Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association

PubMed Central

2012-01-01

Background The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map. Results Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the MAP S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human M. avium subsp. hominissuis strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of MAP (JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level. Conclusions Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the M. avium complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of MAP. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for M. avium complex strains based on these genome sequences. PMID:22409516

Approaches for in silico finishing of microbial genome sequences

PubMed Central

Kremer, Frederico Schmitt; McBride, Alan John Alexander; Pinto, Luciano da Silva

2017-01-01

Abstract The introduction of next-generation sequencing (NGS) had a significant effect on the availability of genomic information, leading to an increase in the number of sequenced genomes from a large spectrum of organisms. Unfortunately, due to the limitations implied by the short-read sequencing platforms, most of these newly sequenced genomes remained as “drafts”, incomplete representations of the whole genetic content. The previous genome sequencing studies indicated that finishing a genome sequenced by NGS, even bacteria, may require additional sequencing to fill the gaps, making the entire process very expensive. As such, several in silico approaches have been developed to optimize the genome assemblies and facilitate the finishing process. The present review aims to explore some free (open source, in many cases) tools that are available to facilitate genome finishing. PMID:28898352

Approaches for in silico finishing of microbial genome sequences.

PubMed

Kremer, Frederico Schmitt; McBride, Alan John Alexander; Pinto, Luciano da Silva

The introduction of next-generation sequencing (NGS) had a significant effect on the availability of genomic information, leading to an increase in the number of sequenced genomes from a large spectrum of organisms. Unfortunately, due to the limitations implied by the short-read sequencing platforms, most of these newly sequenced genomes remained as "drafts", incomplete representations of the whole genetic content. The previous genome sequencing studies indicated that finishing a genome sequenced by NGS, even bacteria, may require additional sequencing to fill the gaps, making the entire process very expensive. As such, several in silico approaches have been developed to optimize the genome assemblies and facilitate the finishing process. The present review aims to explore some free (open source, in many cases) tools that are available to facilitate genome finishing.

The Ensembl genome database project.

PubMed

Hubbard, T; Barker, D; Birney, E; Cameron, G; Chen, Y; Clark, L; Cox, T; Cuff, J; Curwen, V; Down, T; Durbin, R; Eyras, E; Gilbert, J; Hammond, M; Huminiecki, L; Kasprzyk, A; Lehvaslaiho, H; Lijnzaad, P; Melsopp, C; Mongin, E; Pettett, R; Pocock, M; Potter, S; Rust, A; Schmidt, E; Searle, S; Slater, G; Smith, J; Spooner, W; Stabenau, A; Stalker, J; Stupka, E; Ureta-Vidal, A; Vastrik, I; Clamp, M

2002-01-01

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of the human genome sequence, with confirmed gene predictions that have been integrated with external data sources, and is available as either an interactive web site or as flat files. It is also an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements from sequence analysis to data storage and visualisation. The Ensembl site is one of the leading sources of human genome sequence annotation and provided much of the analysis for publication by the international human genome project of the draft genome. The Ensembl system is being installed around the world in both companies and academic sites on machines ranging from supercomputers to laptops.

GDC 2: Compression of large collections of genomes

PubMed Central

Deorowicz, Sebastian; Danek, Agnieszka; Niemiec, Marcin

2015-01-01

The fall of prices of the high-throughput genome sequencing changes the landscape of modern genomics. A number of large scale projects aimed at sequencing many human genomes are in progress. Genome sequencing also becomes an important aid in the personalized medicine. One of the significant side effects of this change is a necessity of storage and transfer of huge amounts of genomic data. In this paper we deal with the problem of compression of large collections of complete genomic sequences. We propose an algorithm that is able to compress the collection of 1092 human diploid genomes about 9,500 times. This result is about 4 times better than what is offered by the other existing compressors. Moreover, our algorithm is very fast as it processes the data with speed 200 MB/s on a modern workstation. In a consequence the proposed algorithm allows storing the complete genomic collections at low cost, e.g., the examined collection of 1092 human genomes needs only about 700 MB when compressed, what can be compared to about 6.7 TB of uncompressed FASTA files. The source code is available at http://sun.aei.polsl.pl/REFRESH/index.php?page=projects&project=gdc&subpage=about. PMID:26108279

GDC 2: Compression of large collections of genomes.

