoop MapReduce framework within the query engine. This software is open source and freely available from the SeqWare project (http://seqware.sourceforge.net). The SeqWare Query Engine provided an easy way to make the U87MG genome accessible to programmers and non-programmers alike. This enabled a faster and more open exploration of results, quicker tuning of parameters for heuristic variant calling filters, and a common data interface to simplify development of analytical tools. The range of data types supported, the ease of querying and integrating with existing tools, and the robust scalability of the underlying cloud-based technologies make SeqWare Query Engine a nature fit for storing and searching ever-growing genome sequence datasets.

SeqWare Query Engine: storing and searching sequence data in the cloud

PubMed Central

2010-01-01

Background Since the introduction of next-generation DNA sequencers the rapid increase in sequencer throughput, and associated drop in costs, has resulted in more than a dozen human genomes being resequenced over the last few years. These efforts are merely a prelude for a future in which genome resequencing will be commonplace for both biomedical research and clinical applications. The dramatic increase in sequencer output strains all facets of computational infrastructure, especially databases and query interfaces. The advent of cloud computing, and a variety of powerful tools designed to process petascale datasets, provide a compelling solution to these ever increasing demands. Results In this work, we present the SeqWare Query Engine which has been created using modern cloud computing technologies and designed to support databasing information from thousands of genomes. Our backend implementation was built using the highly scalable, NoSQL HBase database from the Hadoop project. We also created a web-based frontend that provides both a programmatic and interactive query interface and integrates with widely used genome browsers and tools. Using the query engine, users can load and query variants (SNVs, indels, translocations, etc) with a rich level of annotations including coverage and functional consequences. As a proof of concept we loaded several whole genome datasets including the U87MG cell line. We also used a glioblastoma multiforme tumor/normal pair to both profile performance and provide an example of using the Hadoop MapReduce framework within the query engine. This software is open source and freely available from the SeqWare project (http://seqware.sourceforge.net). Conclusions The SeqWare Query Engine provided an easy way to make the U87MG genome accessible to programmers and non-programmers alike. This enabled a faster and more open exploration of results, quicker tuning of parameters for heuristic variant calling filters, and a common data interface to simplify development of analytical tools. The range of data types supported, the ease of querying and integrating with existing tools, and the robust scalability of the underlying cloud-based technologies make SeqWare Query Engine a nature fit for storing and searching ever-growing genome sequence datasets. PMID:21210981

Hybrid error correction and de novo assembly of single-molecule sequencing reads

PubMed Central

Koren, Sergey; Schatz, Michael C.; Walenz, Brian P.; Martin, Jeffrey; Howard, Jason; Ganapathy, Ganeshkumar; Wang, Zhong; Rasko, David A.; McCombie, W. Richard; Jarvis, Erich D.; Phillippy, Adam M.

2012-01-01

Emerging single-molecule sequencing instruments can generate multi-kilobase sequences with the potential to dramatically improve genome and transcriptome assembly. However, the high error rate of single-molecule reads is challenging, and has limited their use to resequencing bacteria. To address this limitation, we introduce a novel correction algorithm and assembly strategy that utilizes shorter, high-identity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on Pacbio RS reads of phage, prokaryotic, and eukaryotic whole genomes, including the novel genome of the parrot Melopsittacus undulatus, as well as for RNA-seq reads of the corn (Zea mays) transcriptome. Our approach achieves over 99.9% read correction accuracy and produces substantially better assemblies than current sequencing strategies: in the best example, quintupling the median contig size relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly. PMID:22750884

Whole-genome random sequencing and assembly of Haemophilus influenzae Rd

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

Fleischmann, R.D.; Adams, M.D.; White, O.

1995-07-28

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism. 46 refs., 4 figs., 4 tabs.

Cloud computing for comparative genomics

PubMed Central

2010-01-01

Background Large comparative genomics studies and tools are becoming increasingly more compute-expensive as the number of available genome sequences continues to rise. The capacity and cost of local computing infrastructures are likely to become prohibitive with the increase, especially as the breadth of questions continues to rise. Alternative computing architectures, in particular cloud computing environments, may help alleviate this increasing pressure and enable fast, large-scale, and cost-effective comparative genomics strategies going forward. To test this, we redesigned a typical comparative genomics algorithm, the reciprocal smallest distance algorithm (RSD), to run within Amazon's Elastic Computing Cloud (EC2). We then employed the RSD-cloud for ortholog calculations across a wide selection of fully sequenced genomes. Results We ran more than 300,000 RSD-cloud processes within the EC2. These jobs were farmed simultaneously to 100 high capacity compute nodes using the Amazon Web Service Elastic Map Reduce and included a wide mix of large and small genomes. The total computation time took just under 70 hours and cost a total of $6,302 USD. Conclusions The effort to transform existing comparative genomics algorithms from local compute infrastructures is not trivial. However, the speed and flexibility of cloud computing environments provides a substantial boost with manageable cost. The procedure designed to transform the RSD algorithm into a cloud-ready application is readily adaptable to similar comparative genomics problems. PMID:20482786

Cloud computing for comparative genomics.

PubMed

Wall, Dennis P; Kudtarkar, Parul; Fusaro, Vincent A; Pivovarov, Rimma; Patil, Prasad; Tonellato, Peter J

2010-05-18

Large comparative genomics studies and tools are becoming increasingly more compute-expensive as the number of available genome sequences continues to rise. The capacity and cost of local computing infrastructures are likely to become prohibitive with the increase, especially as the breadth of questions continues to rise. Alternative computing architectures, in particular cloud computing environments, may help alleviate this increasing pressure and enable fast, large-scale, and cost-effective comparative genomics strategies going forward. To test this, we redesigned a typical comparative genomics algorithm, the reciprocal smallest distance algorithm (RSD), to run within Amazon's Elastic Computing Cloud (EC2). We then employed the RSD-cloud for ortholog calculations across a wide selection of fully sequenced genomes. We ran more than 300,000 RSD-cloud processes within the EC2. These jobs were farmed simultaneously to 100 high capacity compute nodes using the Amazon Web Service Elastic Map Reduce and included a wide mix of large and small genomes. The total computation time took just under 70 hours and cost a total of $6,302 USD. The effort to transform existing comparative genomics algorithms from local compute infrastructures is not trivial. However, the speed and flexibility of cloud computing environments provides a substantial boost with manageable cost. The procedure designed to transform the RSD algorithm into a cloud-ready application is readily adaptable to similar comparative genomics problems.

High-throughput comparison, functional annotation, and metabolic modeling of plant genomes using the PlantSEED resource

USDA-ARS?s Scientific Manuscript database

The increasing number of sequenced plant genomes is placing new demands on the methods applied to analyze, annotate, and model these genomes. Today's annotation pipelines result in inconsistent gene assignments that complicate comparative analyses and prevent efficient construction of metabolic mode...

The perennial ryegrass GenomeZipper: targeted use of genome resources for comparative grass genomics.

PubMed

Pfeifer, Matthias; Martis, Mihaela; Asp, Torben; Mayer, Klaus F X; Lübberstedt, Thomas; Byrne, Stephen; Frei, Ursula; Studer, Bruno

2013-02-01

Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources have yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne) genome on the basis of conserved synteny to barley (Hordeum vulgare) and the model grass genome Brachypodium (Brachypodium distachyon) as well as rice (Oryza sativa) and sorghum (Sorghum bicolor). A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macrocollinearity and was then utilized to anchor a collection of perennial ryegrass genes in silico to their predicted genome positions. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci, which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species.

The Perennial Ryegrass GenomeZipper: Targeted Use of Genome Resources for Comparative Grass Genomics1[C][W

PubMed Central

Pfeifer, Matthias; Martis, Mihaela; Asp, Torben; Mayer, Klaus F.X.; Lübberstedt, Thomas; Byrne, Stephen; Frei, Ursula; Studer, Bruno

2013-01-01

Whole-genome sequences established for model and major crop species constitute a key resource for advanced genomic research. For outbreeding forage and turf grass species like ryegrasses (Lolium spp.), such resources have yet to be developed. Here, we present a model of the perennial ryegrass (Lolium perenne) genome on the basis of conserved synteny to barley (Hordeum vulgare) and the model grass genome Brachypodium (Brachypodium distachyon) as well as rice (Oryza sativa) and sorghum (Sorghum bicolor). A transcriptome-based genetic linkage map of perennial ryegrass served as a scaffold to establish the chromosomal arrangement of syntenic genes from model grass species. This scaffold revealed a high degree of synteny and macrocollinearity and was then utilized to anchor a collection of perennial ryegrass genes in silico to their predicted genome positions. This resulted in the unambiguous assignment of 3,315 out of 8,876 previously unmapped genes to the respective chromosomes. In total, the GenomeZipper incorporates 4,035 conserved grass gene loci, which were used for the first genome-wide sequence divergence analysis between perennial ryegrass, barley, Brachypodium, rice, and sorghum. The perennial ryegrass GenomeZipper is an ordered, information-rich genome scaffold, facilitating map-based cloning and genome assembly in perennial ryegrass and closely related Poaceae species. It also represents a milestone in describing synteny between perennial ryegrass and fully sequenced model grass genomes, thereby increasing our understanding of genome organization and evolution in the most important temperate forage and turf grass species. PMID:23184232

Multilocus sequence typing of total-genome-sequenced bacteria.

PubMed

Larsen, Mette V; Cosentino, Salvatore; Rasmussen, Simon; Friis, Carsten; Hasman, Henrik; Marvig, Rasmus Lykke; Jelsbak, Lars; Sicheritz-Pontén, Thomas; Ussery, David W; Aarestrup, Frank M; Lund, Ole

2012-04-01

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

CEP152 is a genome maintenance protein disrupted in Seckel syndrome

PubMed Central

Kalay, Ersan; Yigit, Gökhan; Aslan, Yakup; Brown, Karen E; Pohl, Esther; Bicknell, Louise S; Kayserili, Hülya; Li, Yun; Tüysüz, Beyhan; Nürnberg, Gudrun; Kiess, Wieland; Koegl, Manfred; Baessmann, Ingelore; Buruk, Kurtulus; Toraman, Bayram; Kayipmaz, Saadettin; Kul, Sibel; Ikbal, Mevlit; Turner, Daniel J; Taylor, Martin S; Aerts, Jan; Scott, Carol; Milstein, Karen; Dollfus, Helene; Wieczorek, Dagmar; Brunner, Han G; Hurles, Matthew; Jackson, Andrew P; Rauch, Anita; Nürnberg, Peter; Karagüzel, Ahmet; Wollnik, Bernd

2012-01-01

Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation. PMID:21131973

HIA: a genome mapper using hybrid index-based sequence alignment.

PubMed

Choi, Jongpill; Park, Kiejung; Cho, Seong Beom; Chung, Myungguen

2015-01-01

A number of alignment tools have been developed to align sequencing reads to the human reference genome. The scale of information from next-generation sequencing (NGS) experiments, however, is increasing rapidly. Recent studies based on NGS technology have routinely produced exome or whole-genome sequences from several hundreds or thousands of samples. To accommodate the increasing need of analyzing very large NGS data sets, it is necessary to develop faster, more sensitive and accurate mapping tools. HIA uses two indices, a hash table index and a suffix array index. The hash table performs direct lookup of a q-gram, and the suffix array performs very fast lookup of variable-length strings by exploiting binary search. We observed that combining hash table and suffix array (hybrid index) is much faster than the suffix array method for finding a substring in the reference sequence. Here, we defined the matching region (MR) is a longest common substring between a reference and a read. And, we also defined the candidate alignment regions (CARs) as a list of MRs that is close to each other. The hybrid index is used to find candidate alignment regions (CARs) between a reference and a read. We found that aligning only the unmatched regions in the CAR is much faster than aligning the whole CAR. In benchmark analysis, HIA outperformed in mapping speed compared with the other aligners, without significant loss of mapping accuracy. Our experiments show that the hybrid of hash table and suffix array is useful in terms of speed for mapping NGS sequencing reads to the human reference genome sequence. In conclusion, our tool is appropriate for aligning massive data sets generated by NGS sequencing.

RetroTector online, a rational tool for analysis of retroviral elements in small and medium size vertebrate genomic sequences

PubMed Central

Sperber, Göran; Lövgren, Anders; Eriksson, Nils-Einar; Benachenhou, Farid; Blomberg, Jonas

2009-01-01

Background The rapid accumulation of genomic information in databases necessitates rapid and specific algorithms for extracting biologically meaningful information. More or less complete retroviral sequences, also called proviral or endogenous retroviral sequences; ERVs, constitutes at least 5% of vertebrate genomes. After infecting the host, these retroviruses have integrated in germ line cells, and have then been carried in genomes for at least several 100 million years. A better understanding of structure and function of these sequences can have profound biological and medical consequences. Methods RetroTector© (ReTe) is a platform-independent Java program for identification and characterization of proviral sequences in vertebrate genomes. The full ReTe requires a local installation with a MySQL database. Although not overly complicated, the installation may take some time. A "light" version of ReTe, (RetroTector online; ROL) which does not require specific installation procedures is provided, via the World Wide Web. Results ROL was implemented under the Batchelor web interface (A Lövgren et al). It allows both GenBank accession number, file and FASTA cut-and-paste admission of sequences (5 to 10 000 kilobases). Up to ten submissions can be done simultaneously, allowing batch analysis of <= 100 Megabases. Jobs are shown in an IP-number specific list. Results are text files, and can be viewed with the program, RetroTectorViewer.jar (at the same site), which has the full graphical capabilities of the basic ReTe program. A detailed analysis of any retroviral sequences found in the submitted sequence is graphically presented, exportable in standard formats. With the current server, a complete analysis of a 1 Megabase sequence is complete in 10 minutes. It is possible to mask nonretroviral repetitive sequences in the submitted sequence, using host genome specific "brooms", which increase specificity. Discussion Proviral sequences can be hard to recognize, especially if the integration occurred many million years ago. Precise delineation of LTR, gag, pro, pol and env can be difficult, requiring manual work. ROL is a way of simplifying these tasks. Conclusion ROL provides 1. annotation and presentation of known retroviral sequences, 2. detection of proviral chains in unknown genomic sequences, with up to 100 Mbase per submission. PMID:19534753

RetroTector online, a rational tool for analysis of retroviral elements in small and medium size vertebrate genomic sequences.

PubMed

Sperber, Göran; Lövgren, Anders; Eriksson, Nils-Einar; Benachenhou, Farid; Blomberg, Jonas

2009-06-16

The rapid accumulation of genomic information in databases necessitates rapid and specific algorithms for extracting biologically meaningful information. More or less complete retroviral sequences, also called proviral or endogenous retroviral sequences; ERVs, constitutes at least 5% of vertebrate genomes. After infecting the host, these retroviruses have integrated in germ line cells, and have then been carried in genomes for at least several 100 million years. A better understanding of structure and function of these sequences can have profound biological and medical consequences. RetroTector (ReTe) is a platform-independent Java program for identification and characterization of proviral sequences in vertebrate genomes. The full ReTe requires a local installation with a MySQL database. Although not overly complicated, the installation may take some time. A "light" version of ReTe, (RetroTector online; ROL) which does not require specific installation procedures is provided, via the World Wide Web. ROL http://www.fysiologi.neuro.uu.se/jbgs/ was implemented under the Batchelor web interface (A Lövgren et al). It allows both GenBank accession number, file and FASTA cut-and-paste admission of sequences (5 to 10,000 kilobases). Up to ten submissions can be done simultaneously, allowing batch analysis of

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.

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/.

Foreign Plastid Sequences in Plant Mitochondria are Frequently Acquired Via Mitochondrion-to-Mitochondrion Horizontal Transfer

PubMed Central

Gandini, C. L.; Sanchez-Puerta, M. V.

2017-01-01

Angiosperm mitochondrial genomes (mtDNA) exhibit variable quantities of alien sequences. Many of these sequences are acquired by intracellular gene transfer (IGT) from the plastid. In addition, frequent events of horizontal gene transfer (HGT) between mitochondria of different species also contribute to their expanded genomes. In contrast, alien sequences are rarely found in plastid genomes. Most of the plant-to-plant HGT events involve mitochondrion-to-mitochondrion transfers. Occasionally, foreign sequences in mtDNAs are plastid-derived (MTPT), raising questions about their origin, frequency, and mechanism of transfer. The rising number of complete mtDNAs allowed us to address these questions. We identified 15 new foreign MTPTs, increasing significantly the number of those previously reported. One out of five of the angiosperm species analyzed contained at least one foreign MTPT, suggesting a remarkable frequency of HGT among plants. By analyzing the flanking regions of the foreign MTPTs, we found strong evidence for mt-to-mt transfers in 65% of the cases. We hypothesize that plastid sequences were initially acquired by the native mtDNA via IGT and then transferred to a distantly-related plant via mitochondrial HGT, rather than directly from a foreign plastid to the mitochondrial genome. Finally, we describe three novel putative cases of mitochondrial-derived sequences among angiosperm plastomes. PMID:28262720

Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

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

Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Furthermore, our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.« less

Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

DOE PAGES

Immonen, Taina T.; Conway, Jessica M.; Romero-Severson, Ethan O.; ...

2015-12-22

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation processmore » including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Furthermore, our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.« less

Microbial genomic island discovery, visualization and analysis.

PubMed

Bertelli, Claire; Tilley, Keith E; Brinkman, Fiona S L

2018-06-03

Horizontal gene transfer (also called lateral gene transfer) is a major mechanism for microbial genome evolution, enabling rapid adaptation and survival in specific niches. Genomic islands (GIs), commonly defined as clusters of bacterial or archaeal genes of probable horizontal origin, are of particular medical, environmental and/or industrial interest, as they disproportionately encode virulence factors and some antimicrobial resistance genes and may harbor entire metabolic pathways that confer a specific adaptation (solvent resistance, symbiosis properties, etc). As large-scale analyses of microbial genomes increases, such as for genomic epidemiology investigations of infectious disease outbreaks in public health, there is increased appreciation of the need to accurately predict and track GIs. Over the past decade, numerous computational tools have been developed to tackle the challenges inherent in accurate GI prediction. We review here the main types of GI prediction methods and discuss their advantages and limitations for a routine analysis of microbial genomes in this era of rapid whole-genome sequencing. An assessment is provided of 20 GI prediction software methods that use sequence-composition bias to identify the GIs, using a reference GI data set from 104 genomes obtained using an independent comparative genomics approach. Finally, we present guidelines to assist researchers in effectively identifying these key genomic regions.

DOE Joint Genome Institute 2008 Progress Report

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

Gilbert, David

2009-03-12

While initially a virtual institute, the driving force behind the creation of the DOE Joint Genome Institute in Walnut Creek, California in the Fall of 1999 was the Department of Energy's commitment to sequencing the human genome. With the publication in 2004 of a trio of manuscripts describing the finished 'DOE Human Chromosomes', the Institute successfully completed its human genome mission. In the time between the creation of the Department of Energy Joint Genome Institute (DOE JGI) and completion of the Human Genome Project, sequencing and its role in biology spread to fields extending far beyond what could be imaginedmore » when the Human Genome Project first began. Accordingly, the targets of the DOE JGI's sequencing activities changed, moving from a single human genome to the genomes of large numbers of microbes, plants, and other organisms, and the community of users of DOE JGI data similarly expanded and diversified. Transitioning into operating as a user facility, the DOE JGI modeled itself after other DOE user facilities, such as synchrotron light sources and supercomputer facilities, empowering the science of large numbers of investigators working in areas of relevance to energy and the environment. The JGI's approach to being a user facility is based on the concept that by focusing state-of-the-art sequencing and analysis capabilities on the best peer-reviewed ideas drawn from a broad community of scientists, the DOE JGI will effectively encourage creative approaches to DOE mission areas and produce important science. This clearly has occurred, only partially reflected in the fact that the DOE JGI has played a major role in more than 45 papers published in just the past three years alone in Nature and Science. The involvement of a large and engaged community of users working on important problems has helped maximize the impact of JGI science. A seismic technological change is presently underway at the JGI. The Sanger capillary-based sequencing process that dominated how sequencing was done in the last decade is being replaced by a variety of new processes and sequencing instruments. The JGI, with an increasing number of next-generation sequencers, whose throughput is 100- to 1,000-fold greater than the Sanger capillary-based sequencers, is increasingly focused in new directions on projects of scale and complexity not previously attempted. These new directions for the JGI come, in part, from the 2008 National Research Council report on the goals of the National Plant Genome Initiative as well as the 2007 National Research Council report on the New Science of Metagenomics. Both reports outline a crucial need for systematic large-scale surveys of the plant and microbial components of the biosphere as well as an increasing need for large-scale analysis capabilities to meet the challenge of converting sequence data into knowledge. The JGI is extensively discussed in both reports as vital to progress in these fields of major national interest. JGI's future plan for plants and microbes includes a systematic approach for investigation of these organisms at a scale requiring the special capabilities of the JGI to generate, manage, and analyze the datasets. JGI will generate and provide not only community access to these plant and microbial datasets, but also the tools for analyzing them. These activities will produce essential knowledge that will be needed if we are to be able to respond to the world's energy and environmental challenges. As the JGI Plant and Microbial programs advance, the JGI as a user facility is also evolving. The Institute has been highly successful in bending its technical and analytical skills to help users solve large complex problems of major importance, and that effort will continue unabated. The JGI will increasingly move from a central focus on 'one-off' user projects coming from small user communities to much larger scale projects driven by systematic and problem-focused approaches to selection of sequencing targets. Entire communities of scientists working in a particular field, such as feedstock improvement or biomass degradation, will be users of this information. Despite this new emphasis, an investigator-initiated user program will remain. This program in the future will replace small projects that increasingly can be accomplished without the involvement of JGI, with imaginative large-scale 'Grand Challenge' projects of foundational relevance to energy and the environment that require a new scale of sequencing and analysis capabilities. Close interactions with the DOE Bioenergy Research Centers, and with other DOE institutions that may follow, will also play a major role in shaping aspects of how the JGI operates as a user facility. Based on increased availability of high-throughput sequencing, the JGI will increasingly provide to users, in addition to DNA sequencing, an array of both pre- and post-sequencing value-added capabilities to accelerate their science.« less

Mapping-by-sequencing in complex polyploid genomes using genic sequence capture: a case study to map yellow rust resistance in hexaploid wheat.

PubMed

Gardiner, Laura-Jayne; Bansept-Basler, Pauline; Olohan, Lisa; Joynson, Ryan; Brenchley, Rachel; Hall, Neil; O'Sullivan, Donal M; Hall, Anthony

2016-08-01

Previously we extended the utility of mapping-by-sequencing by combining it with sequence capture and mapping sequence data to pseudo-chromosomes that were organized using wheat-Brachypodium synteny. This, with a bespoke haplotyping algorithm, enabled us to map the flowering time locus in the diploid wheat Triticum monococcum L. identifying a set of deleted genes (Gardiner et al., 2014). Here, we develop this combination of gene enrichment and sliding window mapping-by-synteny analysis to map the Yr6 locus for yellow stripe rust resistance in hexaploid wheat. A 110 MB NimbleGen capture probe set was used to enrich and sequence a doubled haploid mapping population of hexaploid wheat derived from an Avalon and Cadenza cross. The Yr6 locus was identified by mapping to the POPSEQ chromosomal pseudomolecules using a bespoke pipeline and algorithm (Chapman et al., 2015). Furthermore the same locus was identified using newly developed pseudo-chromosome sequences as a mapping reference that are based on the genic sequence used for sequence enrichment. The pseudo-chromosomes allow us to demonstrate the application of mapping-by-sequencing to even poorly defined polyploidy genomes where chromosomes are incomplete and sub-genome assemblies are collapsed. This analysis uniquely enabled us to: compare wheat genome annotations; identify the Yr6 locus - defining a smaller genic region than was previously possible; associate the interval with one wheat sub-genome and increase the density of SNP markers associated. Finally, we built the pipeline in iPlant, making it a user-friendly community resource for phenotype mapping. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Applications of nanotechnology, next generation sequencing and microarrays in biomedical research.

PubMed

Elingaramil, Sauli; Li, Xiaolong; He, Nongyue

2013-07-01

Next-generation sequencing technologies, microarrays and advances in bio nanotechnology have had an enormous impact on research within a short time frame. This impact appears certain to increase further as many biomedical institutions are now acquiring these prevailing new technologies. Beyond conventional sampling of genome content, wide-ranging applications are rapidly evolving for next-generation sequencing, microarrays and nanotechnology. To date, these technologies have been applied in a variety of contexts, including whole-genome sequencing, targeted re sequencing and discovery of transcription factor binding sites, noncoding RNA expression profiling and molecular diagnostics. This paper thus discusses current applications of nanotechnology, next-generation sequencing technologies and microarrays in biomedical research and highlights the transforming potential these technologies offer.

Phylogenetics of modern birds in the era of genomics

PubMed Central

Edwards, Scott V; Bryan Jennings, W; Shedlock, Andrew M

2005-01-01

In the 14 years since the first higher-level bird phylogenies based on DNA sequence data, avian phylogenetics has witnessed the advent and maturation of the genomics era, the completion of the chicken genome and a suite of technologies that promise to add considerably to the agenda of avian phylogenetics. In this review, we summarize current approaches and data characteristics of recent higher-level bird studies and suggest a number of as yet untested molecular and analytical approaches for the unfolding tree of life for birds. A variety of comparative genomics strategies, including adoption of objective quality scores for sequence data, analysis of contiguous DNA sequences provided by large-insert genomic libraries, and the systematic use of retroposon insertions and other rare genomic changes all promise an integrated phylogenetics that is solidly grounded in genome evolution. The avian genome is an excellent testing ground for such approaches because of the more balanced representation of single-copy and repetitive DNA regions than in mammals. Although comparative genomics has a number of obvious uses in avian phylogenetics, its application to large numbers of taxa poses a number of methodological and infrastructural challenges, and can be greatly facilitated by a ‘community genomics’ approach in which the modest sequencing throughputs of single PI laboratories are pooled to produce larger, complementary datasets. Although the polymerase chain reaction era of avian phylogenetics is far from complete, the comparative genomics era—with its ability to vastly increase the number and type of molecular characters and to provide a genomic context for these characters—will usher in a host of new perspectives and opportunities for integrating genome evolution and avian phylogenetics. PMID:16024355

Genome sequence of the Japanese oak silk moth, Antheraea yamamai: the first draft genome in the family Saturniidae

PubMed Central

Kim, Seong-Ryul; Kwak, Woori; Kim, Hyaekang; Kim, Kee-Young; Kim, Su-Bae; Choi, Kwang-Ho; Kim, Seong-Wan; Hwang, Jae-Sam; Kim, Minjee; Kim, Iksoo; Goo, Tae-Won

2018-01-01

Abstract Background Antheraea yamamai, also known as the Japanese oak silk moth, is a wild species of silk moth. Silk produced by A. yamamai, referred to as tensan silk, shows different characteristics such as thickness, compressive elasticity, and chemical resistance compared with common silk produced from the domesticated silkworm, Bombyx mori. Its unique characteristics have led to its use in many research fields including biotechnology and medical science, and the scientific as well as economic importance of the wild silk moth continues to gradually increase. However, no genomic information for the wild silk moth, including A. yamamai, is currently available. Findings In order to construct the A. yamamai genome, a total of 147G base pairs using Illumina and Pacbio sequencing platforms were generated, providing 210-fold coverage based on the 700-Mb estimated genome size of A. yamamai. The assembled genome of A. yamamai was 656 Mb (>2 kb) with 3675 scaffolds, and the N50 length of assembly was 739 Kb with a 34.07% GC ratio. Identified repeat elements covered 37.33% of the total genome, and the completeness of the constructed genome assembly was estimated to be 96.7% by Benchmarking Universal Single-Copy Orthologs v2 analysis. A total of 15 481 genes were identified using Evidence Modeler based on the gene prediction results obtained from 3 different methods (ab initio, RNA-seq-based, known-gene-based) and manual curation. Conclusions Here we present the genome sequence of A. yamamai, the first genome sequence of the wild silk moth. These results provide valuable genomic information, which will help enrich our understanding of the molecular mechanisms relating to not only specific phenotypes such as wild silk itself but also the genomic evolution of Saturniidae. PMID:29186418

A world of opportunities with nanopore sequencing.

PubMed

Leggett, Richard M; Clark, Matthew D

2017-11-28

Oxford Nanopore Technologies' MinION sequencer was launched in pre-release form in 2014 and represents an exciting new sequencing paradigm. The device offers multi-kilobase reads and a streamed mode of operation that allows processing of reads as they are generated. Crucially, it is an extremely compact device that is powered from the USB port of a laptop computer, enabling it to be taken out of the lab and facilitating previously impossible in-field sequencing experiments to be undertaken. Many of the initial publications concerning the platform focused on provision of tools to access and analyse the new sequence formats and then demonstrating the assembly of microbial genomes. More recently, as throughput and accuracy have increased, it has been possible to begin work involving more complex genomes and metagenomes. With the release of the high-throughput GridION X5 and PromethION platforms, the sequencing of large genomes will become more cost efficient, and enable the leveraging of extremely long (>100 kb) reads for resolution of complex genomic structures. This review provides a brief overview of nanopore sequencing technology, describes the growing range of nanopore bioinformatics tools, and highlights some of the most influential publications that have emerged over the last 2 years. Finally, we look to the future and the potential the platform has to disrupt work in human, microbiome, and plant genomics. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Genome Improvement at JGI-HAGSC

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

Grimwood, Jane; Schmutz, Jeremy J.; Myers, Richard M.

Since the completion of the sequencing of the human genome, the Joint Genome Institute (JGI) has rapidly expanded its scientific goals in several DOE mission-relevant areas. At the JGI-HAGSC, we have kept pace with this rapid expansion of projects with our focus on assessing, assembling, improving and finishing eukaryotic whole genome shotgun (WGS) projects for which the shotgun sequence is generated at the Production Genomic Facility (JGI-PGF). We follow this by combining the draft WGS with genomic resources generated at JGI-HAGSC or in collaborator laboratories (including BAC end sequences, genetic maps and FLcDNA sequences) to produce an improved draft sequence.more » For eukaryotic genomes important to the DOE mission, we then add further information from directed experiments to produce reference genomic sequences that are publicly available for any scientific researcher. Also, we have continued our program for producing BAC-based finished sequence, both for adding information to JGI genome projects and for small BAC-based sequencing projects proposed through any of the JGI sequencing programs. We have now built our computational expertise in WGS assembly and analysis and have moved eukaryotic genome assembly from the JGI-PGF to JGI-HAGSC. We have concentrated our assembly development work on large plant genomes and complex fungal and algal genomes.« less

Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

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

McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.

2005-08-26

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. Amore » minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.« less

Reference-quality genome sequence of Aegilops tauschii, the source of wheat D genome, shows that recombination shapes genome structure and evolution

USDA-ARS?s Scientific Manuscript database

Aegilops tauschii is the diploid progenitor of the D genome of hexaploid wheat and an important genetic resource for wheat. A reference-quality sequence for the Ae. tauschii genome was produced with a combination of ordered-clone sequencing, whole-genome shotgun sequencing, and BioNano optical geno...

Building a genome analysis pipeline to predict disease risk and prevent disease.

PubMed

Bromberg, Y

2013-11-01

Reduced costs and increased speed and accuracy of sequencing can bring the genome-based evaluation of individual disease risk to the bedside. While past efforts have identified a number of actionable mutations, the bulk of genetic risk remains hidden in sequence data. The biggest challenge facing genomic medicine today is the development of new techniques to predict the specifics of a given human phenome (set of all expressed phenotypes) encoded by each individual variome (full set of genome variants) in the context of the given environment. Numerous tools exist for the computational identification of the functional effects of a single variant. However, the pipelines taking advantage of full genomic, exomic, transcriptomic (and other) sequences have only recently become a reality. This review looks at the building of methodologies for predicting "variome"-defined disease risk. It also discusses some of the challenges for incorporating such a pipeline into everyday medical practice. © 2013. Published by Elsevier Ltd. All rights reserved.

Bacterial genomes in epidemiology—present and future

PubMed Central

Croucher, Nicholas J.; Harris, Simon R.; Grad, Yonatan H.; Hanage, William P.

2013-01-01

Sequence data are well established in the reconstruction of the phylogenetic and demographic scenarios that have given rise to outbreaks of viral pathogens. The application of similar methods to bacteria has been hindered in the main by the lack of high-resolution nucleotide sequence data from quality samples. Developing and already available genomic methods have greatly increased the amount of data that can be used to characterize an isolate and its relationship to others. However, differences in sequencing platforms and data analysis mean that these enhanced data come with a cost in terms of portability: results from one laboratory may not be directly comparable with those from another. Moreover, genomic data for many bacteria bear the mark of a history including extensive recombination, which has the potential to greatly confound phylogenetic and coalescent analyses. Here, we discuss the exacting requirements of genomic epidemiology, and means by which the distorting signal of recombination can be minimized to permit the leverage of growing datasets of genomic data from bacterial pathogens. PMID:23382424

Identification of a precursor genomic segment that provided a sequence unique to glycophorin B and E genes

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

Onda, M.; Kudo, S.; Fukuda, M.

Human glycophorin A, B, and E (GPA, GPB, and GPE) genes belong to a gene family located at the long arm of chromosome 4. These three genes are homologous from the 5'-flanking sequence to the Alu sequence, which is 1 kb downstream from the exon encoding the transmembrane domain. Analysis of the Alu sequence and flanking direct repeat sequences suggested that the GPA gene most closely resembles the ancestral gene, whereas the GPB and GPE gene arose by homologous recombination within the Alu sequence, acquiring 3' sequences from an unrelated precursor genomic segment. Here the authors describe the identification ofmore » this putative precursor genomic segment. A human genomic library was screened by using the sequence of the 3' region of the GPB gene as a probe. The genomic clones isolated were found to contain an Alu sequence that appeared to be involved in the recombination. Downstream from the Alu sequence, the nucleotide sequence of the precursor genomic segment is almost identical to that of the GPB or GPE gene. In contrast, the upstream sequence of the genomic segment differs entirely from that of the GPA, GPB, and GPE genes. Conservation of the direct repeats flanking the Alu sequence of the genomic segment strongly suggests that the sequence of this genomic segment has been maintained during evolution. This identified genomic segment was found to reside downstream from the GPA gene by both gene mapping and in situ chromosomal localization. The precursor genomic segment was also identified in the orangutan genome, which is known to lack GPB and GPE genes. These results indicate that one of the duplicated ancestral glycophorin genes acquired a unique 3' sequence by unequal crossing-over through its Alu sequence and the further downstream Alu sequence present in the duplicated gene. Further duplication and divergence of this gene yielded the GPB and GPE genes. 37 refs., 5 figs.« less

Draft Genome Sequences of Escherichia coli Isolates from Wounded Military Personnel.

PubMed

Arivett, Brock A; Ream, Dave C; Fiester, Steven E; Kidane, Destaalem; Actis, Luis A

2016-08-11

Members of the Escherichia coli bacterial family have been grouped as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens because of their extensive drug resistance phenotypes and increasing threat to human health. The genomes of six extended-spectrum β-lactamase (ESBL)-producing E. coli strains isolated from wounded military personnel were sequenced and annotated. Copyright © 2016 Arivett et al.

Insights on genome size evolution from a miniature inverted repeat transposon driving a satellite DNA.

PubMed

Scalvenzi, Thibault; Pollet, Nicolas

2014-12-01

The genome size in eukaryotes does not correlate well with the number of genes they contain. We can observe this so-called C-value paradox in amphibian species. By analyzing an amphibian genome we asked how repetitive DNA can impact genome size and architecture. We describe here our discovery of a Tc1/mariner miniature inverted-repeat transposon family present in Xenopus frogs. These transposons named miDNA4 are unique since they contain a satellite DNA motif. We found that miDNA4 measured 331 bp, contained 25 bp long inverted terminal repeat sequences and a sequence motif of 119 bp present as a unique copy or as an array of 2-47 copies. We characterized the structure, dynamics, impact and evolution of the miDNA4 family and its satellite DNA in Xenopus frog genomes. This led us to propose a model for the evolution of these two repeated sequences and how they can synergize to increase genome size. Copyright © 2014 Elsevier Inc. All rights reserved.

Leptospiral Pathogenomics

PubMed Central

Lehmann, Jason S.; Matthias, Michael A.; Vinetz, Joseph M.; Fouts, Derrick E.

2014-01-01

Leptospirosis, caused by pathogenic spirochetes belonging to the genus Leptospira, is a zoonosis with important impacts on human and animal health worldwide. Research on the mechanisms of Leptospira pathogenesis has been hindered due to slow growth of infectious strains, poor transformability, and a paucity of genetic tools. As a result of second generation sequencing technologies, there has been an acceleration of leptospiral genome sequencing efforts in the past decade, which has enabled a concomitant increase in functional genomics analyses of Leptospira pathogenesis. A pathogenomics approach, by coupling of pan-genomic analysis of multiple isolates with sequencing of experimentally attenuated highly pathogenic Leptospira, has resulted in the functional inference of virulence factors. The global Leptospira Genome Project supported by the U.S. National Institute of Allergy and Infectious Diseases to which key scientific contributions have been made from the international leptospirosis research community has provided a new roadmap for comprehensive studies of Leptospira and leptospirosis well into the future. This review describes functional genomics approaches to apply the data generated by the Leptospira Genome Project towards deepening our knowledge of virulence factors of Leptospira using the emerging discipline of pathogenomics. PMID:25437801

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

PubMed

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

PubMed Central

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop plants with large and complex genomes. PMID:29250096

Atrx promotes heterochromatin formation at retrotransposons

PubMed Central

Sadic, Dennis; Schmidt, Katharina; Groh, Sophia; Kondofersky, Ivan; Ellwart, Joachim; Fuchs, Christiane; Theis, Fabian J; Schotta, Gunnar

2015-01-01

More than 50% of mammalian genomes consist of retrotransposon sequences. Silencing of retrotransposons by heterochromatin is essential to ensure genomic stability and transcriptional integrity. Here, we identified a short sequence element in intracisternal A particle (IAP) retrotransposons that is sufficient to trigger heterochromatin formation. We used this sequence in a genome-wide shRNA screen and identified the chromatin remodeler Atrx as a novel regulator of IAP silencing. Atrx binds to IAP elements and is necessary for efficient heterochromatin formation. In addition, Atrx facilitates a robust and largely inaccessible heterochromatin structure as Atrx knockout cells display increased chromatin accessibility at retrotransposons and non-repetitive heterochromatic loci. In summary, we demonstrate a direct role of Atrx in the establishment and robust maintenance of heterochromatin. PMID:26012739

Mapping Second Chromosome Mutations to Defined Genomic Regions in Drosophila melanogaster

PubMed Central

Kahsai, Lily; Cook, Kevin R.

2017-01-01

Hundreds of Drosophila melanogaster stocks are currently maintained at the Bloomington Drosophila Stock Center with mutations that have not been associated with sequence-defined genes. They have been preserved because they have interesting loss-of-function phenotypes. The experimental value of these mutations would be increased by tying them to specific genomic intervals so that geneticists can more easily associate them with annotated genes. Here, we report the mapping of 85 second chromosome complementation groups in the Bloomington collection to specific, small clusters of contiguous genes or individual genes in the sequenced genome. This information should prove valuable to Drosophila geneticists interested in processes associated with particular phenotypes and those searching for mutations affecting specific sequence-defined genes. PMID:29066472

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

The zebrafish reference genome sequence and its relationship to the human genome.

PubMed

Howe, Kerstin; Clark, Matthew D; Torroja, Carlos F; Torrance, James; Berthelot, Camille; Muffato, Matthieu; Collins, John E; Humphray, Sean; McLaren, Karen; Matthews, Lucy; McLaren, Stuart; Sealy, Ian; Caccamo, Mario; Churcher, Carol; Scott, Carol; Barrett, Jeffrey C; Koch, Romke; Rauch, Gerd-Jörg; White, Simon; Chow, William; Kilian, Britt; Quintais, Leonor T; Guerra-Assunção, José A; Zhou, Yi; Gu, Yong; Yen, Jennifer; Vogel, Jan-Hinnerk; Eyre, Tina; Redmond, Seth; Banerjee, Ruby; Chi, Jianxiang; Fu, Beiyuan; Langley, Elizabeth; Maguire, Sean F; Laird, Gavin K; Lloyd, David; Kenyon, Emma; Donaldson, Sarah; Sehra, Harminder; Almeida-King, Jeff; Loveland, Jane; Trevanion, Stephen; Jones, Matt; Quail, Mike; Willey, Dave; Hunt, Adrienne; Burton, John; Sims, Sarah; McLay, Kirsten; Plumb, Bob; Davis, Joy; Clee, Chris; Oliver, Karen; Clark, Richard; Riddle, Clare; Elliot, David; Eliott, David; Threadgold, Glen; Harden, Glenn; Ware, Darren; Begum, Sharmin; Mortimore, Beverley; Mortimer, Beverly; Kerry, Giselle; Heath, Paul; Phillimore, Benjamin; Tracey, Alan; Corby, Nicole; Dunn, Matthew; Johnson, Christopher; Wood, Jonathan; Clark, Susan; Pelan, Sarah; Griffiths, Guy; Smith, Michelle; Glithero, Rebecca; Howden, Philip; Barker, Nicholas; Lloyd, Christine; Stevens, Christopher; Harley, Joanna; Holt, Karen; Panagiotidis, Georgios; Lovell, Jamieson; Beasley, Helen; Henderson, Carl; Gordon, Daria; Auger, Katherine; Wright, Deborah; Collins, Joanna; Raisen, Claire; Dyer, Lauren; Leung, Kenric; Robertson, Lauren; Ambridge, Kirsty; Leongamornlert, Daniel; McGuire, Sarah; Gilderthorp, Ruth; Griffiths, Coline; Manthravadi, Deepa; Nichol, Sarah; Barker, Gary; Whitehead, Siobhan; Kay, Michael; Brown, Jacqueline; Murnane, Clare; Gray, Emma; Humphries, Matthew; Sycamore, Neil; Barker, Darren; Saunders, David; Wallis, Justene; Babbage, Anne; Hammond, Sian; Mashreghi-Mohammadi, Maryam; Barr, Lucy; Martin, Sancha; Wray, Paul; Ellington, Andrew; Matthews, Nicholas; Ellwood, Matthew; Woodmansey, Rebecca; Clark, Graham; Cooper, James D; Cooper, James; Tromans, Anthony; Grafham, Darren; Skuce, Carl; Pandian, Richard; Andrews, Robert; Harrison, Elliot; Kimberley, Andrew; Garnett, Jane; Fosker, Nigel; Hall, Rebekah; Garner, Patrick; Kelly, Daniel; Bird, Christine; Palmer, Sophie; Gehring, Ines; Berger, Andrea; Dooley, Christopher M; Ersan-Ürün, Zübeyde; Eser, Cigdem; Geiger, Horst; Geisler, Maria; Karotki, Lena; Kirn, Anette; Konantz, Judith; Konantz, Martina; Oberländer, Martina; Rudolph-Geiger, Silke; Teucke, Mathias; Lanz, Christa; Raddatz, Günter; Osoegawa, Kazutoyo; Zhu, Baoli; Rapp, Amanda; Widaa, Sara; Langford, Cordelia; Yang, Fengtang; Schuster, Stephan C; Carter, Nigel P; Harrow, Jennifer; Ning, Zemin; Herrero, Javier; Searle, Steve M J; Enright, Anton; Geisler, Robert; Plasterk, Ronald H A; Lee, Charles; Westerfield, Monte; de Jong, Pieter J; Zon, Leonard I; Postlethwait, John H; Nüsslein-Volhard, Christiane; Hubbard, Tim J P; Roest Crollius, Hugues; Rogers, Jane; Stemple, Derek L

2013-04-25

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

The zebrafish reference genome sequence and its relationship to the human genome

PubMed Central

Howe, Kerstin; Clark, Matthew D.; Torroja, Carlos F.; Torrance, James; Berthelot, Camille; Muffato, Matthieu; Collins, John E.; Humphray, Sean; McLaren, Karen; Matthews, Lucy; McLaren, Stuart; Sealy, Ian; Caccamo, Mario; Churcher, Carol; Scott, Carol; Barrett, Jeffrey C.; Koch, Romke; Rauch, Gerd-Jörg; White, Simon; Chow, William; Kilian, Britt; Quintais, Leonor T.; Guerra-Assunção, José A.; Zhou, Yi; Gu, Yong; Yen, Jennifer; Vogel, Jan-Hinnerk; Eyre, Tina; Redmond, Seth; Banerjee, Ruby; Chi, Jianxiang; Fu, Beiyuan; Langley, Elizabeth; Maguire, Sean F.; Laird, Gavin K.; Lloyd, David; Kenyon, Emma; Donaldson, Sarah; Sehra, Harminder; Almeida-King, Jeff; Loveland, Jane; Trevanion, Stephen; Jones, Matt; Quail, Mike; Willey, Dave; Hunt, Adrienne; Burton, John; Sims, Sarah; McLay, Kirsten; Plumb, Bob; Davis, Joy; Clee, Chris; Oliver, Karen; Clark, Richard; Riddle, Clare; Eliott, David; Threadgold, Glen; Harden, Glenn; Ware, Darren; Mortimer, Beverly; Kerry, Giselle; Heath, Paul; Phillimore, Benjamin; Tracey, Alan; Corby, Nicole; Dunn, Matthew; Johnson, Christopher; Wood, Jonathan; Clark, Susan; Pelan, Sarah; Griffiths, Guy; Smith, Michelle; Glithero, Rebecca; Howden, Philip; Barker, Nicholas; Stevens, Christopher; Harley, Joanna; Holt, Karen; Panagiotidis, Georgios; Lovell, Jamieson; Beasley, Helen; Henderson, Carl; Gordon, Daria; Auger, Katherine; Wright, Deborah; Collins, Joanna; Raisen, Claire; Dyer, Lauren; Leung, Kenric; Robertson, Lauren; Ambridge, Kirsty; Leongamornlert, Daniel; McGuire, Sarah; Gilderthorp, Ruth; Griffiths, Coline; Manthravadi, Deepa; Nichol, Sarah; Barker, Gary; Whitehead, Siobhan; Kay, Michael; Brown, Jacqueline; Murnane, Clare; Gray, Emma; Humphries, Matthew; Sycamore, Neil; Barker, Darren; Saunders, David; Wallis, Justene; Babbage, Anne; Hammond, Sian; Mashreghi-Mohammadi, Maryam; Barr, Lucy; Martin, Sancha; Wray, Paul; Ellington, Andrew; Matthews, Nicholas; Ellwood, Matthew; Woodmansey, Rebecca; Clark, Graham; Cooper, James; Tromans, Anthony; Grafham, Darren; Skuce, Carl; Pandian, Richard; Andrews, Robert; Harrison, Elliot; Kimberley, Andrew; Garnett, Jane; Fosker, Nigel; Hall, Rebekah; Garner, Patrick; Kelly, Daniel; Bird, Christine; Palmer, Sophie; Gehring, Ines; Berger, Andrea; Dooley, Christopher M.; Ersan-Ürün, Zübeyde; Eser, Cigdem; Geiger, Horst; Geisler, Maria; Karotki, Lena; Kirn, Anette; Konantz, Judith; Konantz, Martina; Oberländer, Martina; Rudolph-Geiger, Silke; Teucke, Mathias; Osoegawa, Kazutoyo; Zhu, Baoli; Rapp, Amanda; Widaa, Sara; Langford, Cordelia; Yang, Fengtang; Carter, Nigel P.; Harrow, Jennifer; Ning, Zemin; Herrero, Javier; Searle, Steve M. J.; Enright, Anton; Geisler, Robert; Plasterk, Ronald H. A.; Lee, Charles; Westerfield, Monte; de Jong, Pieter J.; Zon, Leonard I.; Postlethwait, John H.; Nüsslein-Volhard, Christiane; Hubbard, Tim J. P.; Crollius, Hugues Roest; Rogers, Jane; Stemple, Derek L.

2013-01-01

Zebrafish have become a popular organism for the study of vertebrate gene function1,2. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease3–5. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes6, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination. PMID:23594743

Snake Genome Sequencing: Results and Future Prospects

PubMed Central

Kerkkamp, Harald M. I.; Kini, R. Manjunatha; Pospelov, Alexey S.; Vonk, Freek J.; Henkel, Christiaan V.; Richardson, Michael K.

2016-01-01

Snake genome sequencing is in its infancy—very much behind the progress made in sequencing the genomes of humans, model organisms and pathogens relevant to biomedical research, and agricultural species. We provide here an overview of some of the snake genome projects in progress, and discuss the biological findings, with special emphasis on toxinology, from the small number of draft snake genomes already published. We discuss the future of snake genomics, pointing out that new sequencing technologies will help overcome the problem of repetitive sequences in assembling snake genomes. Genome sequences are also likely to be valuable in examining the clustering of toxin genes on the chromosomes, in designing recombinant antivenoms and in studying the epigenetic regulation of toxin gene expression. PMID:27916957

Snake Genome Sequencing: Results and Future Prospects.

PubMed

Kerkkamp, Harald M I; Kini, R Manjunatha; Pospelov, Alexey S; Vonk, Freek J; Henkel, Christiaan V; Richardson, Michael K

2016-12-01

Snake genome sequencing is in its infancy-very much behind the progress made in sequencing the genomes of humans, model organisms and pathogens relevant to biomedical research, and agricultural species. We provide here an overview of some of the snake genome projects in progress, and discuss the biological findings, with special emphasis on toxinology, from the small number of draft snake genomes already published. We discuss the future of snake genomics, pointing out that new sequencing technologies will help overcome the problem of repetitive sequences in assembling snake genomes. Genome sequences are also likely to be valuable in examining the clustering of toxin genes on the chromosomes, in designing recombinant antivenoms and in studying the epigenetic regulation of toxin gene expression.

The genome sequence of the emerging common midwife toad virus identifies an evolutionary intermediate within ranaviruses.

PubMed

Mavian, Carla; López-Bueno, Alberto; Balseiro, Ana; Casais, Rosa; Alcamí, Antonio; Alejo, Alí

2012-04-01

Worldwide amphibian population declines have been ascribed to global warming, increasing pollution levels, and other factors directly related to human activities. These factors may additionally be favoring the emergence of novel pathogens. In this report, we have determined the complete genome sequence of the emerging common midwife toad ranavirus (CMTV), which has caused fatal disease in several amphibian species across Europe. Phylogenetic and gene content analyses of the first complete genomic sequence from a ranavirus isolated in Europe show that CMTV is an amphibian-like ranavirus (ALRV). However, the CMTV genome structure is novel and represents an intermediate evolutionary stage between the two previously described ALRV groups. We find that CMTV clusters with several other ranaviruses isolated from different hosts and locations which might also be included in this novel ranavirus group. This work sheds light on the phylogenetic relationships within this complex group of emerging, disease-causing viruses.

Sequencing and comparative genomic analysis of 1227 Felis catus cDNA sequences enriched for developmental, clinical and nutritional phenotypes

PubMed Central

2012-01-01

Background The feline genome is valuable to the veterinary and model organism genomics communities because the cat is an obligate carnivore and a model for endangered felids. The initial public release of the Felis catus genome assembly provided a framework for investigating the genomic basis of feline biology. However, the entire set of protein coding genes has not been elucidated. Results We identified and characterized 1227 protein coding feline sequences, of which 913 map to public sequences and 314 are novel. These sequences have been deposited into NCBI's genbank database and complement public genomic resources by providing additional protein coding sequences that fill in some of the gaps in the feline genome assembly. Through functional and comparative genomic analyses, we gained an understanding of the role of these sequences in feline development, nutrition and health. Specifically, we identified 104 orthologs of human genes associated with Mendelian disorders. We detected negative selection within sequences with gene ontology annotations associated with intracellular trafficking, cytoskeleton and muscle functions. We detected relatively less negative selection on protein sequences encoding extracellular networks, apoptotic pathways and mitochondrial gene ontology annotations. Additionally, we characterized feline cDNA sequences that have mouse orthologs associated with clinical, nutritional and developmental phenotypes. Together, this analysis provides an overview of the value of our cDNA sequences and enhances our understanding of how the feline genome is similar to, and different from other mammalian genomes. Conclusions The cDNA sequences reported here expand existing feline genomic resources by providing high-quality sequences annotated with comparative genomic information providing functional, clinical, nutritional and orthologous gene information. PMID:22257742

Whole Genome Complete Resequencing of Bacillus subtilis Natto by Combining Long Reads with High-Quality Short Reads

PubMed Central

Kamada, Mayumi; Hase, Sumitaka; Sato, Kengo; Toyoda, Atsushi; Fujiyama, Asao; Sakakibara, Yasubumi

2014-01-01

De novo microbial genome sequencing reached a turning point with third-generation sequencing (TGS) platforms, and several microbial genomes have been improved by TGS long reads. Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and it has a function in the production of the traditional Japanese fermented food “natto.” The B. subtilis natto BEST195 genome was previously sequenced with short reads, but it included some incomplete regions. We resequenced the BEST195 genome using a PacBio RS sequencer, and we successfully obtained a complete genome sequence from one scaffold without any gaps, and we also applied Illumina MiSeq short reads to enhance quality. Compared with the previous BEST195 draft genome and Marburg 168 genome, we found that incomplete regions in the previous genome sequence were attributed to GC-bias and repetitive sequences, and we also identified some novel genes that are found only in the new genome. PMID:25329997

Sequencing intractable DNA to close microbial genomes.

PubMed

Hurt, Richard A; Brown, Steven D; Podar, Mircea; Palumbo, Anthony V; Elias, Dwayne A

2012-01-01

Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

Making sense of deep sequencing

PubMed Central

Goldman, D.; Domschke, K.

2016-01-01

This review, the first of an occasional series, tries to make sense of the concepts and uses of deep sequencing of polynucleic acids (DNA and RNA). Deep sequencing, synonymous with next-generation sequencing, high-throughput sequencing and massively parallel sequencing, includes whole genome sequencing but is more often and diversely applied to specific parts of the genome captured in different ways, for example the highly expressed portion of the genome known as the exome and portions of the genome that are epigenetically marked either by DNA methylation, the binding of proteins including histones, or that are in different configurations and thus more or less accessible to enzymes that cleave DNA. Deep sequencing of RNA (RNASeq) reverse-transcribed to complementary DNA is invaluable for measuring RNA expression and detecting changes in RNA structure. Important concepts in deep sequencing include the length and depth of sequence reads, mapping and assembly of reads, sequencing error, haplotypes, and the propensity of deep sequencing, as with other types of ‘big data’, to generate large numbers of errors, requiring monitoring for methodologic biases and strategies for replication and validation. Deep sequencing yields a unique genetic fingerprint that can be used to identify a person, and a trove of predictors of genetic medical diseases. Deep sequencing to identify epigenetic events including changes in DNA methylation and RNA expression can reveal the history and impact of environmental exposures. Because of the power of sequencing to identify and deliver biomedically significant information about a person and their blood relatives, it creates ethical dilemmas and practical challenges in research and clinical care, for example the decision and procedures to report incidental findings that will increasingly and frequently be discovered. PMID:24925306

First complete genome sequence of infectious laryngotracheitis virus

PubMed Central

2011-01-01

Background Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. To date, only one complete genomic sequence of ILTV has been reported. This sequence was generated by concatenating partial sequences from six different ILTV strains. Thus, the full genomic sequence of a single (individual) strain of ILTV has not been determined previously. This study aimed to use high throughput sequencing technology to determine the complete genomic sequence of a live attenuated vaccine strain of ILTV. Results The complete genomic sequence of the Serva vaccine strain of ILTV was determined, annotated and compared to the concatenated ILTV reference sequence. The genome size of the Serva strain was 152,628 bp, with a G + C content of 48%. A total of 80 predicted open reading frames were identified. The Serva strain had 96.5% DNA sequence identity with the concatenated ILTV sequence. Notably, the concatenated ILTV sequence was found to lack four large regions of sequence, including 528 bp and 594 bp of sequence in the UL29 and UL36 genes, respectively, and two copies of a 1,563 bp sequence in the repeat regions. Considerable differences in the size of the predicted translation products of 4 other genes (UL54, UL30, UL37 and UL38) were also identified. More than 530 single-nucleotide polymorphisms (SNPs) were identified. Most SNPs were located within three genomic regions, corresponding to sequence from the SA-2 ILTV vaccine strain in the concatenated ILTV sequence. Conclusions This is the first complete genomic sequence of an individual ILTV strain. This sequence will facilitate future comparative genomic studies of ILTV by providing an appropriate reference sequence for the sequence analysis of other ILTV strains. PMID:21501528

One Size Doesn't Fit All - RefEditor: Building Personalized Diploid Reference Genome to Improve Read Mapping and Genotype Calling in Next Generation Sequencing Studies

PubMed Central

Yuan, Shuai; Johnston, H. Richard; Zhang, Guosheng; Li, Yun; Hu, Yi-Juan; Qin, Zhaohui S.

2015-01-01

With rapid decline of the sequencing cost, researchers today rush to embrace whole genome sequencing (WGS), or whole exome sequencing (WES) approach as the next powerful tool for relating genetic variants to human diseases and phenotypes. A fundamental step in analyzing WGS and WES data is mapping short sequencing reads back to the reference genome. This is an important issue because incorrectly mapped reads affect the downstream variant discovery, genotype calling and association analysis. Although many read mapping algorithms have been developed, the majority of them uses the universal reference genome and do not take sequence variants into consideration. Given that genetic variants are ubiquitous, it is highly desirable if they can be factored into the read mapping procedure. In this work, we developed a novel strategy that utilizes genotypes obtained a priori to customize the universal haploid reference genome into a personalized diploid reference genome. The new strategy is implemented in a program named RefEditor. When applying RefEditor to real data, we achieved encouraging improvements in read mapping, variant discovery and genotype calling. Compared to standard approaches, RefEditor can significantly increase genotype calling consistency (from 43% to 61% at 4X coverage; from 82% to 92% at 20X coverage) and reduce Mendelian inconsistency across various sequencing depths. Because many WGS and WES studies are conducted on cohorts that have been genotyped using array-based genotyping platforms previously or concurrently, we believe the proposed strategy will be of high value in practice, which can also be applied to the scenario where multiple NGS experiments are conducted on the same cohort. The RefEditor sources are available at https://github.com/superyuan/refeditor. PMID:26267278

Insights from 20 years of bacterial genome sequencing

DOE PAGES

Land, Miriam L.; Hauser, Loren; Jun, Se-Ran; ...

2015-02-27

Since the first two complete bacterial genome sequences were published in 1995, the science of bacteria has dramatically changed. Using third-generation DNA sequencing, it is possible to completely sequence a bacterial genome in a few hours and identify some types of methylation sites along the genome as well. Sequencing of bacterial genome sequences is now a standard procedure, and the information from tens of thousands of bacterial genomes has had a major impact on our views of the bacterial world. In this review, we explore a series of questions to highlight some insights that comparative genomics has produced. To date,more » there are genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. However, the distribution is quite skewed towards a few phyla that contain model organisms. But the breadth is continuing to improve, with projects dedicated to filling in less characterized taxonomic groups. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides bacteria with immunity against viruses, which outnumber bacteria by tenfold. How fast can we go? Second-generation sequencing has produced a large number of draft genomes (close to 90 % of bacterial genomes in GenBank are currently not complete); third-generation sequencing can potentially produce a finished genome in a few hours, and at the same time provide methlylation sites along the entire chromosome. The diversity of bacterial communities is extensive as is evident from the genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. Genome sequencing can help in classifying an organism, and in the case where multiple genomes of the same species are available, it is possible to calculate the pan- and core genomes; comparison of more than 2000 Escherichia coli genomes finds an E. coli core genome of about 3100 gene families and a total of about 89,000 different gene families. Why do we care about bacterial genome sequencing? There are many practical applications, such as genome-scale metabolic modeling, biosurveillance, bioforensics, and infectious disease epidemiology. In the near future, high-throughput sequencing of patient metagenomic samples could revolutionize medicine in terms of speed and accuracy of finding pathogens and knowing how to treat them.« less

Insights from 20 years of bacterial genome sequencing

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

Land, Miriam L.; Hauser, Loren; Jun, Se-Ran

Since the first two complete bacterial genome sequences were published in 1995, the science of bacteria has dramatically changed. Using third-generation DNA sequencing, it is possible to completely sequence a bacterial genome in a few hours and identify some types of methylation sites along the genome as well. Sequencing of bacterial genome sequences is now a standard procedure, and the information from tens of thousands of bacterial genomes has had a major impact on our views of the bacterial world. In this review, we explore a series of questions to highlight some insights that comparative genomics has produced. To date,more » there are genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. However, the distribution is quite skewed towards a few phyla that contain model organisms. But the breadth is continuing to improve, with projects dedicated to filling in less characterized taxonomic groups. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides bacteria with immunity against viruses, which outnumber bacteria by tenfold. How fast can we go? Second-generation sequencing has produced a large number of draft genomes (close to 90 % of bacterial genomes in GenBank are currently not complete); third-generation sequencing can potentially produce a finished genome in a few hours, and at the same time provide methlylation sites along the entire chromosome. The diversity of bacterial communities is extensive as is evident from the genome sequences available from 50 different bacterial phyla and 11 different archaeal phyla. Genome sequencing can help in classifying an organism, and in the case where multiple genomes of the same species are available, it is possible to calculate the pan- and core genomes; comparison of more than 2000 Escherichia coli genomes finds an E. coli core genome of about 3100 gene families and a total of about 89,000 different gene families. Why do we care about bacterial genome sequencing? There are many practical applications, such as genome-scale metabolic modeling, biosurveillance, bioforensics, and infectious disease epidemiology. In the near future, high-throughput sequencing of patient metagenomic samples could revolutionize medicine in terms of speed and accuracy of finding pathogens and knowing how to treat them.« less

GenomeRNAi: a database for cell-based RNAi phenotypes.

PubMed

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

GenomeRNAi: a database for cell-based RNAi phenotypes

PubMed Central

Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

2007-01-01

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at PMID:17135194

Survey and analysis of simple sequence repeats in the Laccaria bicolor genome, with development of microsatellite markers

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

Labbe, Jessy L; Murat, Claude; Morin, Emmanuelle

It is becoming clear that simple sequence repeats (SSRs) play a significant role in fungal genome organization, and they are a large source of genetic markers for population genetics and meiotic maps. We identified SSRs in the Laccaria bicolor genome by in silico survey and analyzed their distribution in the different genomic regions. We also compared the abundance and distribution of SSRs in L. bicolor with those of the following fungal genomes: Phanerochaete chrysosporium, Coprinopsis cinerea, Ustilago maydis, Cryptococcus neoformans, Aspergillus nidulans, Magnaporthe grisea, Neurospora crassa and Saccharomyces cerevisiae. Using the MISA computer program, we detected 277,062 SSRs in themore » L. bicolor genome representing 8% of the assembled genomic sequence. Among the analyzed basidiomycetes, L. bicolor exhibited the highest SSR density although no correlation between relative abundance and the genome sizes was observed. In most genomes the short motifs (mono- to trinucleotides) were more abundant than the longer repeated SSRs. Generally, in each organism, the occurrence, relative abundance, and relative density of SSRs decreased as the repeat unit increased. Furthermore, each organism had its own common and longest SSRs. In the L. bicolor genome, most of the SSRs were located in intergenic regions (73.3%) and the highest SSR density was observed in transposable elements (TEs; 6,706 SSRs/Mb). However, 81% of the protein-coding genes contained SSRs in their exons, suggesting that SSR polymorphism may alter gene phenotypes. Within a L. bicolor offspring, sequence polymorphism of 78 SSRs was mainly detected in non-TE intergenic regions. Unlike previously developed microsatellite markers, these new ones are spread throughout the genome; these markers could have immediate applications in population genetics.« less

Gene calling and bacterial genome annotation with BG7.

PubMed

Tobes, Raquel; Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Kovach, Evdokim; Alekhin, Alexey; Pareja, Eduardo

2015-01-01

New massive sequencing technologies are providing many bacterial genome sequences from diverse taxa but a refined annotation of these genomes is crucial for obtaining scientific findings and new knowledge. Thus, bacterial genome annotation has emerged as a key point to investigate in bacteria. Any efficient tool designed specifically to annotate bacterial genomes sequenced with massively parallel technologies has to consider the specific features of bacterial genomes (absence of introns and scarcity of nonprotein-coding sequence) and of next-generation sequencing (NGS) technologies (presence of errors and not perfectly assembled genomes). These features make it convenient to focus on coding regions and, hence, on protein sequences that are the elements directly related with biological functions. In this chapter we describe how to annotate bacterial genomes with BG7, an open-source tool based on a protein-centered gene calling/annotation paradigm. BG7 is specifically designed for the annotation of bacterial genomes sequenced with NGS. This tool is sequence error tolerant maintaining their capabilities for the annotation of highly fragmented genomes or for annotating mixed sequences coming from several genomes (as those obtained through metagenomics samples). BG7 has been designed with scalability as a requirement, with a computing infrastructure completely based on cloud computing (Amazon Web Services).

Quantifying the Number of Independent Organelle DNA Insertions in Genome Evolution and Human Health

PubMed Central

Martin, William F.

2017-01-01

Fragments of organelle genomes are often found as insertions in nuclear DNA. These fragments of mitochondrial DNA (numts) and plastid DNA (nupts) are ubiquitous components of eukaryotic genomes. They are, however, often edited out during the genome assembly process, leading to systematic underestimation of their frequency. Numts and nupts, once inserted, can become further fragmented through subsequent insertion of mobile elements or other recombinational events that disrupt the continuity of the inserted sequence relative to the genuine organelle DNA copy. Because numts and nupts are typically identified through sequence comparison tools such as BLAST, disruption of insertions into smaller fragments can lead to systematic overestimation of numt and nupt frequencies. Accurate identification of numts and nupts is important, however, both for better understanding of their role during evolution, and for monitoring their increasingly evident role in human disease. Human populations are polymorphic for 141 numt loci, five numts are causal to genetic disease, and cancer genomic studies are revealing an abundance of numts associated with tumor progression. Here, we report investigation of salient parameters involved in obtaining accurate estimates of numt and nupt numbers in genome sequence data. Numts and nupts from 44 sequenced eukaryotic genomes reveal lineage-specific differences in the number, relative age and frequency of insertional events as well as lineage-specific dynamics of their postinsertional fragmentation. Our findings outline the main technical parameters influencing accurate identification and frequency estimation of numts in genomic studies pertinent to both evolution and human health. PMID:28444372

Genomics-assisted breeding for boosting crop improvement in pigeonpea (Cajanus cajan)

PubMed Central

Pazhamala, Lekha; Saxena, Rachit K.; Singh, Vikas K.; Sameerkumar, C. V.; Kumar, Vinay; Sinha, Pallavi; Patel, Kishan; Obala, Jimmy; Kaoneka, Seleman R.; Tongoona, P.; Shimelis, Hussein A.; Gangarao, N. V. P. R.; Odeny, Damaris; Rathore, Abhishek; Dharmaraj, P. S.; Yamini, K. N.; Varshney, Rajeev K.

2015-01-01

Pigeonpea is an important pulse crop grown predominantly in the tropical and sub-tropical regions of the world. Although pigeonpea growing area has considerably increased, yield has remained stagnant for the last six decades mainly due to the exposure of the crop to various biotic and abiotic constraints. In addition, low level of genetic variability and limited genomic resources have been serious impediments to pigeonpea crop improvement through modern breeding approaches. In recent years, however, due to the availability of next generation sequencing and high-throughput genotyping technologies, the scenario has changed tremendously. The reduced sequencing costs resulting in the decoding of the pigeonpea genome has led to the development of various genomic resources including molecular markers, transcript sequences and comprehensive genetic maps. Mapping of some important traits including resistance to Fusarium wilt and sterility mosaic disease, fertility restoration, determinacy with other agronomically important traits have paved the way for applying genomics-assisted breeding (GAB) through marker assisted selection as well as genomic selection (GS). This would accelerate the development and improvement of both varieties and hybrids in pigeonpea. Particularly for hybrid breeding programme, mitochondrial genomes of cytoplasmic male sterile (CMS) lines, maintainers and hybrids have been sequenced to identify genes responsible for cytoplasmic male sterility. Furthermore, several diagnostic molecular markers have been developed to assess the purity of commercial hybrids. In summary, pigeonpea has become a genomic resources-rich crop and efforts have already been initiated to integrate these resources in pigeonpea breeding. PMID:25741349

GWAS and fine-mapping of 35 production, reproduction, and conformation traits with imputed sequences of 27K Holstein bulls

USDA-ARS?s Scientific Manuscript database

Imputation has been routinely applied to ascertain sequence variants in large genotyped populations based on reference populations of sequenced animals. With the implementation of the 1000 Bull Genomes Project and increasing numbers of animals sequenced, fine-mapping of causal variants is becoming f...

Complete sequence and comparative analysis of the chloroplast genome of Plinia trunciflora

PubMed Central

Eguiluz, Maria; Yuyama, Priscila Mary; Guzman, Frank; Rodrigues, Nureyev Ferreira; Margis, Rogerio

2017-01-01

Abstract Plinia trunciflora is a Brazilian native fruit tree from the Myrtaceae family, also known as jaboticaba. This species has great potential by its fruit production. Due to the high content of essential oils in their leaves and of anthocyanins in the fruits, there is also an increasing interest by the pharmaceutical industry. Nevertheless, there are few studies focusing on its molecular biology and genetic characterization. We herein report the complete chloroplast (cp) genome of P. trunciflora using high-throughput sequencing and compare it to other previously sequenced Myrtaceae genomes. The cp genome of P. trunciflora is 159,512 bp in size, comprising inverted repeats of 26,414 bp and single-copy regions of 88,097 bp (LSC) and 18,587 bp (SSC). The genome contains 111 single-copy genes (77 protein-coding, 30 tRNA and four rRNA genes). Phylogenetic analysis using 57 cp protein-coding genes demonstrated that P. trunciflora, Eugenia uniflora and Acca sellowiana form a cluster with closer relationship to Syzygium cumini than with Eucalyptus. The complete cp sequence reported here can be used in evolutionary and population genetics studies, contributing to resolve the complex taxonomy of this species and fill the gap in genetic characterization. PMID:29111566

LTR Retrotransposons Contribute to Genomic Gigantism in Plethodontid Salamanders

PubMed Central

Sun, Cheng; Shepard, Donald B.; Chong, Rebecca A.; López Arriaza, José; Hall, Kathryn; Castoe, Todd A.; Feschotte, Cédric; Pollock, David D.; Mueller, Rachel Lockridge

2012-01-01

Among vertebrates, most of the largest genomes are found within the salamanders, a clade of amphibians that includes 613 species. Salamander genome sizes range from ∼14 to ∼120 Gb. Because genome size is correlated with nucleus and cell sizes, as well as other traits, morphological evolution in salamanders has been profoundly affected by genomic gigantism. However, the molecular mechanisms driving genomic expansion in this clade remain largely unknown. Here, we present the first comparative analysis of transposable element (TE) content in salamanders. Using high-throughput sequencing, we generated genomic shotgun data for six species from the Plethodontidae, the largest family of salamanders. We then developed a pipeline to mine TE sequences from shotgun data in taxa with limited genomic resources, such as salamanders. Our summaries of overall TE abundance and diversity for each species demonstrate that TEs make up a substantial portion of salamander genomes, and that all of the major known types of TEs are represented in salamanders. The most abundant TE superfamilies found in the genomes of our six focal species are similar, despite substantial variation in genome size. However, our results demonstrate a major difference between salamanders and other vertebrates: salamander genomes contain much larger amounts of long terminal repeat (LTR) retrotransposons, primarily Ty3/gypsy elements. Thus, the extreme increase in genome size that occurred in salamanders was likely accompanied by a shift in TE landscape. These results suggest that increased proliferation of LTR retrotransposons was a major molecular mechanism contributing to genomic expansion in salamanders. PMID:22200636

Toward allotetraploid cotton genome assembly: integration of a high-density molecular genetic linkage map with DNA sequence information

PubMed Central

2012-01-01

Background Cotton is the world’s most important natural textile fiber and a significant oilseed crop. Decoding cotton genomes will provide the ultimate reference and resource for research and utilization of the species. Integration of high-density genetic maps with genomic sequence information will largely accelerate the process of whole-genome assembly in cotton. Results In this paper, we update a high-density interspecific genetic linkage map of allotetraploid cultivated cotton. An additional 1,167 marker loci have been added to our previously published map of 2,247 loci. Three new marker types, InDel (insertion-deletion) and SNP (single nucleotide polymorphism) developed from gene information, and REMAP (retrotransposon-microsatellite amplified polymorphism), were used to increase map density. The updated map consists of 3,414 loci in 26 linkage groups covering 3,667.62 cM with an average inter-locus distance of 1.08 cM. Furthermore, genome-wide sequence analysis was finished using 3,324 informative sequence-based markers and publicly-available Gossypium DNA sequence information. A total of 413,113 EST and 195 BAC sequences were physically anchored and clustered by 3,324 sequence-based markers. Of these, 14,243 ESTs and 188 BACs from different species of Gossypium were clustered and specifically anchored to the high-density genetic map. A total of 2,748 candidate unigenes from 2,111 ESTs clusters and 63 BACs were mined for functional annotation and classification. The 337 ESTs/genes related to fiber quality traits were integrated with 132 previously reported cotton fiber quality quantitative trait loci, which demonstrated the important roles in fiber quality of these genes. Higher-level sequence conservation between different cotton species and between the A- and D-subgenomes in tetraploid cotton was found, indicating a common evolutionary origin for orthologous and paralogous loci in Gossypium. Conclusion This study will serve as a valuable genomic resource for tetraploid cotton genome assembly, for cloning genes related to superior agronomic traits, and for further comparative genomic analyses in Gossypium. PMID:23046547

PCR Amplification Strategies towards full-length HIV-1 Genome sequencing.

PubMed

Liu, Chao Chun; Ji, Hezhao

2018-06-26

The advent of next generation sequencing has enabled greater resolution of viral diversity and improved feasibility of full viral genome sequencing allowing routine HIV-1 full genome sequencing in both research and diagnostic settings. Regardless of the sequencing platform selected, successful PCR amplification of the HIV-1 genome is essential for sequencing template preparation. As such, full HIV-1 genome amplification is a crucial step in dictating the successful and reliable sequencing downstream. Here we reviewed existing PCR protocols leading to HIV-1 full genome sequencing. In addition to the discussion on basic considerations on relevant PCR design, the advantages as well as the pitfalls of published protocols were reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Reverse Vaccinology: Developing Vaccines in the Era of Genomics

PubMed Central

Sette, Alessandro; Rappuoli, Rino

2012-01-01

The sequence of microbial genomes made all potential antigens of each pathogen available for vaccine development. This increased by orders of magnitude potential vaccine targets in bacteria, parasites, and large viruses and revealed virtually all their CD4+ and CD8+ T cell epitopes. The genomic information was first used for the development of a vaccine against serogroup B meningococcus, and it is now being used for several other bacterial vaccines. In this review, we will first summarize the impact that genome sequencing has had on vaccine development, and then we will analyze how the genomic information can help further our understanding of immunity to infection or vaccination and lead to the design of better vaccines by diving into the world of T cell immunity. PMID:21029963

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.

Choice of Reference Sequence and Assembler for Alignment of Listeria monocytogenes Short-Read Sequence Data Greatly Influences Rates of Error in SNP Analyses

PubMed Central

Pightling, Arthur W.; Petronella, Nicholas; Pagotto, Franco

2014-01-01

The wide availability of whole-genome sequencing (WGS) and an abundance of open-source software have made detection of single-nucleotide polymorphisms (SNPs) in bacterial genomes an increasingly accessible and effective tool for comparative analyses. Thus, ensuring that real nucleotide differences between genomes (i.e., true SNPs) are detected at high rates and that the influences of errors (such as false positive SNPs, ambiguously called sites, and gaps) are mitigated is of utmost importance. The choices researchers make regarding the generation and analysis of WGS data can greatly influence the accuracy of short-read sequence alignments and, therefore, the efficacy of such experiments. We studied the effects of some of these choices, including: i) depth of sequencing coverage, ii) choice of reference-guided short-read sequence assembler, iii) choice of reference genome, and iv) whether to perform read-quality filtering and trimming, on our ability to detect true SNPs and on the frequencies of errors. We performed benchmarking experiments, during which we assembled simulated and real Listeria monocytogenes strain 08-5578 short-read sequence datasets of varying quality with four commonly used assemblers (BWA, MOSAIK, Novoalign, and SMALT), using reference genomes of varying genetic distances, and with or without read pre-processing (i.e., quality filtering and trimming). We found that assemblies of at least 50-fold coverage provided the most accurate results. In addition, MOSAIK yielded the fewest errors when reads were aligned to a nearly identical reference genome, while using SMALT to align reads against a reference sequence that is ∼0.82% distant from 08-5578 at the nucleotide level resulted in the detection of the greatest numbers of true SNPs and the fewest errors. Finally, we show that whether read pre-processing improves SNP detection depends upon the choice of reference sequence and assembler. In total, this study demonstrates that researchers should test a variety of conditions to achieve optimal results. PMID:25144537

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs.

PubMed

Yang, Jun-Bo; Li, De-Zhu; Li, Hong-Tao

2014-09-01

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle-scale barcodes. Next-generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high-quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long-range PCR and sequenced using next-generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early-diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome-scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms. © 2014 John Wiley & Sons Ltd.

Enhanced Methods for Local Ancestry Assignment in Sequenced Admixed Individuals

PubMed Central

Brown, Robert; Pasaniuc, Bogdan

2014-01-01

Inferring the ancestry at each locus in the genome of recently admixed individuals (e.g., Latino Americans) plays a major role in medical and population genetic inferences, ranging from finding disease-risk loci, to inferring recombination rates, to mapping missing contigs in the human genome. Although many methods for local ancestry inference have been proposed, most are designed for use with genotyping arrays and fail to make use of the full spectrum of data available from sequencing. In addition, current haplotype-based approaches are very computationally demanding, requiring large computational time for moderately large sample sizes. Here we present new methods for local ancestry inference that leverage continent-specific variants (CSVs) to attain increased performance over existing approaches in sequenced admixed genomes. A key feature of our approach is that it incorporates the admixed genomes themselves jointly with public datasets, such as 1000 Genomes, to improve the accuracy of CSV calling. We use simulations to show that our approach attains accuracy similar to widely used computationally intensive haplotype-based approaches with large decreases in runtime. Most importantly, we show that our method recovers comparable local ancestries, as the 1000 Genomes consensus local ancestry calls in the real admixed individuals from the 1000 Genomes Project. We extend our approach to account for low-coverage sequencing and show that accurate local ancestry inference can be attained at low sequencing coverage. Finally, we generalize CSVs to sub-continental population-specific variants (sCSVs) and show that in some cases it is possible to determine the sub-continental ancestry for short chromosomal segments on the basis of sCSVs. PMID:24743331

Sequencing and assembly of the 22-gb loblolly pine genome.

PubMed

Zimin, Aleksey; Stevens, Kristian A; Crepeau, Marc W; Holtz-Morris, Ann; Koriabine, Maxim; Marçais, Guillaume; Puiu, Daniela; Roberts, Michael; Wegrzyn, Jill L; de Jong, Pieter J; Neale, David B; Salzberg, Steven L; Yorke, James A; Langley, Charles H

2014-03-01

Conifers are the predominant gymnosperm. The size and complexity of their genomes has presented formidable technical challenges for whole-genome shotgun sequencing and assembly. We employed novel strategies that allowed us to determine the loblolly pine (Pinus taeda) reference genome sequence, the largest genome assembled to date. Most of the sequence data were derived from whole-genome shotgun sequencing of a single megagametophyte, the haploid tissue of a single pine seed. Although that constrained the quantity of available DNA, the resulting haploid sequence data were well-suited for assembly. The haploid sequence was augmented with multiple linking long-fragment mate pair libraries from the parental diploid DNA. For the longest fragments, we used novel fosmid DiTag libraries. Sequences from the linking libraries that did not match the megagametophyte were identified and removed. Assembly of the sequence data were aided by condensing the enormous number of paired-end reads into a much smaller set of longer "super-reads," rendering subsequent assembly with an overlap-based assembly algorithm computationally feasible. To further improve the contiguity and biological utility of the genome sequence, additional scaffolding methods utilizing independent genome and transcriptome assemblies were implemented. The combination of these strategies resulted in a draft genome sequence of 20.15 billion bases, with an N50 scaffold size of 66.9 kbp.

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum.

PubMed

VanBuren, Robert; Bryant, Doug; Edger, Patrick P; Tang, Haibao; Burgess, Diane; Challabathula, Dinakar; Spittle, Kristi; Hall, Richard; Gu, Jenny; Lyons, Eric; Freeling, Michael; Bartels, Dorothea; Ten Hallers, Boudewijn; Hastie, Alex; Michael, Todd P; Mockler, Todd C

2015-11-26

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.

The first genome sequences of human bocaviruses from Vietnam

PubMed Central

Thanh, Tran Tan; Van, Hoang Minh Tu; Hong, Nguyen Thi Thu; Nhu, Le Nguyen Truc; Anh, Nguyen To; Tuan, Ha Manh; Hien, Ho Van; Tuong, Nguyen Manh; Kien, Trinh Trung; Khanh, Truong Huu; Nhan, Le Nguyen Thanh; Hung, Nguyen Thanh; Chau, Nguyen Van Vinh; Thwaites, Guy; van Doorn, H. Rogier; Tan, Le Van

2017-01-01

As part of an ongoing effort to generate complete genome sequences of hand, foot and mouth disease-causing enteroviruses directly from clinical specimens, two complete coding sequences and two partial genomic sequences of human bocavirus 1 (n=3) and 2 (n=1) were co-amplified and sequenced, representing the first genome sequences of human bocaviruses from Vietnam. The sequences may aid future study aiming at understanding the evolution of the virus. PMID:28090592

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PubMed

Nakano, Kazuma; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Minami, Maiko; Nakanishi, Tetsuhiro; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

2017-07-01

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II's sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

Genomic Enzymology: Web Tools for Leveraging Protein Family Sequence–Function Space and Genome Context to Discover Novel Functions

PubMed Central

2017-01-01

The exponentially increasing number of protein and nucleic acid sequences provides opportunities to discover novel enzymes, metabolic pathways, and metabolites/natural products, thereby adding to our knowledge of biochemistry and biology. The challenge has evolved from generating sequence information to mining the databases to integrating and leveraging the available information, i.e., the availability of “genomic enzymology” web tools. Web tools that allow identification of biosynthetic gene clusters are widely used by the natural products/synthetic biology community, thereby facilitating the discovery of novel natural products and the enzymes responsible for their biosynthesis. However, many novel enzymes with interesting mechanisms participate in uncharacterized small-molecule metabolic pathways; their discovery and functional characterization also can be accomplished by leveraging information in protein and nucleic acid databases. This Perspective focuses on two genomic enzymology web tools that assist the discovery novel metabolic pathways: (1) Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) for generating sequence similarity networks to visualize and analyze sequence–function space in protein families and (2) Enzyme Function Initiative-Genome Neighborhood Tool (EFI-GNT) for generating genome neighborhood networks to visualize and analyze the genome context in microbial and fungal genomes. Both tools have been adapted to other applications to facilitate target selection for enzyme discovery and functional characterization. As the natural products community has demonstrated, the enzymology community needs to embrace the essential role of web tools that allow the protein and genome sequence databases to be leveraged for novel insights into enzymological problems. PMID:28826221

Microbes, metagenomes and marine mammals: enabling the next generation of scientist to enter the genomic era

PubMed Central

2013-01-01

Background The revolution in DNA sequencing technology continues unabated, and is affecting all aspects of the biological and medical sciences. The training and recruitment of the next generation of researchers who are able to use and exploit the new technology is severely lacking and potentially negatively influencing research and development efforts to advance genome biology. Here we present a cross-disciplinary course that provides undergraduate students with practical experience in running a next generation sequencing instrument through to the analysis and annotation of the generated DNA sequences. Results Many labs across world are installing next generation sequencing technology and we show that the undergraduate students produce quality sequence data and were excited to participate in cutting edge research. The students conducted the work flow from DNA extraction, library preparation, running the sequencing instrument, to the extraction and analysis of the data. They sequenced microbes, metagenomes, and a marine mammal, the Californian sea lion, Zalophus californianus. The students met sequencing quality controls, had no detectable contamination in the targeted DNA sequences, provided publication quality data, and became part of an international collaboration to investigate carcinomas in carnivores. Conclusions Students learned important skills for their future education and career opportunities, and a perceived increase in students’ ability to conduct independent scientific research was measured. DNA sequencing is rapidly expanding in the life sciences. Teaching undergraduates to use the latest technology to sequence genomic DNA ensures they are ready to meet the challenges of the genomic era and allows them to participate in annotating the tree of life. PMID:24007365

The Complete Mitochondrial Genome of Gossypium hirsutum and Evolutionary Analysis of Higher Plant Mitochondrial Genomes

PubMed Central

Su, Aiguo; Geng, Jianing; Grover, Corrinne E.; Hu, Songnian; Hua, Jinping

2013-01-01

Background Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes. Methodology/Principal Findings We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes. Conclusion The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species. PMID:23940520

The complete mitochondrial genome of Gossypium hirsutum and evolutionary analysis of higher plant mitochondrial genomes.

PubMed

Liu, Guozheng; Cao, Dandan; Li, Shuangshuang; Su, Aiguo; Geng, Jianing; Grover, Corrinne E; Hu, Songnian; Hua, Jinping

2013-01-01

Mitochondria are the main manufacturers of cellular ATP in eukaryotes. The plant mitochondrial genome contains large number of foreign DNA and repeated sequences undergone frequently intramolecular recombination. Upland Cotton (Gossypium hirsutum L.) is one of the main natural fiber crops and also an important oil-producing plant in the world. Sequencing of the cotton mitochondrial (mt) genome could be helpful for the evolution research of plant mt genomes. We utilized 454 technology for sequencing and combined with Fosmid library of the Gossypium hirsutum mt genome screening and positive clones sequencing and conducted a series of evolutionary analysis on Cycas taitungensis and 24 angiosperms mt genomes. After data assembling and contigs joining, the complete mitochondrial genome sequence of G. hirsutum was obtained. The completed G.hirsutum mt genome is 621,884 bp in length, and contained 68 genes, including 35 protein genes, four rRNA genes and 29 tRNA genes. Five gene clusters are found conserved in all plant mt genomes; one and four clusters are specifically conserved in monocots and dicots, respectively. Homologous sequences are distributed along the plant mt genomes and species closely related share the most homologous sequences. For species that have both mt and chloroplast genome sequences available, we checked the location of cp-like migration and found several fragments closely linked with mitochondrial genes. The G. hirsutum mt genome possesses most of the common characters of higher plant mt genomes. The existence of syntenic gene clusters, as well as the conservation of some intergenic sequences and genic content among the plant mt genomes suggest that evolution of mt genomes is consistent with plant taxonomy but independent among different species.

Whole-genome sequence-based analysis of thyroid function.

PubMed

Taylor, Peter N; Porcu, Eleonora; Chew, Shelby; Campbell, Purdey J; Traglia, Michela; Brown, Suzanne J; Mullin, Benjamin H; Shihab, Hashem A; Min, Josine; Walter, Klaudia; Memari, Yasin; Huang, Jie; Barnes, Michael R; Beilby, John P; Charoen, Pimphen; Danecek, Petr; Dudbridge, Frank; Forgetta, Vincenzo; Greenwood, Celia; Grundberg, Elin; Johnson, Andrew D; Hui, Jennie; Lim, Ee M; McCarthy, Shane; Muddyman, Dawn; Panicker, Vijay; Perry, John R B; Bell, Jordana T; Yuan, Wei; Relton, Caroline; Gaunt, Tom; Schlessinger, David; Abecasis, Goncalo; Cucca, Francesco; Surdulescu, Gabriela L; Woltersdorf, Wolfram; Zeggini, Eleftheria; Zheng, Hou-Feng; Toniolo, Daniela; Dayan, Colin M; Naitza, Silvia; Walsh, John P; Spector, Tim; Davey Smith, George; Durbin, Richard; Richards, J Brent; Sanna, Serena; Soranzo, Nicole; Timpson, Nicholas J; Wilson, Scott G

2015-03-06

Normal thyroid function is essential for health, but its genetic architecture remains poorly understood. Here, for the heritable thyroid traits thyrotropin (TSH) and free thyroxine (FT4), we analyse whole-genome sequence data from the UK10K project (N=2,287). Using additional whole-genome sequence and deeply imputed data sets, we report meta-analysis results for common variants (MAF≥1%) associated with TSH and FT4 (N=16,335). For TSH, we identify a novel variant in SYN2 (MAF=23.5%, P=6.15 × 10(-9)) and a new independent variant in PDE8B (MAF=10.4%, P=5.94 × 10(-14)). For FT4, we report a low-frequency variant near B4GALT6/SLC25A52 (MAF=3.2%, P=1.27 × 10(-9)) tagging a rare TTR variant (MAF=0.4%, P=2.14 × 10(-11)). All common variants explain ≥20% of the variance in TSH and FT4. Analysis of rare variants (MAF<1%) using sequence kernel association testing reveals a novel association with FT4 in NRG1. Our results demonstrate that increased coverage in whole-genome sequence association studies identifies novel variants associated with thyroid function.

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

Non-invasive prenatal testing using massively parallel sequencing of maternal plasma DNA: from molecular karyotyping to fetal whole-genome sequencing.

PubMed

Lo, Y M Dennis

2013-12-01

The discovery of cell-free fetal DNA in maternal plasma in 1997 has stimulated a rapid development of non-invasive prenatal testing. The recent advent of massively parallel sequencing has allowed the analysis of circulating cell-free fetal DNA to be performed with unprecedented sensitivity and precision. Fetal trisomies 21, 18 and 13 are now robustly detectable in maternal plasma and such analyses have been available clinically since 2011. Fetal genome-wide molecular karyotyping and whole-genome sequencing have now been demonstrated in a number of proof-of-concept studies. Genome-wide and targeted sequencing of maternal plasma has been shown to allow the non-invasive prenatal testing of β-thalassaemia and can potentially be generalized to other monogenic diseases. It is thus expected that plasma DNA-based non-invasive prenatal testing will play an increasingly important role in future obstetric care. It is thus timely and important that the ethical, social and legal issues of non-invasive prenatal testing be discussed actively by all parties involved in prenatal care. Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

The Pinus taeda genome is characterized by diverse and highly diverged repetitive sequences

PubMed Central

2010-01-01

Background In today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24). The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda. Results We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS) sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (≥ 75% nucleotide identity) elsewhere in the genome, but only 23% have identical copies (99% identity). The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. Conclusions This study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is a feasible goal. PMID:20609256

Illuminating the Black Box of Genome Sequence Assembly: A Free Online Tool to Introduce Students to Bioinformatics

ERIC Educational Resources Information Center

Taylor, D. Leland; Campbell, A. Malcolm; Heyer, Laurie J.

2013-01-01

Next-generation sequencing technologies have greatly reduced the cost of sequencing genomes. With the current sequencing technology, a genome is broken into fragments and sequenced, producing millions of "reads." A computer algorithm pieces these reads together in the genome assembly process. PHAST is a set of online modules…

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.

Human Genomic Loci Important in Common Infectious Diseases: Role of High-Throughput Sequencing and Genome-Wide Association Studies

PubMed Central

Sserwadda, Ivan; Amujal, Marion; Namatovu, Norah

2018-01-01

HIV/AIDS, tuberculosis (TB), and malaria are 3 major global public health threats that undermine development in many resource-poor settings. Recently, the notion that positive selection during epidemics or longer periods of exposure to common infectious diseases may have had a major effect in modifying the constitution of the human genome is being interrogated at a large scale in many populations around the world. This positive selection from infectious diseases increases power to detect associations in genome-wide association studies (GWASs). High-throughput sequencing (HTS) has transformed both the management of infectious diseases and continues to enable large-scale functional characterization of host resistance/susceptibility alleles and loci; a paradigm shift from single candidate gene studies. Application of genome sequencing technologies and genomics has enabled us to interrogate the host-pathogen interface for improving human health. Human populations are constantly locked in evolutionary arms races with pathogens; therefore, identification of common infectious disease-associated genomic variants/markers is important in therapeutic, vaccine development, and screening susceptible individuals in a population. This review describes a range of host-pathogen genomic loci that have been associated with disease susceptibility and resistant patterns in the era of HTS. We further highlight potential opportunities for these genetic markers. PMID:29755620

Ensembl Genomes: an integrative resource for genome-scale data from non-vertebrate species.

PubMed

Kersey, Paul J; Staines, Daniel M; Lawson, Daniel; Kulesha, Eugene; Derwent, Paul; Humphrey, Jay C; Hughes, Daniel S T; Keenan, Stephan; Kerhornou, Arnaud; Koscielny, Gautier; Langridge, Nicholas; McDowall, Mark D; Megy, Karine; Maheswari, Uma; Nuhn, Michael; Paulini, Michael; Pedro, Helder; Toneva, Iliana; Wilson, Derek; Yates, Andrew; Birney, Ewan

2012-01-01

Ensembl Genomes (http://www.ensemblgenomes.org) is an integrative resource for genome-scale data from non-vertebrate species. The project exploits and extends technology (for genome annotation, analysis and dissemination) developed in the context of the (vertebrate-focused) Ensembl project and provides a complementary set of resources for non-vertebrate species through a consistent set of programmatic and interactive interfaces. These provide access to data including reference sequence, gene models, transcriptional data, polymorphisms and comparative analysis. Since its launch in 2009, Ensembl Genomes has undergone rapid expansion, with the goal of providing coverage of all major experimental organisms, and additionally including taxonomic reference points to provide the evolutionary context in which genes can be understood. Against the backdrop of a continuing increase in genome sequencing activities in all parts of the tree of life, we seek to work, wherever possible, with the communities actively generating and using data, and are participants in a growing range of collaborations involved in the annotation and analysis of genomes.

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

Exome-wide DNA capture and next generation sequencing in domestic and wild species.

PubMed

Cosart, Ted; Beja-Pereira, Albano; Chen, Shanyuan; Ng, Sarah B; Shendure, Jay; Luikart, Gordon

2011-07-05

Gene-targeted and genome-wide markers are crucial to advance evolutionary biology, agriculture, and biodiversity conservation by improving our understanding of genetic processes underlying adaptation and speciation. Unfortunately, for eukaryotic species with large genomes it remains costly to obtain genome sequences and to develop genome resources such as genome-wide SNPs. A method is needed to allow gene-targeted, next-generation sequencing that is flexible enough to include any gene or number of genes, unlike transcriptome sequencing. Such a method would allow sequencing of many individuals, avoiding ascertainment bias in subsequent population genetic analyses.We demonstrate the usefulness of a recent technology, exon capture, for genome-wide, gene-targeted marker discovery in species with no genome resources. We use coding gene sequences from the domestic cow genome sequence (Bos taurus) to capture (enrich for), and subsequently sequence, thousands of exons of B. taurus, B. indicus, and Bison bison (wild bison). Our capture array has probes for 16,131 exons in 2,570 genes, including 203 candidate genes with known function and of interest for their association with disease and other fitness traits. We successfully sequenced and mapped exon sequences from across the 29 autosomes and X chromosome in the B. taurus genome sequence. Exon capture and high-throughput sequencing identified thousands of putative SNPs spread evenly across all reference chromosomes, in all three individuals, including hundreds of SNPs in our targeted candidate genes. This study shows exon capture can be customized for SNP discovery in many individuals and for non-model species without genomic resources. Our captured exome subset was small enough for affordable next-generation sequencing, and successfully captured exons from a divergent wild species using the domestic cow genome as reference.

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.

Mosaic Graphs and Comparative Genomics in Phage Communities

PubMed Central

Belcaid, Mahdi; Bergeron, Anne

2010-01-01

Abstract Comparing the genomes of two closely related viruses often produces mosaics where nearly identical sequences alternate with sequences that are unique to each genome. When several closely related genomes are compared, the unique sequences are likely to be shared with third genomes, leading to virus mosaic communities. Here we present comparative analysis of sets of Staphylococcus aureus phages that share large identical sequences with up to three other genomes, and with different partners along their genomes. We introduce mosaic graphs to represent these complex recombination events, and use them to illustrate the breath and depth of sequence sharing: some genomes are almost completely made up of shared sequences, while genomes that share very large identical sequences can adopt alternate functional modules. Mosaic graphs also allow us to identify breakpoints that could eventually be used for the construction of recombination networks. These findings have several implications on phage metagenomics assembly, on the horizontal gene transfer paradigm, and more generally on the understanding of the composition and evolutionary dynamics of virus communities. PMID:20874413

Evolution of biological complexity

PubMed Central

Adami, Christoph; Ofria, Charles; Collier, Travis C.

2000-01-01

To make a case for or against a trend in the evolution of complexity in biological evolution, complexity needs to be both rigorously defined and measurable. A recent information-theoretic (but intuitively evident) definition identifies genomic complexity with the amount of information a sequence stores about its environment. We investigate the evolution of genomic complexity in populations of digital organisms and monitor in detail the evolutionary transitions that increase complexity. We show that, because natural selection forces genomes to behave as a natural “Maxwell Demon,” within a fixed environment, genomic complexity is forced to increase. PMID:10781045

Structural genomics: keeping up with expanding knowledge of the protein universe.

PubMed

Grabowski, Marek; Joachimiak, Andrzej; Otwinowski, Zbyszek; Minor, Wladek

2007-06-01

Structural characterization of the protein universe is the main mission of Structural Genomics (SG) programs. However, progress in gene sequencing technology, set in motion in the 1990s, has resulted in rapid expansion of protein sequence space--a twelvefold increase in the past seven years. For the SG field, this creates new challenges and necessitates a re-assessment of its strategies. Nevertheless, despite the growth of sequence space, at present nearly half of the content of the Swiss-Prot database and over 40% of Pfam protein families can be structurally modeled based on structures determined so far, with SG projects making an increasingly significant contribution. The SG contribution of new Pfam structures nearly doubled from 27.2% in 2003 to 51.6% in 2006.

Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding.

PubMed

Lan, Freeman; Demaree, Benjamin; Ahmed, Noorsher; Abate, Adam R

2017-07-01

The application of single-cell genome sequencing to large cell populations has been hindered by technical challenges in isolating single cells during genome preparation. Here we present single-cell genomic sequencing (SiC-seq), which uses droplet microfluidics to isolate, fragment, and barcode the genomes of single cells, followed by Illumina sequencing of pooled DNA. We demonstrate ultra-high-throughput sequencing of >50,000 cells per run in a synthetic community of Gram-negative and Gram-positive bacteria and fungi. The sequenced genomes can be sorted in silico based on characteristic sequences. We use this approach to analyze the distributions of antibiotic-resistance genes, virulence factors, and phage sequences in microbial communities from an environmental sample. The ability to routinely sequence large populations of single cells will enable the de-convolution of genetic heterogeneity in diverse cell populations.

Resources and costs for microbial sequence analysis evaluated using virtual machines and cloud computing.

PubMed

Angiuoli, Samuel V; White, James R; Matalka, Malcolm; White, Owen; Fricke, W Florian

2011-01-01

The widespread popularity of genomic applications is threatened by the "bioinformatics bottleneck" resulting from uncertainty about the cost and infrastructure needed to meet increasing demands for next-generation sequence analysis. Cloud computing services have been discussed as potential new bioinformatics support systems but have not been evaluated thoroughly. We present benchmark costs and runtimes for common microbial genomics applications, including 16S rRNA analysis, microbial whole-genome shotgun (WGS) sequence assembly and annotation, WGS metagenomics and large-scale BLAST. Sequence dataset types and sizes were selected to correspond to outputs typically generated by small- to midsize facilities equipped with 454 and Illumina platforms, except for WGS metagenomics where sampling of Illumina data was used. Automated analysis pipelines, as implemented in the CloVR virtual machine, were used in order to guarantee transparency, reproducibility and portability across different operating systems, including the commercial Amazon Elastic Compute Cloud (EC2), which was used to attach real dollar costs to each analysis type. We found considerable differences in computational requirements, runtimes and costs associated with different microbial genomics applications. While all 16S analyses completed on a single-CPU desktop in under three hours, microbial genome and metagenome analyses utilized multi-CPU support of up to 120 CPUs on Amazon EC2, where each analysis completed in under 24 hours for less than $60. Representative datasets were used to estimate maximum data throughput on different cluster sizes and to compare costs between EC2 and comparable local grid servers. Although bioinformatics requirements for microbial genomics depend on dataset characteristics and the analysis protocols applied, our results suggests that smaller sequencing facilities (up to three Roche/454 or one Illumina GAIIx sequencer) invested in 16S rRNA amplicon sequencing, microbial single-genome and metagenomics WGS projects can achieve cost-efficient bioinformatics support using CloVR in combination with Amazon EC2 as an alternative to local computing centers.

Resources and Costs for Microbial Sequence Analysis Evaluated Using Virtual Machines and Cloud Computing

PubMed Central

Angiuoli, Samuel V.; White, James R.; Matalka, Malcolm; White, Owen; Fricke, W. Florian

2011-01-01

Background The widespread popularity of genomic applications is threatened by the “bioinformatics bottleneck” resulting from uncertainty about the cost and infrastructure needed to meet increasing demands for next-generation sequence analysis. Cloud computing services have been discussed as potential new bioinformatics support systems but have not been evaluated thoroughly. Results We present benchmark costs and runtimes for common microbial genomics applications, including 16S rRNA analysis, microbial whole-genome shotgun (WGS) sequence assembly and annotation, WGS metagenomics and large-scale BLAST. Sequence dataset types and sizes were selected to correspond to outputs typically generated by small- to midsize facilities equipped with 454 and Illumina platforms, except for WGS metagenomics where sampling of Illumina data was used. Automated analysis pipelines, as implemented in the CloVR virtual machine, were used in order to guarantee transparency, reproducibility and portability across different operating systems, including the commercial Amazon Elastic Compute Cloud (EC2), which was used to attach real dollar costs to each analysis type. We found considerable differences in computational requirements, runtimes and costs associated with different microbial genomics applications. While all 16S analyses completed on a single-CPU desktop in under three hours, microbial genome and metagenome analyses utilized multi-CPU support of up to 120 CPUs on Amazon EC2, where each analysis completed in under 24 hours for less than $60. Representative datasets were used to estimate maximum data throughput on different cluster sizes and to compare costs between EC2 and comparable local grid servers. Conclusions Although bioinformatics requirements for microbial genomics depend on dataset characteristics and the analysis protocols applied, our results suggests that smaller sequencing facilities (up to three Roche/454 or one Illumina GAIIx sequencer) invested in 16S rRNA amplicon sequencing, microbial single-genome and metagenomics WGS projects can achieve cost-efficient bioinformatics support using CloVR in combination with Amazon EC2 as an alternative to local computing centers. PMID:22028928

Next-generation sequencing for targeted discovery of rare mutations in rice

USDA-ARS?s Scientific Manuscript database

Advances in DNA sequencing (i.e., next-generation sequencing, NGS) have greatly increased the power and efficiency of detecting rare mutations in large mutant populations. Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach for identifying gene mutations resulting fro...

Large-Scale Sequencing: The Future of Genomic Sciences Colloquium

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

Margaret Riley; Merry Buckley

2009-01-01

Genetic sequencing and the various molecular techniques it has enabled have revolutionized the field of microbiology. Examining and comparing the genetic sequences borne by microbes - including bacteria, archaea, viruses, and microbial eukaryotes - provides researchers insights into the processes microbes carry out, their pathogenic traits, and new ways to use microorganisms in medicine and manufacturing. Until recently, sequencing entire microbial genomes has been laborious and expensive, and the decision to sequence the genome of an organism was made on a case-by-case basis by individual researchers and funding agencies. Now, thanks to new technologies, the cost and effort of sequencingmore » is within reach for even the smallest facilities, and the ability to sequence the genomes of a significant fraction of microbial life may be possible. The availability of numerous microbial genomes will enable unprecedented insights into microbial evolution, function, and physiology. However, the current ad hoc approach to gathering sequence data has resulted in an unbalanced and highly biased sampling of microbial diversity. A well-coordinated, large-scale effort to target the breadth and depth of microbial diversity would result in the greatest impact. The American Academy of Microbiology convened a colloquium to discuss the scientific benefits of engaging in a large-scale, taxonomically-based sequencing project. A group of individuals with expertise in microbiology, genomics, informatics, ecology, and evolution deliberated on the issues inherent in such an effort and generated a set of specific recommendations for how best to proceed. The vast majority of microbes are presently uncultured and, thus, pose significant challenges to such a taxonomically-based approach to sampling genome diversity. However, we have yet to even scratch the surface of the genomic diversity among cultured microbes. A coordinated sequencing effort of cultured organisms is an appropriate place to begin, since not only are their genomes available, but they are also accompanied by data on environment and physiology that can be used to understand the resulting data. As single cell isolation methods improve, there should be a shift toward incorporating uncultured organisms and communities into this effort. Efforts to sequence cultivated isolates should target characterized isolates from culture collections for which biochemical data are available, as well as other cultures of lasting value from personal collections. The genomes of type strains should be among the first targets for sequencing, but creative culture methods, novel cell isolation, and sorting methods would all be helpful in obtaining organisms we have not yet been able to cultivate for sequencing. The data that should be provided for strains targeted for sequencing will depend on the phylogenetic context of the organism and the amount of information available about its nearest relatives. Annotation is an important part of transforming genome sequences into useful resources, but it represents the most significant bottleneck to the field of comparative genomics right now and must be addressed. Furthermore, there is a need for more consistency in both annotation and achieving annotation data. As new annotation tools become available over time, re-annotation of genomes should be implemented, taking advantage of advancements in annotation techniques in order to capitalize on the genome sequences and increase both the societal and scientific benefit of genomics work. Given the proper resources, the knowledge and ability exist to be able to select model systems, some simple, some less so, and dissect them so that we may understand the processes and interactions at work in them. Colloquium participants suggest a five-pronged, coordinated initiative to exhaustively describe six different microbial ecosystems, designed to describe all the gene diversity, across genomes. In this effort, sequencing should be complemented by other experimental data, particularly transcriptomics and metabolomics data, all of which should be gathered and curated continuously. Systematic genomics efforts like the ones outlined in this document would significantly broaden our view of biological diversity and have major effects on science. This has to be backed up with examples. Considering these potential impacts and the need for acquiescence from both the public and scientists to get such projects funded and functioning, education and training will be crucial. New collaborations within the scientific community will also be necessary.« less

Newborn Sequencing in Genomic Medicine and Public Health

PubMed Central

Agrawal, Pankaj B.; Bailey, Donald B.; Beggs, Alan H.; Brenner, Steven E.; Brower, Amy M.; Cakici, Julie A.; Ceyhan-Birsoy, Ozge; Chan, Kee; Chen, Flavia; Currier, Robert J.; Dukhovny, Dmitry; Green, Robert C.; Harris-Wai, Julie; Holm, Ingrid A.; Iglesias, Brenda; Joseph, Galen; Kingsmore, Stephen F.; Koenig, Barbara A.; Kwok, Pui-Yan; Lantos, John; Leeder, Steven J.; Lewis, Megan A.; McGuire, Amy L.; Milko, Laura V.; Mooney, Sean D.; Parad, Richard B.; Pereira, Stacey; Petrikin, Joshua; Powell, Bradford C.; Powell, Cynthia M.; Puck, Jennifer M.; Rehm, Heidi L.; Risch, Neil; Roche, Myra; Shieh, Joseph T.; Veeraraghavan, Narayanan; Watson, Michael S.; Willig, Laurel; Yu, Timothy W.; Urv, Tiina; Wise, Anastasia L.

2017-01-01

The rapid development of genomic sequencing technologies has decreased the cost of genetic analysis to the extent that it seems plausible that genome-scale sequencing could have widespread availability in pediatric care. Genomic sequencing provides a powerful diagnostic modality for patients who manifest symptoms of monogenic disease and an opportunity to detect health conditions before their development. However, many technical, clinical, ethical, and societal challenges should be addressed before such technology is widely deployed in pediatric practice. This article provides an overview of the Newborn Sequencing in Genomic Medicine and Public Health Consortium, which is investigating the application of genome-scale sequencing in newborns for both diagnosis and screening. PMID:28096516

Genome Science: A Video Tour of the Washington University Genome Sequencing Center for High School and Undergraduate Students

PubMed Central

2005-01-01

Sequencing of the human genome has ushered in a new era of biology. The technologies developed to facilitate the sequencing of the human genome are now being applied to the sequencing of other genomes. In 2004, a partnership was formed between Washington University School of Medicine Genome Sequencing Center's Outreach Program and Washington University Department of Biology Science Outreach to create a video tour depicting the processes involved in large-scale sequencing. “Sequencing a Genome: Inside the Washington University Genome Sequencing Center” is a tour of the laboratory that follows the steps in the sequencing pipeline, interspersed with animated explanations of the scientific procedures used at the facility. Accompanying interviews with the staff illustrate different entry levels for a career in genome science. This video project serves as an example of how research and academic institutions can provide teachers and students with access and exposure to innovative technologies at the forefront of biomedical research. Initial feedback on the video from undergraduate students, high school teachers, and high school students provides suggestions for use of this video in a classroom setting to supplement present curricula. PMID:16341256

Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly

PubMed Central

Austin, Christopher M; Hammer, Michael P; Lee, Yin Peng; Gan, Han Ming

2018-01-01

Abstract Background Some of the most widely recognized coral reef fishes are clownfish or anemonefish, members of the family Pomacentridae (subfamily: Amphiprioninae). They are popular aquarium species due to their bright colours, adaptability to captivity, and fascinating behavior. Their breeding biology (sequential hermaphrodites) and symbiotic mutualism with sea anemones have attracted much scientific interest. Moreover, there are some curious geographic-based phenotypes that warrant investigation. Leveraging on the advancement in Nanopore long read technology, we report the first hybrid assembly of the clown anemonefish (Amphiprion ocellaris) genome utilizing Illumina and Nanopore reads, further demonstrating the substantial impact of modest long read sequencing data sets on improving genome assembly statistics. Results We generated 43 Gb of short Illumina reads and 9 Gb of long Nanopore reads, representing approximate genome coverage of 54× and 11×, respectively, based on the range of estimated k-mer-predicted genome sizes of between 791 and 967 Mbp. The final assembled genome is contained in 6404 scaffolds with an accumulated length of 880 Mb (96.3% BUSCO-calculated genome completeness). Compared with the Illumina-only assembly, the hybrid approach generated 94% fewer scaffolds with an 18-fold increase in N50 length (401 kb) and increased the genome completeness by an additional 16%. A total of 27 240 high-quality protein-coding genes were predicted from the clown anemonefish, 26 211 (96%) of which were annotated functionally with information from either sequence homology or protein signature searches. Conclusions We present the first genome of any anemonefish and demonstrate the value of low coverage (∼11×) long Nanopore read sequencing in improving both genome assembly contiguity and completeness. The near-complete assembly of the A. ocellaris genome will be an invaluable molecular resource for supporting a range of genetic, genomic, and phylogenetic studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae. PMID:29342277

Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly.

PubMed

Tan, Mun Hua; Austin, Christopher M; Hammer, Michael P; Lee, Yin Peng; Croft, Laurence J; Gan, Han Ming

2018-03-01

Some of the most widely recognized coral reef fishes are clownfish or anemonefish, members of the family Pomacentridae (subfamily: Amphiprioninae). They are popular aquarium species due to their bright colours, adaptability to captivity, and fascinating behavior. Their breeding biology (sequential hermaphrodites) and symbiotic mutualism with sea anemones have attracted much scientific interest. Moreover, there are some curious geographic-based phenotypes that warrant investigation. Leveraging on the advancement in Nanopore long read technology, we report the first hybrid assembly of the clown anemonefish (Amphiprion ocellaris) genome utilizing Illumina and Nanopore reads, further demonstrating the substantial impact of modest long read sequencing data sets on improving genome assembly statistics. We generated 43 Gb of short Illumina reads and 9 Gb of long Nanopore reads, representing approximate genome coverage of 54× and 11×, respectively, based on the range of estimated k-mer-predicted genome sizes of between 791 and 967 Mbp. The final assembled genome is contained in 6404 scaffolds with an accumulated length of 880 Mb (96.3% BUSCO-calculated genome completeness). Compared with the Illumina-only assembly, the hybrid approach generated 94% fewer scaffolds with an 18-fold increase in N50 length (401 kb) and increased the genome completeness by an additional 16%. A total of 27 240 high-quality protein-coding genes were predicted from the clown anemonefish, 26 211 (96%) of which were annotated functionally with information from either sequence homology or protein signature searches. We present the first genome of any anemonefish and demonstrate the value of low coverage (∼11×) long Nanopore read sequencing in improving both genome assembly contiguity and completeness. The near-complete assembly of the A. ocellaris genome will be an invaluable molecular resource for supporting a range of genetic, genomic, and phylogenetic studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae.

Initial sequencing and comparative analysis of the mouse genome

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

Waterston, Robert H.; Lindblad-Toh, Kerstin; Birney, Ewan

2002-12-15

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of themore » genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.« less

Tapping the promise of genomics in species with complex, nonmodel genomes.

PubMed

Hirsch, Candice N; Buell, C Robin

2013-01-01

Genomics is enabling a renaissance in all disciplines of plant biology. However, many plant genomes are complex and remain recalcitrant to current genomic technologies. The complexities of these nonmodel plant genomes are attributable to gene and genome duplication, heterozygosity, ploidy, and/or repetitive sequences. Methods are available to simplify the genome and reduce these barriers, including inbreeding and genome reduction, making these species amenable to current sequencing and assembly methods. Some, but not all, of the complexities in nonmodel genomes can be bypassed by sequencing the transcriptome rather than the genome. Additionally, comparative genomics approaches, which leverage phylogenetic relatedness, can aid in the interpretation of complex genomes. Although there are limitations in accessing complex nonmodel plant genomes using current sequencing technologies, genome manipulation and resourceful analyses can allow access to even the most recalcitrant plant genomes.

Genome-wide characterization of centromeric satellites from multiple mammalian genomes.

PubMed

Alkan, Can; Cardone, Maria Francesca; Catacchio, Claudia Rita; Antonacci, Francesca; O'Brien, Stephen J; Ryder, Oliver A; Purgato, Stefania; Zoli, Monica; Della Valle, Giuliano; Eichler, Evan E; Ventura, Mario

2011-01-01

Despite its importance in cell biology and evolution, the centromere has remained the final frontier in genome assembly and annotation due to its complex repeat structure. However, isolation and characterization of the centromeric repeats from newly sequenced species are necessary for a complete understanding of genome evolution and function. In recent years, various genomes have been sequenced, but the characterization of the corresponding centromeric DNA has lagged behind. Here, we present a computational method (RepeatNet) to systematically identify higher-order repeat structures from unassembled whole-genome shotgun sequence and test whether these sequence elements correspond to functional centromeric sequences. We analyzed genome datasets from six species of mammals representing the diversity of the mammalian lineage, namely, horse, dog, elephant, armadillo, opossum, and platypus. We define candidate monomer satellite repeats and demonstrate centromeric localization for five of the six genomes. Our analysis revealed the greatest diversity of centromeric sequences in horse and dog in contrast to elephant and armadillo, which showed high-centromeric sequence homogeneity. We could not isolate centromeric sequences within the platypus genome, suggesting that centromeres in platypus are not enriched in satellite DNA. Our method can be applied to the characterization of thousands of other vertebrate genomes anticipated for sequencing in the near future, providing an important tool for annotation of centromeres.

Parents' interest in whole-genome sequencing of newborns.

PubMed

Goldenberg, Aaron J; Dodson, Daniel S; Davis, Matthew M; Tarini, Beth A

2014-01-01

The aim of this study was to assess parents' interest in whole-genome sequencing for newborns. We conducted a survey of a nationally representative sample of 1,539 parents about their interest in whole-genome sequencing of newborns. Participants were randomly presented with one of two scenarios that differed in the venue of testing: one offered whole-genome sequencing through a state newborn screening program, whereas the other offered whole-genome sequencing in a pediatrician's office. Overall interest in having future newborns undergo whole-genome sequencing was generally high among parents. If whole-genome sequencing were offered through a state's newborn-screening program, 74% of parents were either definitely or somewhat interested in utilizing this technology. If offered in a pediatrician's office, 70% of parents were either definitely or somewhat interested. Parents in both groups most frequently identified test accuracy and the ability to prevent a child from developing a disease as "very important" in making a decision to have a newborn's whole genome sequenced. These data may help health departments and children's health-care providers anticipate parents' level of interest in genomic screening for newborns. As whole-genome sequencing is integrated into clinical and public health services, these findings may inform the development of educational strategies and outreach messages for parents.

Coding Complete Genome for the Mogiana Tick Virus, a Jingmenvirus Isolated from Ticks in Brazil

DTIC Science & Technology

2017-05-04

sequences for all four genome segments. We downloaded the raw Illumina sequence reads from the NCBI Short Read Archive (GenBank...MGTV genome segments through sequence similarity (BLASTN) to the published genome of Jingmen tick virus (JMTV) isolate SY84 (GenBank: KJ001579-KJ001582...2014. Standards for sequencing viral genomes in the era of high-throughput sequencing . MBio 5:e01360–14. 8. Bankevich A, Nurk S, Antipov

Genome-Wide Characterization and Linkage Mapping of Simple Sequence Repeats in Mei (Prunus mume Sieb. et Zucc.)

PubMed Central

Sun, Lidan; Yang, Weiru; Zhang, Qixiang; Cheng, Tangren; Pan, Huitang; Xu, Zongda; Zhang, Jie; Chen, Chuguang

2013-01-01

Because of its popularity as an ornamental plant in East Asia, mei (Prunus mume Sieb. et Zucc.) has received increasing attention in genetic and genomic research with the recent shotgun sequencing of its genome. Here, we performed the genome-wide characterization of simple sequence repeats (SSRs) in the mei genome and detected a total of 188,149 SSRs occurring at a frequency of 794 SSR/Mb. Mononucleotide repeats were the most common type of SSR in genomic regions, followed by di- and tetranucleotide repeats. Most of the SSRs in coding sequences (CDS) were composed of tri- or hexanucleotide repeat motifs, but mononucleotide repeats were always the most common in intergenic regions. Genome-wide comparison of SSR patterns among the mei, strawberry (Fragaria vesca), and apple (Malus×domestica) genomes showed mei to have the highest density of SSRs, slightly higher than that of strawberry (608 SSR/Mb) and almost twice as high as that of apple (398 SSR/Mb). Mononucleotide repeats were the dominant SSR motifs in the three Rosaceae species. Using 144 SSR markers, we constructed a 670 cM-long linkage map of mei delimited into eight linkage groups (LGs), with an average marker distance of 5 cM. Seventy one scaffolds covering about 27.9% of the assembled mei genome were anchored to the genetic map, depending on which the macro-colinearity between the mei genome and Prunus T×E reference map was identified. The framework map of mei constructed provides a first step into subsequent high-resolution genetic mapping and marker-assisted selection for this ornamental species. PMID:23555708

Global MLST of Salmonella Typhi Revisited in Post-genomic Era: Genetic Conservation, Population Structure, and Comparative Genomics of Rare Sequence Types.

PubMed

Yap, Kien-Pong; Ho, Wing S; Gan, Han M; Chai, Lay C; Thong, Kwai L

2016-01-01

Typhoid fever, caused by Salmonella enterica serovar Typhi, remains an important public health burden in Southeast Asia and other endemic countries. Various genotyping methods have been applied to study the genetic variations of this human-restricted pathogen. Multilocus sequence typing (MLST) is one of the widely accepted methods, and recently, there is a growing interest in the re-application of MLST in the post-genomic era. In this study, we provide the global MLST distribution of S. Typhi utilizing both publicly available 1,826 S. Typhi genome sequences in addition to performing conventional MLST on S. Typhi strains isolated from various endemic regions spanning over a century. Our global MLST analysis confirms the predominance of two sequence types (ST1 and ST2) co-existing in the endemic regions. Interestingly, S. Typhi strains with ST8 are currently confined within the African continent. Comparative genomic analyses of ST8 and other rare STs with genomes of ST1/ST2 revealed unique mutations in important virulence genes such as flhB, sipC, and tviD that may explain the variations that differentiate between seemingly successful (widespread) and unsuccessful (poor dissemination) S. Typhi populations. Large scale whole-genome phylogeny demonstrated evidence of phylogeographical structuring and showed that ST8 may have diverged from the earlier ancestral population of ST1 and ST2, which later lost some of its fitness advantages, leading to poor worldwide dissemination. In response to the unprecedented increase in genomic data, this study demonstrates and highlights the utility of large-scale genome-based MLST as a quick and effective approach to narrow the scope of in-depth comparative genomic analysis and consequently provide new insights into the fine scale of pathogen evolution and population structure.

Bovine Genome Database: supporting community annotation and analysis of the Bos taurus genome

PubMed Central

2010-01-01

Background A goal of the Bovine Genome Database (BGD; http://BovineGenome.org) has been to support the Bovine Genome Sequencing and Analysis Consortium (BGSAC) in the annotation and analysis of the bovine genome. We were faced with several challenges, including the need to maintain consistent quality despite diversity in annotation expertise in the research community, the need to maintain consistent data formats, and the need to minimize the potential duplication of annotation effort. With new sequencing technologies allowing many more eukaryotic genomes to be sequenced, the demand for collaborative annotation is likely to increase. Here we present our approach, challenges and solutions facilitating a large distributed annotation project. Results and Discussion BGD has provided annotation tools that supported 147 members of the BGSAC in contributing 3,871 gene models over a fifteen-week period, and these annotations have been integrated into the bovine Official Gene Set. Our approach has been to provide an annotation system, which includes a BLAST site, multiple genome browsers, an annotation portal, and the Apollo Annotation Editor configured to connect directly to our Chado database. In addition to implementing and integrating components of the annotation system, we have performed computational analyses to create gene evidence tracks and a consensus gene set, which can be viewed on individual gene pages at BGD. Conclusions We have provided annotation tools that alleviate challenges associated with distributed annotation. Our system provides a consistent set of data to all annotators and eliminates the need for annotators to format data. Involving the bovine research community in genome annotation has allowed us to leverage expertise in various areas of bovine biology to provide biological insight into the genome sequence. PMID:21092105

The MLH1 ATPase domain is needed for suppressing aberrant formation of interstitial telomeric sequences.

PubMed

Jia, Pingping; Chai, Weihang

2018-05-01

Genome instability gives rise to cancer. MLH1, commonly known for its important role in mismatch repair (MMR), DNA damage signaling and double-strand break (DSB) repair, safeguards genome stability. Recently we have reported a novel role of MLH1 in preventing aberrant formation of interstitial telomeric sequences (ITSs) at intra-chromosomal regions. Deficiency in MLH1, in particular its N-terminus, leads to an increase of ITSs. Here, we identify that the ATPase activity in the MLH1 N-terminal domain is important for suppressing the formation of ITSs. The ATPase activity is also needed for recruiting MLH1 to DSBs. Moreover, defective ATPase activity of MLH1 causes an increase in micronuclei formation. Our results highlight the crucial role of MLH1's ATPase domain in preventing the aberrant formation of telomeric sequences at the intra-chromosomal regions and preserving genome stability. Copyright © 2018 Elsevier B.V. All rights reserved.

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system.

PubMed

Oba, Mami; Tsuchiaka, Shinobu; Omatsu, Tsutomu; Katayama, Yukie; Otomaru, Konosuke; Hirata, Teppei; Aoki, Hiroshi; Murata, Yoshiteru; Makino, Shinji; Nagai, Makoto; Mizutani, Tetsuya

2018-01-08

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples. Copyright © 2017 Elsevier Inc. All rights reserved.

On the Sequence-Directed Nature of Human Gene Mutation: The Role of Genomic Architecture and the Local DNA Sequence Environment in Mediating Gene Mutations Underlying Human Inherited Disease

PubMed Central

Cooper, David N.; Bacolla, Albino; Férec, Claude; Vasquez, Karen M.; Kehrer-Sawatzki, Hildegard; Chen, Jian-Min

2011-01-01

Different types of human gene mutation may vary in size, from structural variants (SVs) to single base-pair substitutions, but what they all have in common is that their nature, size and location are often determined either by specific characteristics of the local DNA sequence environment or by higher-order features of the genomic architecture. The human genome is now recognized to contain ‘pervasive architectural flaws’ in that certain DNA sequences are inherently mutation-prone by virtue of their base composition, sequence repetitivity and/or epigenetic modification. Here we explore how the nature, location and frequency of different types of mutation causing inherited disease are shaped in large part, and often in remarkably predictable ways, by the local DNA sequence environment. The mutability of a given gene or genomic region may also be influenced indirectly by a variety of non-canonical (non-B) secondary structures whose formation is facilitated by the underlying DNA sequence. Since these non-B DNA structures can interfere with subsequent DNA replication and repair, and may serve to increase mutation frequencies in generalized fashion (i.e. both in the context of subtle mutations and SVs), they have the potential to serve as a unifying concept in studies of mutational mechanisms underlying human inherited disease. PMID:21853507

Consequences of Normalizing Transcriptomic and Genomic Libraries of Plant Genomes Using a Duplex-Specific Nuclease and Tetramethylammonium Chloride

PubMed Central

Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

2013-01-01

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce. PMID:23409088

Consequences of normalizing transcriptomic and genomic libraries of plant genomes using a duplex-specific nuclease and tetramethylammonium chloride.

PubMed

Matvienko, Marta; Kozik, Alexander; Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

2013-01-01

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce.

Personal Genome Sequencing in Ostensibly Healthy Individuals and the PeopleSeq Consortium

PubMed Central

Linderman, Michael D.; Nielsen, Daiva E.; Green, Robert C.

2016-01-01

Thousands of ostensibly healthy individuals have had their exome or genome sequenced, but a much smaller number of these individuals have received any personal genomic results from that sequencing. We term those projects in which ostensibly healthy participants can receive sequencing-derived genetic findings and may also have access to their genomic data as participatory predispositional personal genome sequencing (PPGS). Here we are focused on genome sequencing applied in a pre-symptomatic context and so define PPGS to exclude diagnostic genome sequencing intended to identify the molecular cause of suspected or diagnosed genetic disease. In this report we describe the design of completed and underway PPGS projects, briefly summarize the results reported to date and introduce the PeopleSeq Consortium, a newly formed collaboration of PPGS projects designed to collect much-needed longitudinal outcome data. PMID:27023617

Fungal Genomics for Energy and Environment

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

Grigoriev, Igor V.

2013-03-11

Genomes of fungi relevant to energy and environment are in focus of the Fungal Genomic Program at the US Department of Energy Joint Genome Institute (JGI). One of its projects, the Genomics Encyclopedia of Fungi, targets fungi related to plant health (symbionts, pathogens, and biocontrol agents) and biorefinery processes (cellulose degradation, sugar fermentation, industrial hosts) by means of genome sequencing and analysis. New chapters of the Encyclopedia can be opened with user proposals to the JGI Community Sequencing Program (CSP). Another JGI project, the 1000 fungal genomes, explores fungal diversity on genome level at scale and is open for usersmore » to nominate new species for sequencing. Over 200 fungal genomes have been sequenced by JGI to date and released through MycoCosm (www.jgi.doe.gov/fungi), a fungal web-portal, which integrates sequence and functional data with genome analysis tools for user community. Sequence analysis supported by functional genomics leads to developing parts list for complex systems ranging from ecosystems of biofuel crops to biorefineries. Recent examples of such parts suggested by comparative genomics and functional analysis in these areas are presented here.« less

Associations of Perceived Norms With Intentions to Learn Genomic Sequencing Results: Roles for Attitudes and Ambivalence

PubMed Central

Reid, Allecia E.; Taber, Jennifer M.; Ferrer, Rebecca A.; Biesecker, Barbara B.; Lewis, Katie L.; Biesecker, Leslie G.; Klein, William M. P.

2018-01-01

Objective Genomic sequencing is becoming increasingly accessible, highlighting the need to understand the social and psychological factors that drive interest in receiving testing results. These decisions may depend on perceived descriptive norms (how most others behave) and injunctive norms (what is approved of by others). We predicted that descriptive norms would be directly associated with intentions to learn genomic sequencing results, whereas injunctive norms would be associated indirectly, via attitudes. These differential associations with intentions versus attitudes were hypothesized to be strongest when individuals held ambivalent attitudes toward obtaining results. Methods Participants enrolled in a genomic sequencing trial (n=372) reported intentions to learn medically actionable, non-medically actionable, and carrier sequencing results. Descriptive norms items referenced other study participants. Injunctive norms were analyzed separately for close friends and family members. Attitudes, attitudinal ambivalence, and sociodemographic covariates were also assessed. Results In structural equation models, both descriptive norms and friend injunctive norms were associated with intentions to receive all sequencing results (ps<.004). Attitudes consistently mediated all friend injunctive norms-intentions associations, but not the descriptive norms-intentions associations. Attitudinal ambivalence moderated the association between friend injunctive norms (p≤.001), but not descriptive norms (p=.16), and attitudes. Injunctive norms were significantly associated with attitudes when ambivalence was high, but were unrelated when ambivalence was low. Results replicated for family injunctive norms. Conclusions Descriptive and injunctive norms play roles in genomic sequencing decisions. Considering mediators and moderators of these processes enhances ability to optimize use of normative information to support informed decision making. PMID:29745680

The First Isolation and Whole Genome Sequencing of Murray Valley Encephalitis Virus from Cerebrospinal Fluid of a Patient with Encephalitis.

PubMed

Russell, Jessica S; Caly, Leon; Kostecki, Renata; McGuinness, Sarah L; Carter, Glen; Bulach, Dieter; Seemann, Torsten; Stinear, Tim P; Baird, Rob; Catton, Mike; Druce, Julian

2018-06-11

Murray Valley Encephalitis virus (MVEV) is a mosquito-borne Flavivirus. Clinical presentation is rare but severe, with a case fatality rate of 15⁻30%. Here we report a case of MVEV from the cerebrospinal fluid (CSF) of a patient in the Northern Territory in Australia. Initial diagnosis was performed using both MVEV-specific real-time, and Pan- Flavivirus conventional, Polymerase Chain Reaction (PCR), with confirmation by Sanger sequencing. Subsequent isolation, the first from CSF, was conducted in Vero cells and the observed cytopathic effect was confirmed by increasing viral titre in the real-time PCR. Isolation allowed for full genome sequencing using the Scriptseq V2 RNASeq library preparation kit. A consensus genome for VIDRL-MVE was generated and phylogenetic analysis identified it as Genotype 2. This is the first reported isolation, and full genome sequencing of MVEV from CSF. It is also the first time Genotype 2 has been identified in humans. As such, this case has significant implications for public health surveillance, epidemiology, and the understanding of MVEV evolution.

Serendipitous discovery of Wolbachia genomes in multiple Drosophila species.

PubMed

Salzberg, Steven L; Dunning Hotopp, Julie C; Delcher, Arthur L; Pop, Mihai; Smith, Douglas R; Eisen, Michael B; Nelson, William C

2005-01-01

The Trace Archive is a repository for the raw, unanalyzed data generated by large-scale genome sequencing projects. The existence of this data offers scientists the possibility of discovering additional genomic sequences beyond those originally sequenced. In particular, if the source DNA for a sequencing project came from a species that was colonized by another organism, then the project may yield substantial amounts of genomic DNA, including near-complete genomes, from the symbiotic or parasitic organism. By searching the publicly available repository of DNA sequencing trace data, we discovered three new species of the bacterial endosymbiont Wolbachia pipientis in three different species of fruit fly: Drosophila ananassae, D. simulans, and D. mojavensis. We extracted all sequences with partial matches to a previously sequenced Wolbachia strain and assembled those sequences using customized software. For one of the three new species, the data recovered were sufficient to produce an assembly that covers more than 95% of the genome; for a second species the data produce the equivalent of a 'light shotgun' sampling of the genome, covering an estimated 75-80% of the genome; and for the third species the data cover approximately 6-7% of the genome. The results of this study reveal an unexpected benefit of depositing raw data in a central genome sequence repository: new species can be discovered within this data. The differences between these three new Wolbachia genomes and the previously sequenced strain revealed numerous rearrangements and insertions within each lineage and hundreds of novel genes. The three new genomes, with annotation, have been deposited in GenBank.

Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.

PubMed

Jia, Xianbo; Lin, Xinjian; Chen, Jichen

2017-11-02

Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.

Why Assembling Plant Genome Sequences Is So Challenging

PubMed Central

Claros, Manuel Gonzalo; Bautista, Rocío; Guerrero-Fernández, Darío; Benzerki, Hicham; Seoane, Pedro; Fernández-Pozo, Noé

2012-01-01

In spite of the biological and economic importance of plants, relatively few plant species have been sequenced. Only the genome sequence of plants with relatively small genomes, most of them angiosperms, in particular eudicots, has been determined. The arrival of next-generation sequencing technologies has allowed the rapid and efficient development of new genomic resources for non-model or orphan plant species. But the sequencing pace of plants is far from that of animals and microorganisms. This review focuses on the typical challenges of plant genomes that can explain why plant genomics is less developed than animal genomics. Explanations about the impact of some confounding factors emerging from the nature of plant genomes are given. As a result of these challenges and confounding factors, the correct assembly and annotation of plant genomes is hindered, genome drafts are produced, and advances in plant genomics are delayed. PMID:24832233

Insights from Human/Mouse genome comparisons

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

Pennacchio, Len A.

2003-03-30

Large-scale public genomic sequencing efforts have provided a wealth of vertebrate sequence data poised to provide insights into mammalian biology. These include deep genomic sequence coverage of human, mouse, rat, zebrafish, and two pufferfish (Fugu rubripes and Tetraodon nigroviridis) (Aparicio et al. 2002; Lander et al. 2001; Venter et al. 2001; Waterston et al. 2002). In addition, a high-priority has been placed on determining the genomic sequence of chimpanzee, dog, cow, frog, and chicken (Boguski 2002). While only recently available, whole genome sequence data have provided the unique opportunity to globally compare complete genome contents. Furthermore, the shared evolutionary ancestrymore » of vertebrate species has allowed the development of comparative genomic approaches to identify ancient conserved sequences with functionality. Accordingly, this review focuses on the initial comparison of available mammalian genomes and describes various insights derived from such analysis.« less

CoSMoS: Conserved Sequence Motif Search in the proteome

PubMed Central

Liu, Xiao I; Korde, Neeraj; Jakob, Ursula; Leichert, Lars I

2006-01-01

Background With the ever-increasing number of gene sequences in the public databases, generating and analyzing multiple sequence alignments becomes increasingly time consuming. Nevertheless it is a task performed on a regular basis by researchers in many labs. Results We have now created a database called CoSMoS to find the occurrences and at the same time evaluate the significance of sequence motifs and amino acids encoded in the whole genome of the model organism Escherichia coli K12. We provide a precomputed set of multiple sequence alignments for each individual E. coli protein with all of its homologues in the RefSeq database. The alignments themselves, information about the occurrence of sequence motifs together with information on the conservation of each of the more than 1.3 million amino acids encoded in the E. coli genome can be accessed via the web interface of CoSMoS. Conclusion CoSMoS is a valuable tool to identify highly conserved sequence motifs, to find regions suitable for mutational studies in functional analyses and to predict important structural features in E. coli proteins. PMID:16433915

Integrating Genomic Data Sets for Knowledge Discovery: An Informed Approach to Management of Captive Endangered Species.

PubMed

Irizarry, Kristopher J L; Bryant, Doug; Kalish, Jordan; Eng, Curtis; Schmidt, Peggy L; Barrett, Gini; Barr, Margaret C

2016-01-01

Many endangered captive populations exhibit reduced genetic diversity resulting in health issues that impact reproductive fitness and quality of life. Numerous cost effective genomic sequencing and genotyping technologies provide unparalleled opportunity for incorporating genomics knowledge in management of endangered species. Genomic data, such as sequence data, transcriptome data, and genotyping data, provide critical information about a captive population that, when leveraged correctly, can be utilized to maximize population genetic variation while simultaneously reducing unintended introduction or propagation of undesirable phenotypes. Current approaches aimed at managing endangered captive populations utilize species survival plans (SSPs) that rely upon mean kinship estimates to maximize genetic diversity while simultaneously avoiding artificial selection in the breeding program. However, as genomic resources increase for each endangered species, the potential knowledge available for management also increases. Unlike model organisms in which considerable scientific resources are used to experimentally validate genotype-phenotype relationships, endangered species typically lack the necessary sample sizes and economic resources required for such studies. Even so, in the absence of experimentally verified genetic discoveries, genomics data still provides value. In fact, bioinformatics and comparative genomics approaches offer mechanisms for translating these raw genomics data sets into integrated knowledge that enable an informed approach to endangered species management.

Integrating Genomic Data Sets for Knowledge Discovery: An Informed Approach to Management of Captive Endangered Species

PubMed Central

Irizarry, Kristopher J. L.; Bryant, Doug; Kalish, Jordan; Eng, Curtis; Schmidt, Peggy L.; Barrett, Gini; Barr, Margaret C.

2016-01-01

Many endangered captive populations exhibit reduced genetic diversity resulting in health issues that impact reproductive fitness and quality of life. Numerous cost effective genomic sequencing and genotyping technologies provide unparalleled opportunity for incorporating genomics knowledge in management of endangered species. Genomic data, such as sequence data, transcriptome data, and genotyping data, provide critical information about a captive population that, when leveraged correctly, can be utilized to maximize population genetic variation while simultaneously reducing unintended introduction or propagation of undesirable phenotypes. Current approaches aimed at managing endangered captive populations utilize species survival plans (SSPs) that rely upon mean kinship estimates to maximize genetic diversity while simultaneously avoiding artificial selection in the breeding program. However, as genomic resources increase for each endangered species, the potential knowledge available for management also increases. Unlike model organisms in which considerable scientific resources are used to experimentally validate genotype-phenotype relationships, endangered species typically lack the necessary sample sizes and economic resources required for such studies. Even so, in the absence of experimentally verified genetic discoveries, genomics data still provides value. In fact, bioinformatics and comparative genomics approaches offer mechanisms for translating these raw genomics data sets into integrated knowledge that enable an informed approach to endangered species management. PMID:27376076

Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly of Malassezia sympodialis

PubMed Central

Tellgren-Roth, Christian; Baudo, Charles D.; Kennell, John C.; Sun, Sheng; Billmyre, R. Blake; Schröder, Markus S.; Andersson, Anna; Holm, Tina; Sigurgeirsson, Benjamin; Wu, Guangxi; Sankaranarayanan, Sundar Ram; Siddharthan, Rahul; Sanyal, Kaustuv; Lundeberg, Joakim; Nystedt, Björn; Boekhout, Teun; Dawson, Thomas L.; Heitman, Joseph

2017-01-01

Abstract Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies. PMID:28100699

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

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

VanBuren, Robert; Bryant, Doug; Edger, Patrick P.

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

DOE PAGES

VanBuren, Robert; Bryant, Doug; Edger, Patrick P.; ...

2015-11-11

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less

Food Safety in the Age of Next Generation Sequencing, Bioinformatics, and Open Data Access.

PubMed

Taboada, Eduardo N; Graham, Morag R; Carriço, João A; Van Domselaar, Gary

2017-01-01

Public health labs and food regulatory agencies globally are embracing whole genome sequencing (WGS) as a revolutionary new method that is positioned to replace numerous existing diagnostic and microbial typing technologies with a single new target: the microbial draft genome. The ability to cheaply generate large amounts of microbial genome sequence data, combined with emerging policies of food regulatory and public health institutions making their microbial sequences increasingly available and public, has served to open up the field to the general scientific community. This open data access policy shift has resulted in a proliferation of data being deposited into sequence repositories and of novel bioinformatics software designed to analyze these vast datasets. There also has been a more recent drive for improved data sharing to achieve more effective global surveillance, public health and food safety. Such developments have heightened the need for enhanced analytical systems in order to process and interpret this new type of data in a timely fashion. In this review we outline the emergence of genomics, bioinformatics and open data in the context of food safety. We also survey major efforts to translate genomics and bioinformatics technologies out of the research lab and into routine use in modern food safety labs. We conclude by discussing the challenges and opportunities that remain, including those expected to play a major role in the future of food safety science.

Complete genome sequence of Enterococcus faecium strain TX16 and comparative genomic analysis of Enterococcus faecium genomes

PubMed Central

2012-01-01

Background Enterococci are among the leading causes of hospital-acquired infections in the United States and Europe, with Enterococcus faecalis and Enterococcus faecium being the two most common species isolated from enterococcal infections. In the last decade, the proportion of enterococcal infections caused by E. faecium has steadily increased compared to other Enterococcus species. Although the underlying mechanism for the gradual replacement of E. faecalis by E. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of E. faecium strains in clinical environments. Systematic study of the molecular epidemiology and pathogenesis of E. faecium has been hindered by the lack of closed, complete E. faecium genomes that can be used as references. Results In this study, we report the complete genome sequence of the E. faecium strain TX16, also known as DO, which belongs to multilocus sequence type (ST) 18, and was the first E. faecium strain ever sequenced. Whole genome comparison of the TX16 genome with 21 E. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated (HA) strains (including STs 16, 17, 18, and 78), in addition to strains of non-hospital origin, group in the same clade (referred to as the HA clade) and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated (CA) clade with approximately a 3–4% average nucleotide sequence difference between the two clades at the core genome level. Our study also revealed that many genomic loci in the TX16 genome are unique to the HA clade. 380 ORFs in TX16 are HA-clade specific and antibiotic resistance genes are enriched in HA-clade strains. Mobile elements such as IS16 and transposons were also found almost exclusively in HA strains, as previously reported. Conclusions Our findings along with other studies show that HA clonal lineages harbor specific genetic elements as well as sequence differences in the core genome which may confer selection advantages over the more heterogeneous CA E. faecium isolates. Which of these differences are important for the success of specific E. faecium lineages in the hospital environment remain(s) to be determined. PMID:22769602

BATCH-GE: Batch analysis of Next-Generation Sequencing data for genome editing assessment

PubMed Central

Boel, Annekatrien; Steyaert, Woutert; De Rocker, Nina; Menten, Björn; Callewaert, Bert; De Paepe, Anne; Coucke, Paul; Willaert, Andy

2016-01-01

Targeted mutagenesis by the CRISPR/Cas9 system is currently revolutionizing genetics. The ease of this technique has enabled genome engineering in-vitro and in a range of model organisms and has pushed experimental dimensions to unprecedented proportions. Due to its tremendous progress in terms of speed, read length, throughput and cost, Next-Generation Sequencing (NGS) has been increasingly used for the analysis of CRISPR/Cas9 genome editing experiments. However, the current tools for genome editing assessment lack flexibility and fall short in the analysis of large amounts of NGS data. Therefore, we designed BATCH-GE, an easy-to-use bioinformatics tool for batch analysis of NGS-generated genome editing data, available from https://github.com/WouterSteyaert/BATCH-GE.git. BATCH-GE detects and reports indel mutations and other precise genome editing events and calculates the corresponding mutagenesis efficiencies for a large number of samples in parallel. Furthermore, this new tool provides flexibility by allowing the user to adapt a number of input variables. The performance of BATCH-GE was evaluated in two genome editing experiments, aiming to generate knock-out and knock-in zebrafish mutants. This tool will not only contribute to the evaluation of CRISPR/Cas9-based experiments, but will be of use in any genome editing experiment and has the ability to analyze data from every organism with a sequenced genome. PMID:27461955

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.

It’s More Than Stamp Collecting: How Genome Sequencing Can Unify Biological Research

PubMed Central

Richards, Stephen

2015-01-01

The availability of reference genome sequences, especially the human reference, has revolutionized the study of biology. However, whilst the genomes of some species have been fully sequenced, a wide range of biological problems still cannot be effectively studied for lack of genome sequence information. Here, I identify neglected areas of biology and describe how both targeted species sequencing and more broad taxonomic surveys of the tree of life can address important biological questions. I enumerate the significant benefits that would accrue from sequencing a broader range of taxa, as well as discuss the technical advances in sequencing and assembly methods that would allow for wide-ranging application of whole-genome analysis. Finally, I suggest that in addition to “Big Science” survey initiatives to sequence the tree of life, a modified infrastructure-funding paradigm would better support reference genome sequence generation for research communities most in need. PMID:26003218

It's more than stamp collecting: how genome sequencing can unify biological research.

PubMed

Richards, Stephen

2015-07-01

The availability of reference genome sequences, especially the human reference, has revolutionized the study of biology. However, while the genomes of some species have been fully sequenced, a wide range of biological problems still cannot be effectively studied for lack of genome sequence information. Here, I identify neglected areas of biology and describe how both targeted species sequencing and more broad taxonomic surveys of the tree of life can address important biological questions. I enumerate the significant benefits that would accrue from sequencing a broader range of taxa, as well as discuss the technical advances in sequencing and assembly methods that would allow for wide-ranging application of whole-genome analysis. Finally, I suggest that in addition to 'big science' survey initiatives to sequence the tree of life, a modified infrastructure-funding paradigm would better support reference genome sequence generation for research communities most in need. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

Complete Genome Sequence of Pigmentation Negative Yersinia Pestis strain Cadman Running head: Complete Genome Sequence of Y. pestis strain Cadman

DTIC Science & Technology

2016-10-27

Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland, USA 9 10 11 Running head: Complete Genome Sequence of Y. pestis strain Cadman...1 Complete Genome Sequence of Pigmentation Negative Yersinia pestis strain Cadman 1 2 3 Sean Lovetta, Kitty Chaseb, Galina Korolevaa, Gustavo...we report the genome sequence of Yersinia pestis strain Cadman, an attenuated strain 25 lacking the pgm locus. Y. pestis is the causative agent of

From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

PubMed Central

2014-01-01

Background Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users. Results Here we present an ‘A to Z’ protocol for obtaining complete human mitochondrial (mtDNA) genomes – from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling). Conclusions All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual ‘modules’ can be swapped out to suit available resources. PMID:24460871

Characterization of Aeromonas hydrophila wound pathotypes by comparative genomic and functional analyses of virulence genes.

PubMed

Grim, Christopher J; Kozlova, Elena V; Sha, Jian; Fitts, Eric C; van Lier, Christina J; Kirtley, Michelle L; Joseph, Sandeep J; Read, Timothy D; Burd, Eileen M; Tall, Ben D; Joseph, Sam W; Horneman, Amy J; Chopra, Ashok K; Shak, Joshua R

2013-04-23

Aeromonas hydrophila has increasingly been implicated as a virulent and antibiotic-resistant etiologic agent in various human diseases. In a previously published case report, we described a subject with a polymicrobial wound infection that included a persistent and aggressive strain of A. hydrophila (E1), as well as a more antibiotic-resistant strain of A. hydrophila (E2). To better understand the differences between pathogenic and environmental strains of A. hydrophila, we conducted comparative genomic and functional analyses of virulence-associated genes of these two wound isolates (E1 and E2), the environmental type strain A. hydrophila ATCC 7966(T), and four other isolates belonging to A. aquariorum, A. veronii, A. salmonicida, and A. caviae. Full-genome sequencing of strains E1 and E2 revealed extensive differences between the two and strain ATCC 7966(T). The more persistent wound infection strain, E1, harbored coding sequences for a cytotoxic enterotoxin (Act), a type 3 secretion system (T3SS), flagella, hemolysins, and a homolog of exotoxin A found in Pseudomonas aeruginosa. Corresponding phenotypic analyses with A. hydrophila ATCC 7966(T) and SSU as reference strains demonstrated the functionality of these virulence genes, with strain E1 displaying enhanced swimming and swarming motility, lateral flagella on electron microscopy, the presence of T3SS effector AexU, and enhanced lethality in a mouse model of Aeromonas infection. By combining sequence-based analysis and functional assays, we characterized an A. hydrophila pathotype, exemplified by strain E1, that exhibited increased virulence in a mouse model of infection, likely because of encapsulation, enhanced motility, toxin secretion, and cellular toxicity. Aeromonas hydrophila is a common aquatic bacterium that has increasingly been implicated in serious human infections. While many determinants of virulence have been identified in Aeromonas, rapid identification of pathogenic versus nonpathogenic strains remains a challenge for this genus, as it is for other opportunistic pathogens. This paper demonstrates, by using whole-genome sequencing of clinical Aeromonas strains, followed by corresponding virulence assays, that comparative genomics can be used to identify a virulent subtype of A. hydrophila that is aggressive during human infection and more lethal in a mouse model of infection. This aggressive pathotype contained genes for toxin production, toxin secretion, and bacterial motility that likely enabled its pathogenicity. Our results highlight the potential of whole-genome sequencing to transform microbial diagnostics; with further advances in rapid sequencing and annotation, genomic analysis will be able to provide timely information on the identities and virulence potential of clinically isolated microorganisms.

The Genome Sequencer FLX System--longer reads, more applications, straight forward bioinformatics and more complete data sets.

PubMed

Droege, Marcus; Hill, Brendon

2008-08-31

The Genome Sequencer FLX System (GS FLX), powered by 454 Sequencing, is a next-generation DNA sequencing technology featuring a unique mix of long reads, exceptional accuracy, and ultra-high throughput. It has been proven to be the most versatile of all currently available next-generation sequencing technologies, supporting many high-profile studies in over seven applications categories. GS FLX users have pursued innovative research in de novo sequencing, re-sequencing of whole genomes and target DNA regions, metagenomics, and RNA analysis. 454 Sequencing is a powerful tool for human genetics research, having recently re-sequenced the genome of an individual human, currently re-sequencing the complete human exome and targeted genomic regions using the NimbleGen sequence capture process, and detected low-frequency somatic mutations linked to cancer.

Newborn Sequencing in Genomic Medicine and Public Health.

PubMed

Berg, Jonathan S; Agrawal, Pankaj B; Bailey, Donald B; Beggs, Alan H; Brenner, Steven E; Brower, Amy M; Cakici, Julie A; Ceyhan-Birsoy, Ozge; Chan, Kee; Chen, Flavia; Currier, Robert J; Dukhovny, Dmitry; Green, Robert C; Harris-Wai, Julie; Holm, Ingrid A; Iglesias, Brenda; Joseph, Galen; Kingsmore, Stephen F; Koenig, Barbara A; Kwok, Pui-Yan; Lantos, John; Leeder, Steven J; Lewis, Megan A; McGuire, Amy L; Milko, Laura V; Mooney, Sean D; Parad, Richard B; Pereira, Stacey; Petrikin, Joshua; Powell, Bradford C; Powell, Cynthia M; Puck, Jennifer M; Rehm, Heidi L; Risch, Neil; Roche, Myra; Shieh, Joseph T; Veeraraghavan, Narayanan; Watson, Michael S; Willig, Laurel; Yu, Timothy W; Urv, Tiina; Wise, Anastasia L

2017-02-01

The rapid development of genomic sequencing technologies has decreased the cost of genetic analysis to the extent that it seems plausible that genome-scale sequencing could have widespread availability in pediatric care. Genomic sequencing provides a powerful diagnostic modality for patients who manifest symptoms of monogenic disease and an opportunity to detect health conditions before their development. However, many technical, clinical, ethical, and societal challenges should be addressed before such technology is widely deployed in pediatric practice. This article provides an overview of the Newborn Sequencing in Genomic Medicine and Public Health Consortium, which is investigating the application of genome-scale sequencing in newborns for both diagnosis and screening. Copyright © 2017 by the American Academy of Pediatrics.

Comparative sequence analysis of Sordaria macrospora and Neurospora crassa as a means to improve genome annotation.

PubMed

Nowrousian, Minou; Würtz, Christian; Pöggeler, Stefanie; Kück, Ulrich

2004-03-01

One of the most challenging parts of large scale sequencing projects is the identification of functional elements encoded in a genome. Recently, studies of genomes of up to six different Saccharomyces species have demonstrated that a comparative analysis of genome sequences from closely related species is a powerful approach to identify open reading frames and other functional regions within genomes [Science 301 (2003) 71, Nature 423 (2003) 241]. Here, we present a comparison of selected sequences from Sordaria macrospora to their corresponding Neurospora crassa orthologous regions. Our analysis indicates that due to the high degree of sequence similarity and conservation of overall genomic organization, S. macrospora sequence information can be used to simplify the annotation of the N. crassa genome.

Mapping genomic features to functional traits through microbial whole genome sequences.

PubMed

Zhang, Wei; Zeng, Erliang; Liu, Dan; Jones, Stuart E; Emrich, Scott

2014-01-01

Recently, the utility of trait-based approaches for microbial communities has been identified. Increasing availability of whole genome sequences provide the opportunity to explore the genetic foundations of a variety of functional traits. We proposed a machine learning framework to quantitatively link the genomic features with functional traits. Genes from bacteria genomes belonging to different functional traits were grouped to Cluster of Orthologs (COGs), and were used as features. Then, TF-IDF technique from the text mining domain was applied to transform the data to accommodate the abundance and importance of each COG. After TF-IDF processing, COGs were ranked using feature selection methods to identify their relevance to the functional trait of interest. Extensive experimental results demonstrated that functional trait related genes can be detected using our method. Further, the method has the potential to provide novel biological insights.

A Primer on Infectious Disease Bacterial Genomics

PubMed Central

Petkau, Aaron; Knox, Natalie; Graham, Morag; Van Domselaar, Gary

2016-01-01

SUMMARY The number of large-scale genomics projects is increasing due to the availability of affordable high-throughput sequencing (HTS) technologies. The use of HTS for bacterial infectious disease research is attractive because one whole-genome sequencing (WGS) run can replace multiple assays for bacterial typing, molecular epidemiology investigations, and more in-depth pathogenomic studies. The computational resources and bioinformatics expertise required to accommodate and analyze the large amounts of data pose new challenges for researchers embarking on genomics projects for the first time. Here, we present a comprehensive overview of a bacterial genomics projects from beginning to end, with a particular focus on the planning and computational requirements for HTS data, and provide a general understanding of the analytical concepts to develop a workflow that will meet the objectives and goals of HTS projects. PMID:28590251

Multiplexed fragaria chloroplast genome sequencing

Treesearch

W. Njuguna; A. Liston; R. Cronn; N.V. Bassil

2010-01-01

A method to sequence multiple chloroplast genomes using ultra high throughput sequencing technologies was recently described. Complete chloroplast genome sequences can resolve phylogenetic relationships at low taxonomic levels and identify informative point mutations and indels. The objective of this research was to sequence multiple Fragaria...

Genome Sequence of Lactobacillus salivarius NIAS840, Isolated from Chicken Intestine

PubMed Central

Ham, Jun-Sang; Kim, Hyoun-Wook; Seol, Kuk-Hwan; Jang, Aera; Jeong, Seok-Geun; Oh, Mi-Hwa; Kim, Dong-Hun; Kang, Dae-Kyung; Kim, Geun-Bae; Cha, Chang-Jun

2011-01-01

Lactobacillus salivarius is a well-known lactic acid bacterium to which increasing attention has been paid recently for use as probiotics for humans and animals. L. salivarius NIAS840 was first isolated from broiler chicken feces, displaying antimicrobial activities against multidrug-resistant Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Here, we report the genome sequence of L. salivarius NIAS840 (2,046,557 bp) including a small plasmid and two megaplasmids. PMID:21914873

Human Genome Sequencing in Health and Disease

PubMed Central

Gonzaga-Jauregui, Claudia; Lupski, James R.; Gibbs, Richard A.

2013-01-01

Following the “finished,” euchromatic, haploid human reference genome sequence, the rapid development of novel, faster, and cheaper sequencing technologies is making possible the era of personalized human genomics. Personal diploid human genome sequences have been generated, and each has contributed to our better understanding of variation in the human genome. We have consequently begun to appreciate the vastness of individual genetic variation from single nucleotide to structural variants. Translation of genome-scale variation into medically useful information is, however, in its infancy. This review summarizes the initial steps undertaken in clinical implementation of personal genome information, and describes the application of whole-genome and exome sequencing to identify the cause of genetic diseases and to suggest adjuvant therapies. Better analysis tools and a deeper understanding of the biology of our genome are necessary in order to decipher, interpret, and optimize clinical utility of what the variation in the human genome can teach us. Personal genome sequencing may eventually become an instrument of common medical practice, providing information that assists in the formulation of a differential diagnosis. We outline herein some of the remaining challenges. PMID:22248320

Identification of coding and non-coding mutational hotspots in cancer genomes.

PubMed

Piraino, Scott W; Furney, Simon J

2017-01-05

The identification of mutations that play a causal role in tumour development, so called "driver" mutations, is of critical importance for understanding how cancers form and how they might be treated. Several large cancer sequencing projects have identified genes that are recurrently mutated in cancer patients, suggesting a role in tumourigenesis. While the landscape of coding drivers has been extensively studied and many of the most prominent driver genes are well characterised, comparatively less is known about the role of mutations in the non-coding regions of the genome in cancer development. The continuing fall in genome sequencing costs has resulted in a concomitant increase in the number of cancer whole genome sequences being produced, facilitating systematic interrogation of both the coding and non-coding regions of cancer genomes. To examine the mutational landscapes of tumour genomes we have developed a novel method to identify mutational hotspots in tumour genomes using both mutational data and information on evolutionary conservation. We have applied our methodology to over 1300 whole cancer genomes and show that it identifies prominent coding and non-coding regions that are known or highly suspected to play a role in cancer. Importantly, we applied our method to the entire genome, rather than relying on predefined annotations (e.g. promoter regions) and we highlight recurrently mutated regions that may have resulted from increased exposure to mutational processes rather than selection, some of which have been identified previously as targets of selection. Finally, we implicate several pan-cancer and cancer-specific candidate non-coding regions, which could be involved in tumourigenesis. We have developed a framework to identify mutational hotspots in cancer genomes, which is applicable to the entire genome. This framework identifies known and novel coding and non-coding mutional hotspots and can be used to differentiate candidate driver regions from likely passenger regions susceptible to somatic mutation.

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome

PubMed Central

2009-01-01

Background Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. Results We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. Conclusion We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes. PMID:19656416

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome.

PubMed

Hamberger, Björn; Hall, Dawn; Yuen, Mack; Oddy, Claire; Hamberger, Britta; Keeling, Christopher I; Ritland, Carol; Ritland, Kermit; Bohlmann, Jörg

2009-08-06

Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes.

Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

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

Angelova, Angelina; Park, Sang-Hycuk; Kyndt, John

2013-09-01

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis.more » The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.« less

Evaluation and integration of functional annotation pipelines for newly sequenced organisms: the potato genome as a test case.

PubMed

Amar, David; Frades, Itziar; Danek, Agnieszka; Goldberg, Tatyana; Sharma, Sanjeev K; Hedley, Pete E; Proux-Wera, Estelle; Andreasson, Erik; Shamir, Ron; Tzfadia, Oren; Alexandersson, Erik

2014-12-05

For most organisms, even if their genome sequence is available, little functional information about individual genes or proteins exists. Several annotation pipelines have been developed for functional analysis based on sequence, 'omics', and literature data. However, researchers encounter little guidance on how well they perform. Here, we used the recently sequenced potato genome as a case study. The potato genome was selected since its genome is newly sequenced and it is a non-model plant even if there is relatively ample information on individual potato genes, and multiple gene expression profiles are available. We show that the automatic gene annotations of potato have low accuracy when compared to a "gold standard" based on experimentally validated potato genes. Furthermore, we evaluate six state-of-the-art annotation pipelines and show that their predictions are markedly dissimilar (Jaccard similarity coefficient of 0.27 between pipelines on average). To overcome this discrepancy, we introduce a simple GO structure-based algorithm that reconciles the predictions of the different pipelines. We show that the integrated annotation covers more genes, increases by over 50% the number of highly co-expressed GO processes, and obtains much higher agreement with the gold standard. We find that different annotation pipelines produce different results, and show how to integrate them into a unified annotation that is of higher quality than each single pipeline. We offer an improved functional annotation of both PGSC and ITAG potato gene models, as well as tools that can be applied to additional pipelines and improve annotation in other organisms. This will greatly aid future functional analysis of '-omics' datasets from potato and other organisms with newly sequenced genomes. The new potato annotations are available with this paper.

Uncovering the novel characteristics of Asian honey bee, Apis cerana, by whole genome sequencing.

PubMed

Park, Doori; Jung, Je Won; Choi, Beom-Soon; Jayakodi, Murukarthick; Lee, Jeongsoo; Lim, Jongsung; Yu, Yeisoo; Choi, Yong-Soo; Lee, Myeong-Lyeol; Park, Yoonseong; Choi, Ik-Young; Yang, Tae-Jin; Edwards, Owain R; Nah, Gyoungju; Kwon, Hyung Wook

2015-01-02

The honey bee is an important model system for increasing understanding of molecular and neural mechanisms underlying social behaviors relevant to the agricultural industry and basic science. The western honey bee, Apis mellifera, has served as a model species, and its genome sequence has been published. In contrast, the genome of the Asian honey bee, Apis cerana, has not yet been sequenced. A. cerana has been raised in Asian countries for thousands of years and has brought considerable economic benefits to the apicultural industry. A cerana has divergent biological traits compared to A. mellifera and it has played a key role in maintaining biodiversity in eastern and southern Asia. Here we report the first whole genome sequence of A. cerana. Using de novo assembly methods, we produced a 238 Mbp draft of the A. cerana genome and generated 10,651 genes. A.cerana-specific genes were analyzed to better understand the novel characteristics of this honey bee species. Seventy-two percent of the A. cerana-specific genes had more than one GO term, and 1,696 enzymes were categorized into 125 pathways. Genes involved in chemoreception and immunity were carefully identified and compared to those from other sequenced insect models. These included 10 gustatory receptors, 119 odorant receptors, 10 ionotropic receptors, and 160 immune-related genes. This first report of the whole genome sequence of A. cerana provides resources for comparative sociogenomics, especially in the field of social insect communication. These important tools will contribute to a better understanding of the complex behaviors and natural biology of the Asian honey bee and to anticipate its future evolutionary trajectory.

Whole-genome sequencing in bacteriology: state of the art

PubMed Central

Dark, Michael J

2013-01-01

Over the last ten years, genome sequencing capabilities have expanded exponentially. There have been tremendous advances in sequencing technology, DNA sample preparation, genome assembly, and data analysis. This has led to advances in a number of facets of bacterial genomics, including metagenomics, clinical medicine, bacterial archaeology, and bacterial evolution. This review examines the strengths and weaknesses of techniques in bacterial genome sequencing, upcoming technologies, and assembly techniques, as well as highlighting recent studies that highlight new applications for bacterial genomics. PMID:24143115

A web-based genomic sequence database for the Streptomycetaceae: a tool for systematics and genome mining

USDA-ARS?s Scientific Manuscript database

The ARS Microbial Genome Sequence Database (http://199.133.98.43), a web-based database server, was established utilizing the BIGSdb (Bacterial Isolate Genomics Sequence Database) software package, developed at Oxford University, as a tool to manage multi-locus sequence data for the family Streptomy...

Complete genome sequence of Clavibacter michiganensis subsp. insidiosus R1-1 using PacBio single-molecule real-time technology

USDA-ARS?s Scientific Manuscript database

We report the complete genome sequence of Clavibacter michiganensis subsp. insidiosus R1-1 isolated in Minnesota, USA. The R1-1 genome, generated by de novo assembly of PacBio sequencing data, is the first complete genome sequence available for this subspecies....

Draft Genome Sequence of a Rare Smut Relative, Tilletiaria anomala UBC 951

DOE PAGES

Toome, Merje; Kuo, Alan; Henrissat, Bernard; ...

2014-06-12

We present the draft genome sequence of the smut fungus Tilletiaria anomala UBC 951 (Basidiomycota, Ustilaginomycotina). The sequenced genome size is 18.7 Mb, consisting of 289 scaffolds and a total of 6,810 predicted genes. This is the first genome sequence published for a fungus in the order Georgefisheriales (Exobasidiomycetes).

Insights into Conifer Giga-Genomes1

PubMed Central

De La Torre, Amanda R.; Birol, Inanc; Bousquet, Jean; Ingvarsson, Pär K.; Jansson, Stefan; Jones, Steven J.M.; Keeling, Christopher I.; MacKay, John; Nilsson, Ove; Ritland, Kermit; Street, Nathaniel; Yanchuk, Alvin; Zerbe, Philipp; Bohlmann, Jörg

2014-01-01

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world’s forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20–30 Gb) and the highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: white spruce (Picea glauca), Norway spruce (Picea abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared with other plant genomes and may represent a window into the past of seed plant genomes. This Update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes. PMID:25349325

Insights into conifer giga-genomes.

PubMed

De La Torre, Amanda R; Birol, Inanc; Bousquet, Jean; Ingvarsson, Pär K; Jansson, Stefan; Jones, Steven J M; Keeling, Christopher I; MacKay, John; Nilsson, Ove; Ritland, Kermit; Street, Nathaniel; Yanchuk, Alvin; Zerbe, Philipp; Bohlmann, Jörg

2014-12-01

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world's forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20-30 Gb) and the highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: white spruce (Picea glauca), Norway spruce (Picea abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared with other plant genomes and may represent a window into the past of seed plant genomes. This Update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes. © 2014 American Society of Plant Biologists. All Rights Reserved.

Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus Cuscuta.

PubMed

McNeal, Joel R; Kuehl, Jennifer V; Boore, Jeffrey L; de Pamphilis, Claude W

2007-10-24

Plastid genome content and protein sequence are highly conserved across land plants and their closest algal relatives. Parasitic plants, which obtain some or all of their nutrition through an attachment to a host plant, are often a striking exception. Heterotrophy can lead to relaxed constraint on some plastid genes or even total gene loss. We sequenced plastid genomes of two species in the parasitic genus Cuscuta along with a non-parasitic relative, Ipomoea purpurea, to investigate changes in the plastid genome that may result from transition to the parasitic lifestyle. Aside from loss of all ndh genes, Cuscuta exaltata retains photosynthetic and photorespiratory genes that evolve under strong selective constraint. Cuscuta obtusiflora has incurred substantially more change to its plastid genome, including loss of all genes for the plastid-encoded RNA polymerase. Despite extensive change in gene content and greatly increased rate of overall nucleotide substitution, C. obtusiflora also retains all photosynthetic and photorespiratory genes with only one minor exception. Although Epifagus virginiana, the only other parasitic plant with its plastid genome sequenced to date, has lost a largely overlapping set of transfer-RNA and ribosomal genes as Cuscuta, it has lost all genes related to photosynthesis and maintains a set of genes which are among the most divergent in Cuscuta. Analyses demonstrate photosynthetic genes are under the highest constraint of any genes within the plastid genomes of Cuscuta, indicating a function involving RuBisCo and electron transport through photosystems is still the primary reason for retention of the plastid genome in these species.

Complete plastid genome sequences suggest strong selection for retention of photosynthetic genes in the parasitic plant genus Cuscuta

PubMed Central

McNeal, Joel R; Kuehl, Jennifer V; Boore, Jeffrey L; de Pamphilis, Claude W

2007-01-01

Background Plastid genome content and protein sequence are highly conserved across land plants and their closest algal relatives. Parasitic plants, which obtain some or all of their nutrition through an attachment to a host plant, are often a striking exception. Heterotrophy can lead to relaxed constraint on some plastid genes or even total gene loss. We sequenced plastid genomes of two species in the parasitic genus Cuscuta along with a non-parasitic relative, Ipomoea purpurea, to investigate changes in the plastid genome that may result from transition to the parasitic lifestyle. Results Aside from loss of all ndh genes, Cuscuta exaltata retains photosynthetic and photorespiratory genes that evolve under strong selective constraint. Cuscuta obtusiflora has incurred substantially more change to its plastid genome, including loss of all genes for the plastid-encoded RNA polymerase. Despite extensive change in gene content and greatly increased rate of overall nucleotide substitution, C. obtusiflora also retains all photosynthetic and photorespiratory genes with only one minor exception. Conclusion Although Epifagus virginiana, the only other parasitic plant with its plastid genome sequenced to date, has lost a largely overlapping set of transfer-RNA and ribosomal genes as Cuscuta, it has lost all genes related to photosynthesis and maintains a set of genes which are among the most divergent in Cuscuta. Analyses demonstrate photosynthetic genes are under the highest constraint of any genes within the plastid genomes of Cuscuta, indicating a function involving RuBisCo and electron transport through photosystems is still the primary reason for retention of the plastid genome in these species. PMID:17956636

Quantifying the Number of Independent Organelle DNA Insertions in Genome Evolution and Human Health.

PubMed

Hazkani-Covo, Einat; Martin, William F

2017-05-01

Fragments of organelle genomes are often found as insertions in nuclear DNA. These fragments of mitochondrial DNA (numts) and plastid DNA (nupts) are ubiquitous components of eukaryotic genomes. They are, however, often edited out during the genome assembly process, leading to systematic underestimation of their frequency. Numts and nupts, once inserted, can become further fragmented through subsequent insertion of mobile elements or other recombinational events that disrupt the continuity of the inserted sequence relative to the genuine organelle DNA copy. Because numts and nupts are typically identified through sequence comparison tools such as BLAST, disruption of insertions into smaller fragments can lead to systematic overestimation of numt and nupt frequencies. Accurate identification of numts and nupts is important, however, both for better understanding of their role during evolution, and for monitoring their increasingly evident role in human disease. Human populations are polymorphic for 141 numt loci, five numts are causal to genetic disease, and cancer genomic studies are revealing an abundance of numts associated with tumor progression. Here, we report investigation of salient parameters involved in obtaining accurate estimates of numt and nupt numbers in genome sequence data. Numts and nupts from 44 sequenced eukaryotic genomes reveal lineage-specific differences in the number, relative age and frequency of insertional events as well as lineage-specific dynamics of their postinsertional fragmentation. Our findings outline the main technical parameters influencing accurate identification and frequency estimation of numts in genomic studies pertinent to both evolution and human health. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Draft genome sequence of an aflatoxigenic Aspergillus species, A. bombycis

USDA-ARS?s Scientific Manuscript database

The genome of the A. bombycis Type strain was sequenced using a Personal Genome Machine, followed by annotation of its predicted genes. The genome size for A. bombycis was found to be approximately 37 Mb and contained 12,266 genes. This announcement introduces a sequenced genome for an aflatoxigenic...

GeneWiz browser: An Interactive Tool for Visualizing Sequenced Chromosomes.

PubMed

Hallin, Peter F; Stærfeldt, Hans-Henrik; Rotenberg, Eva; Binnewies, Tim T; Benham, Craig J; Ussery, David W

2009-09-25

We present an interactive web application for visualizing genomic data of prokaryotic chromosomes. The tool (GeneWiz browser) allows users to carry out various analyses such as mapping alignments of homologous genes to other genomes, mapping of short sequencing reads to a reference chromosome, and calculating DNA properties such as curvature or stacking energy along the chromosome. The GeneWiz browser produces an interactive graphic that enables zooming from a global scale down to single nucleotides, without changing the size of the plot. Its ability to disproportionally zoom provides optimal readability and increased functionality compared to other browsers. The tool allows the user to select the display of various genomic features, color setting and data ranges. Custom numerical data can be added to the plot allowing, for example, visualization of gene expression and regulation data. Further, standard atlases are pre-generated for all prokaryotic genomes available in GenBank, providing a fast overview of all available genomes, including recently deposited genome sequences. The tool is available online from http://www.cbs.dtu.dk/services/gwBrowser. Supplemental material including interactive atlases is available online at http://www.cbs.dtu.dk/services/gwBrowser/suppl/.

Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis.

PubMed

Sato, Kengo; Kuroki, Yoko; Kumita, Wakako; Fujiyama, Asao; Toyoda, Atsushi; Kawai, Jun; Iriki, Atsushi; Sasaki, Erika; Okano, Hideyuki; Sakakibara, Yasubumi

2015-11-20

The first draft of the common marmoset (Callithrix jacchus) genome was published by the Marmoset Genome Sequencing and Analysis Consortium. The draft was based on whole-genome shotgun sequencing, and the current assembly version is Callithrix_jacches-3.2.1, but there still exist 187,214 undetermined gap regions and supercontigs and relatively short contigs that are unmapped to chromosomes in the draft genome. We performed resequencing and assembly of the genome of common marmoset by deep sequencing with high-throughput sequencing technology. Several different sequence runs using Illumina sequencing platforms were executed, and 181 Gbp of high-quality bases including mate-pairs with long insert lengths of 3, 8, 20, and 40 Kbp were obtained, that is, approximately 60× coverage. The resequencing significantly improved the MGSAC draft genome sequence. The N50 of the contigs, which is a statistical measure used to evaluate assembly quality, doubled. As a result, 51% of the contigs (total length: 299 Mbp) that were unmapped to chromosomes in the MGSAC draft were merged with chromosomal contigs, and the improved genome sequence helped to detect 5,288 new genes that are homologous to human cDNAs and the gaps in 5,187 transcripts of the Ensembl gene annotations were completely filled.

How Primary Care Providers Talk to Patients about Genome Sequencing Results: Risk, Rationale, and Recommendation.

PubMed

Vassy, Jason L; Davis, J Kelly; Kirby, Christine; Richardson, Ian J; Green, Robert C; McGuire, Amy L; Ubel, Peter A

2018-06-01

Genomics will play an increasingly prominent role in clinical medicine. To describe how primary care physicians (PCPs) discuss and make clinical recommendations about genome sequencing results. Qualitative analysis. PCPs and their generally healthy patients undergoing genome sequencing. Patients received clinical genome reports that included four categories of results: monogenic disease risk variants (if present), carrier status, five pharmacogenetics results, and polygenic risk estimates for eight cardiometabolic traits. Patients' office visits with their PCPs were audio-recorded, and summative content analysis was used to describe how PCPs discussed genomic results. For each genomic result discussed in 48 PCP-patient visits, we identified a "take-home" message (recommendation), categorized as continuing current management, further treatment, further evaluation, behavior change, remembering for future care, or sharing with family members. We analyzed how PCPs came to each recommendation by identifying 1) how they described the risk or importance of the given result and 2) the rationale they gave for translating that risk into a specific recommendation. Quantitative analysis showed that continuing current management was the most commonly coded recommendation across results overall (492/749, 66%) and for each individual result type except monogenic disease risk results. Pharmacogenetics was the most common result type to prompt a recommendation to remember for future care (94/119, 79%); carrier status was the most common type prompting a recommendation to share with family members (45/54, 83%); and polygenic results were the most common type prompting a behavior change recommendation (55/58, 95%). One-fifth of recommendation codes associated with monogenic results were for further evaluation (6/24, 25%). Rationales for these recommendations included patient context, family context, and scientific/clinical limitations of sequencing. PCPs distinguish substantive differences among categories of genome sequencing results and use clinical judgment to justify continuing current management in generally healthy patients with genomic results.

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea

DOE PAGES

Bowers, Robert M.; Kyrpides, Nikos C.; Stepanauskas, Ramunas; ...

2017-08-08

Here, we present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a MetagenomeAssembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Genemore » Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.« less

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea

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

Bowers, Robert M.; Kyrpides, Nikos C.; Stepanauskas, Ramunas

Here, we present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a MetagenomeAssembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Genemore » Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.« less

Partial Shotgun Sequencing of the Boechera stricta Genome Reveals Extensive Microsynteny and Promoter Conservation with Arabidopsis1[W

PubMed Central

Windsor, Aaron J.; Schranz, M. Eric; Formanová, Nataša; Gebauer-Jung, Steffi; Bishop, John G.; Schnabelrauch, Domenica; Kroymann, Juergen; Mitchell-Olds, Thomas

2006-01-01

Comparative genomics provides insight into the evolutionary dynamics that shape discrete sequences as well as whole genomes. To advance comparative genomics within the Brassicaceae, we have end sequenced 23,136 medium-sized insert clones from Boechera stricta, a wild relative of Arabidopsis (Arabidopsis thaliana). A significant proportion of these sequences, 18,797, are nonredundant and display highly significant similarity (BLASTn e-value ≤ 10−30) to low copy number Arabidopsis genomic regions, including more than 9,000 annotated coding sequences. We have used this dataset to identify orthologous gene pairs in the two species and to perform a global comparison of DNA regions 5′ to annotated coding regions. On average, the 500 nucleotides upstream to coding sequences display 71.4% identity between the two species. In a similar analysis, 61.4% identity was observed between 5′ noncoding sequences of Brassica oleracea and Arabidopsis, indicating that regulatory regions are not as diverged among these lineages as previously anticipated. By mapping the B. stricta end sequences onto the Arabidopsis genome, we have identified nearly 2,000 conserved blocks of microsynteny (bracketing 26% of the Arabidopsis genome). A comparison of fully sequenced B. stricta inserts to their homologous Arabidopsis genomic regions indicates that indel polymorphisms >5 kb contribute substantially to the genome size difference observed between the two species. Further, we demonstrate that microsynteny inferred from end-sequence data can be applied to the rapid identification and cloning of genomic regions of interest from nonmodel species. These results suggest that among diploid relatives of Arabidopsis, small- to medium-scale shotgun sequencing approaches can provide rapid and cost-effective benefits to evolutionary and/or functional comparative genomic frameworks. PMID:16607030

Ensembl variation resources

PubMed Central

2010-01-01

Background The maturing field of genomics is rapidly increasing the number of sequenced genomes and producing more information from those previously sequenced. Much of this additional information is variation data derived from sampling multiple individuals of a given species with the goal of discovering new variants and characterising the population frequencies of the variants that are already known. These data have immense value for many studies, including those designed to understand evolution and connect genotype to phenotype. Maximising the utility of the data requires that it be stored in an accessible manner that facilitates the integration of variation data with other genome resources such as gene annotation and comparative genomics. Description The Ensembl project provides comprehensive and integrated variation resources for a wide variety of chordate genomes. This paper provides a detailed description of the sources of data and the methods for creating the Ensembl variation databases. It also explores the utility of the information by explaining the range of query options available, from using interactive web displays, to online data mining tools and connecting directly to the data servers programmatically. It gives a good overview of the variation resources and future plans for expanding the variation data within Ensembl. Conclusions Variation data is an important key to understanding the functional and phenotypic differences between individuals. The development of new sequencing and genotyping technologies is greatly increasing the amount of variation data known for almost all genomes. The Ensembl variation resources are integrated into the Ensembl genome browser and provide a comprehensive way to access this data in the context of a widely used genome bioinformatics system. All Ensembl data is freely available at http://www.ensembl.org and from the public MySQL database server at ensembldb.ensembl.org. PMID:20459805

Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data.

PubMed

Desai, Aarti; Marwah, Veer Singh; Yadav, Akshay; Jha, Vineet; Dhaygude, Kishor; Bangar, Ujwala; Kulkarni, Vivek; Jere, Abhay

2013-01-01

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6-40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis.

PubMed

Morrison, Cheryl L; Iwanowicz, Luke; Work, Thierry M; Fahsbender, Elizabeth; Breitbart, Mya; Adams, Cynthia; Iwanowicz, Deb; Sanders, Lakyn; Ackermann, Mathias; Cornman, Robert S

2018-01-01

Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis

USGS Publications Warehouse

Morrison, Cheryl L.; Iwanowicz, Luke R.; Work, Thierry M.; Fahsbender, Elizabeth; Breitbart, Mya; Adams, Cynthia; Iwanowicz, Deborah; Sanders, Lakyn; Ackermann, Mathias; Cornman, Robert S.

2018-01-01

Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Genome-Wide Typing of Clostridium difficile.

PubMed

Bletz, Stefan; Janezic, Sandra; Harmsen, Dag; Rupnik, Maja; Mellmann, Alexander

2018-06-01

Clostridium difficile , recently renamed Clostridioides difficile , is the most common cause of antibiotic-associated nosocomial gastrointestinal infections worldwide. To differentiate endogenous infections and transmission events, highly discriminatory subtyping is necessary. Today, methods based on whole-genome sequencing data are increasingly used to subtype bacterial pathogens; however, frequently a standardized methodology and typing nomenclature are missing. Here we report a core genome multilocus sequence typing (cgMLST) approach developed for C. difficile Initially, we determined the breadth of the C. difficile population based on all available MLST sequence types with Bayesian inference (BAPS). The resulting BAPS partitions were used in combination with C. difficile clade information to select representative isolates that were subsequently used to define cgMLST target genes. Finally, we evaluated the novel cgMLST scheme with genomes from 3,025 isolates. BAPS grouping ( n = 6 groups) together with the clade information led to a total of 11 representative isolates that were included for cgMLST definition and resulted in 2,270 cgMLST genes that were present in all isolates. Overall, 2,184 to 2,268 cgMLST targets were detected in the genome sequences of 70 outbreak-associated and reference strains, and on average 99.3% cgMLST targets (1,116 to 2,270 targets) were present in 2,954 genomes downloaded from the NCBI database, underlining the representativeness of the cgMLST scheme. Moreover, reanalyzing different cluster scenarios with cgMLST were concordant to published single nucleotide variant analyses. In conclusion, the novel cgMLST is representative for the whole C. difficile population, is highly discriminatory in outbreak situations, and provides a unique nomenclature facilitating interlaboratory exchange. Copyright © 2018 American Society for Microbiology.

Genomic evolution, recombination, and inter-strain diversity of chelonid alphaherpesvirus 5 from Florida and Hawaii green sea turtles with fibropapillomatosis

PubMed Central

Iwanowicz, Luke; Work, Thierry M.; Fahsbender, Elizabeth; Breitbart, Mya; Adams, Cynthia; Iwanowicz, Deb; Sanders, Lakyn; Ackermann, Mathias; Cornman, Robert S.

2018-01-01

Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants. PMID:29479497

From algae to angiosperms–inferring the phylogeny of green plants (Viridiplantae) from 360 plastid genomes

PubMed Central

2014-01-01

Background Next-generation sequencing has provided a wealth of plastid genome sequence data from an increasingly diverse set of green plants (Viridiplantae). Although these data have helped resolve the phylogeny of numerous clades (e.g., green algae, angiosperms, and gymnosperms), their utility for inferring relationships across all green plants is uncertain. Viridiplantae originated 700-1500 million years ago and may comprise as many as 500,000 species. This clade represents a major source of photosynthetic carbon and contains an immense diversity of life forms, including some of the smallest and largest eukaryotes. Here we explore the limits and challenges of inferring a comprehensive green plant phylogeny from available complete or nearly complete plastid genome sequence data. Results We assembled protein-coding sequence data for 78 genes from 360 diverse green plant taxa with complete or nearly complete plastid genome sequences available from GenBank. Phylogenetic analyses of the plastid data recovered well-supported backbone relationships and strong support for relationships that were not observed in previous analyses of major subclades within Viridiplantae. However, there also is evidence of systematic error in some analyses. In several instances we obtained strongly supported but conflicting topologies from analyses of nucleotides versus amino acid characters, and the considerable variation in GC content among lineages and within single genomes affected the phylogenetic placement of several taxa. Conclusions Analyses of the plastid sequence data recovered a strongly supported framework of relationships for green plants. This framework includes: i) the placement of Zygnematophyceace as sister to land plants (Embryophyta), ii) a clade of extant gymnosperms (Acrogymnospermae) with cycads + Ginkgo sister to remaining extant gymnosperms and with gnetophytes (Gnetophyta) sister to non-Pinaceae conifers (Gnecup trees), and iii) within the monilophyte clade (Monilophyta), Equisetales + Psilotales are sister to Marattiales + leptosporangiate ferns. Our analyses also highlight the challenges of using plastid genome sequences in deep-level phylogenomic analyses, and we provide suggestions for future analyses that will likely incorporate plastid genome sequence data for thousands of species. We particularly emphasize the importance of exploring the effects of different partitioning and character coding strategies. PMID:24533922

Rainbow: a tool for large-scale whole-genome sequencing data analysis using cloud computing.

PubMed

Zhao, Shanrong; Prenger, Kurt; Smith, Lance; Messina, Thomas; Fan, Hongtao; Jaeger, Edward; Stephens, Susan

2013-06-27

Technical improvements have decreased sequencing costs and, as a result, the size and number of genomic datasets have increased rapidly. Because of the lower cost, large amounts of sequence data are now being produced by small to midsize research groups. Crossbow is a software tool that can detect single nucleotide polymorphisms (SNPs) in whole-genome sequencing (WGS) data from a single subject; however, Crossbow has a number of limitations when applied to multiple subjects from large-scale WGS projects. The data storage and CPU resources that are required for large-scale whole genome sequencing data analyses are too large for many core facilities and individual laboratories to provide. To help meet these challenges, we have developed Rainbow, a cloud-based software package that can assist in the automation of large-scale WGS data analyses. Here, we evaluated the performance of Rainbow by analyzing 44 different whole-genome-sequenced subjects. Rainbow has the capacity to process genomic data from more than 500 subjects in two weeks using cloud computing provided by the Amazon Web Service. The time includes the import and export of the data using Amazon Import/Export service. The average cost of processing a single sample in the cloud was less than 120 US dollars. Compared with Crossbow, the main improvements incorporated into Rainbow include the ability: (1) to handle BAM as well as FASTQ input files; (2) to split large sequence files for better load balance downstream; (3) to log the running metrics in data processing and monitoring multiple Amazon Elastic Compute Cloud (EC2) instances; and (4) to merge SOAPsnp outputs for multiple individuals into a single file to facilitate downstream genome-wide association studies. Rainbow is a scalable, cost-effective, and open-source tool for large-scale WGS data analysis. For human WGS data sequenced by either the Illumina HiSeq 2000 or HiSeq 2500 platforms, Rainbow can be used straight out of the box. Rainbow is available for third-party implementation and use, and can be downloaded from http://s3.amazonaws.com/jnj_rainbow/index.html.

Rainbow: a tool for large-scale whole-genome sequencing data analysis using cloud computing

PubMed Central

2013-01-01

Background Technical improvements have decreased sequencing costs and, as a result, the size and number of genomic datasets have increased rapidly. Because of the lower cost, large amounts of sequence data are now being produced by small to midsize research groups. Crossbow is a software tool that can detect single nucleotide polymorphisms (SNPs) in whole-genome sequencing (WGS) data from a single subject; however, Crossbow has a number of limitations when applied to multiple subjects from large-scale WGS projects. The data storage and CPU resources that are required for large-scale whole genome sequencing data analyses are too large for many core facilities and individual laboratories to provide. To help meet these challenges, we have developed Rainbow, a cloud-based software package that can assist in the automation of large-scale WGS data analyses. Results Here, we evaluated the performance of Rainbow by analyzing 44 different whole-genome-sequenced subjects. Rainbow has the capacity to process genomic data from more than 500 subjects in two weeks using cloud computing provided by the Amazon Web Service. The time includes the import and export of the data using Amazon Import/Export service. The average cost of processing a single sample in the cloud was less than 120 US dollars. Compared with Crossbow, the main improvements incorporated into Rainbow include the ability: (1) to handle BAM as well as FASTQ input files; (2) to split large sequence files for better load balance downstream; (3) to log the running metrics in data processing and monitoring multiple Amazon Elastic Compute Cloud (EC2) instances; and (4) to merge SOAPsnp outputs for multiple individuals into a single file to facilitate downstream genome-wide association studies. Conclusions Rainbow is a scalable, cost-effective, and open-source tool for large-scale WGS data analysis. For human WGS data sequenced by either the Illumina HiSeq 2000 or HiSeq 2500 platforms, Rainbow can be used straight out of the box. Rainbow is available for third-party implementation and use, and can be downloaded from http://s3.amazonaws.com/jnj_rainbow/index.html. PMID:23802613

Large-scale contamination of microbial isolate genomes by Illumina PhiX control.

PubMed

Mukherjee, Supratim; Huntemann, Marcel; Ivanova, Natalia; Kyrpides, Nikos C; Pati, Amrita

2015-01-01

With the rapid growth and development of sequencing technologies, genomes have become the new go-to for exploring solutions to some of the world's biggest challenges such as searching for alternative energy sources and exploration of genomic dark matter. However, progress in sequencing has been accompanied by its share of errors that can occur during template or library preparation, sequencing, imaging or data analysis. In this study we screened over 18,000 publicly available microbial isolate genome sequences in the Integrated Microbial Genomes database and identified more than 1000 genomes that are contaminated with PhiX, a control frequently used during Illumina sequencing runs. Approximately 10% of these genomes have been published in literature and 129 contaminated genomes were sequenced under the Human Microbiome Project. Raw sequence reads are prone to contamination from various sources and are usually eliminated during downstream quality control steps. Detection of PhiX contaminated genomes indicates a lapse in either the application or effectiveness of proper quality control measures. The presence of PhiX contamination in several publicly available isolate genomes can result in additional errors when such data are used in comparative genomics analyses. Such contamination of public databases have far-reaching consequences in the form of erroneous data interpretation and analyses, and necessitates better measures to proofread raw sequences before releasing them to the broader scientific community.

The Complete Chloroplast and Mitochondrial Genome Sequences of Boea hygrometrica: Insights into the Evolution of Plant Organellar Genomes

PubMed Central

Wang, Xumin; Deng, Xin; Zhang, Xiaowei; Hu, Songnian; Yu, Jun

2012-01-01

The complete nucleotide sequences of the chloroplast (cp) and mitochondrial (mt) genomes of resurrection plant Boea hygrometrica (Bh, Gesneriaceae) have been determined with the lengths of 153,493 bp and 510,519 bp, respectively. The smaller chloroplast genome contains more genes (147) with a 72% coding sequence, and the larger mitochondrial genome have less genes (65) with a coding faction of 12%. Similar to other seed plants, the Bh cp genome has a typical quadripartite organization with a conserved gene in each region. The Bh mt genome has three recombinant sequence repeats of 222 bp, 843 bp, and 1474 bp in length, which divide the genome into a single master circle (MC) and four isomeric molecules. Compared to other angiosperms, one remarkable feature of the Bh mt genome is the frequent transfer of genetic material from the cp genome during recent Bh evolution. We also analyzed organellar genome evolution in general regarding genome features as well as compositional dynamics of sequence and gene structure/organization, providing clues for the understanding of the evolution of organellar genomes in plants. The cp-derived sequences including tRNAs found in angiosperm mt genomes support the conclusion that frequent gene transfer events may have begun early in the land plant lineage. PMID:22291979

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes).

PubMed

Dessimoz, Christophe; Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-09-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references.

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes)

PubMed Central

Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-01-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references. PMID:21712341

Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

PubMed

Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

2003-07-15

Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

Exploration of the Drosophila buzzatii transposable element content suggests underestimation of repeats in Drosophila genomes.

PubMed

Rius, Nuria; Guillén, Yolanda; Delprat, Alejandra; Kapusta, Aurélie; Feschotte, Cédric; Ruiz, Alfredo

2016-05-10

Many new Drosophila genomes have been sequenced in recent years using new-generation sequencing platforms and assembly methods. Transposable elements (TEs), being repetitive sequences, are often misassembled, especially in the genomes sequenced with short reads. Consequently, the mobile fraction of many of the new genomes has not been analyzed in detail or compared with that of other genomes sequenced with different methods, which could shed light into the understanding of genome and TE evolution. Here we compare the TE content of three genomes: D. buzzatii st-1, j-19, and D. mojavensis. We have sequenced a new D. buzzatii genome (j-19) that complements the D. buzzatii reference genome (st-1) already published, and compared their TE contents with that of D. mojavensis. We found an underestimation of TE sequences in Drosophila genus NGS-genomes when compared to Sanger-genomes. To be able to compare genomes sequenced with different technologies, we developed a coverage-based method and applied it to the D. buzzatii st-1 and j-19 genome. Between 10.85 and 11.16 % of the D. buzzatii st-1 genome is made up of TEs, between 7 and 7,5 % of D. buzzatii j-19 genome, while TEs represent 15.35 % of the D. mojavensis genome. Helitrons are the most abundant order in the three genomes. TEs in D. buzzatii are less abundant than in D. mojavensis, as expected according to the genome size and TE content positive correlation. However, TEs alone do not explain the genome size difference. TEs accumulate in the dot chromosomes and proximal regions of D. buzzatii and D. mojavensis chromosomes. We also report a significantly higher TE density in D. buzzatii and D. mojavensis X chromosomes, which is not expected under the current models. Our easy-to-use correction method allowed us to identify recently active families in D. buzzatii st-1 belonging to the LTR-retrotransposon superfamily Gypsy.

Genome sequence of the lager brewing yeast, an interspecies hybrid.

PubMed

Nakao, Yoshihiro; Kanamori, Takeshi; Itoh, Takehiko; Kodama, Yukiko; Rainieri, Sandra; Nakamura, Norihisa; Shimonaga, Tomoko; Hattori, Masahira; Ashikari, Toshihiko

2009-04-01

This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production.

Computing prokaryotic gene ubiquity: rescuing the core from extinction.

PubMed

Charlebois, Robert L; Doolittle, W Ford

2004-12-01

The genomic core concept has found several uses in comparative and evolutionary genomics. Defined as the set of all genes common to (ubiquitous among) all genomes in a phylogenetically coherent group, core size decreases as the number and phylogenetic diversity of the relevant group increases. Here, we focus on methods for defining the size and composition of the core of all genes shared by sequenced genomes of prokaryotes (Bacteria and Archaea). There are few (almost certainly less than 50) genes shared by all of the 147 genomes compared, surely insufficient to conduct all essential functions. Sequencing and annotation errors are responsible for the apparent absence of some genes, while very limited but genuine disappearances (from just one or a few genomes) can account for several others. Core size will continue to decrease as more genome sequences appear, unless the requirement for ubiquity is relaxed. Such relaxation seems consistent with any reasonable biological purpose for seeking a core, but it renders the problem of definition more problematic. We propose an alternative approach (the phylogenetically balanced core), which preserves some of the biological utility of the core concept. Cores, however delimited, preferentially contain informational rather than operational genes; we present a new hypothesis for why this might be so.