PubMed

Deorowicz, Sebastian; Danek, Agnieszka; Niemiec, Marcin

2015-06-25

The fall of prices of the high-throughput genome sequencing changes the landscape of modern genomics. A number of large scale projects aimed at sequencing many human genomes are in progress. Genome sequencing also becomes an important aid in the personalized medicine. One of the significant side effects of this change is a necessity of storage and transfer of huge amounts of genomic data. In this paper we deal with the problem of compression of large collections of complete genomic sequences. We propose an algorithm that is able to compress the collection of 1092 human diploid genomes about 9,500 times. This result is about 4 times better than what is offered by the other existing compressors. Moreover, our algorithm is very fast as it processes the data with speed 200 MB/s on a modern workstation. In a consequence the proposed algorithm allows storing the complete genomic collections at low cost, e.g., the examined collection of 1092 human genomes needs only about 700 MB when compressed, what can be compared to about 6.7 TB of uncompressed FASTA files. The source code is available at http://sun.aei.polsl.pl/REFRESH/index.php?page=projects&project=gdc&subpage=about.

Initial characterization of the large genome of the salamander Ambystoma mexicanum using shotgun and laser capture chromosome sequencing

PubMed Central

Keinath, Melissa C.; Timoshevskiy, Vladimir A.; Timoshevskaya, Nataliya Y.; Tsonis, Panagiotis A.; Voss, S. Randal; Smith, Jeramiah J.

2015-01-01

Vertebrates exhibit substantial diversity in genome size, and some of the largest genomes exist in species that uniquely inform diverse areas of basic and biomedical research. For example, the salamander Ambystoma mexicanum (the Mexican axolotl) is a model organism for studies of regeneration, development and genome evolution, yet its genome is ~10× larger than the human genome. As part of a hierarchical approach toward improving genome resources for the species, we generated 600 Gb of shotgun sequence data and developed methods for sequencing individual laser-captured chromosomes. Based on these data, we estimate that the A. mexicanum genome is ~32 Gb. Notably, as much as 19 Gb of the A. mexicanum genome can potentially be considered single copy, which presumably reflects the evolutionary diversification of mobile elements that accumulated during an ancient episode of genome expansion. Chromosome-targeted sequencing permitted the development of assemblies within the constraints of modern computational platforms, allowed us to place 2062 genes on the two smallest A. mexicanum chromosomes and resolves key events in the history of vertebrate genome evolution. Our analyses show that the capture and sequencing of individual chromosomes is likely to provide valuable information for the systematic sequencing, assembly and scaffolding of large genomes. PMID:26553646

Initial characterization of the large genome of the salamander Ambystoma mexicanum using shotgun and laser capture chromosome sequencing.

PubMed

Keinath, Melissa C; Timoshevskiy, Vladimir A; Timoshevskaya, Nataliya Y; Tsonis, Panagiotis A; Voss, S Randal; Smith, Jeramiah J

2015-11-10

Vertebrates exhibit substantial diversity in genome size, and some of the largest genomes exist in species that uniquely inform diverse areas of basic and biomedical research. For example, the salamander Ambystoma mexicanum (the Mexican axolotl) is a model organism for studies of regeneration, development and genome evolution, yet its genome is ~10× larger than the human genome. As part of a hierarchical approach toward improving genome resources for the species, we generated 600 Gb of shotgun sequence data and developed methods for sequencing individual laser-captured chromosomes. Based on these data, we estimate that the A. mexicanum genome is ~32 Gb. Notably, as much as 19 Gb of the A. mexicanum genome can potentially be considered single copy, which presumably reflects the evolutionary diversification of mobile elements that accumulated during an ancient episode of genome expansion. Chromosome-targeted sequencing permitted the development of assemblies within the constraints of modern computational platforms, allowed us to place 2062 genes on the two smallest A. mexicanum chromosomes and resolves key events in the history of vertebrate genome evolution. Our analyses show that the capture and sequencing of individual chromosomes is likely to provide valuable information for the systematic sequencing, assembly and scaffolding of large genomes.

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

The Peculiar Landscape of Repetitive Sequences in the Olive (Olea europaea L.) Genome

PubMed Central

Barghini, Elena; Natali, Lucia; Cossu, Rosa Maria; Giordani, Tommaso; Pindo, Massimo; Cattonaro, Federica; Scalabrin, Simone; Velasco, Riccardo; Morgante, Michele; Cavallini, Andrea

2014-01-01

Analyzing genome structure in different species allows to gain an insight into the evolution of plant genome size. Olive (Olea europaea L.) has a medium-sized haploid genome of 1.4 Gb, whose structure is largely uncharacterized, despite the growing importance of this tree as oil crop. Next-generation sequencing technologies and different computational procedures have been used to study the composition of the olive genome and its repetitive fraction. A total of 2.03 and 2.3 genome equivalents of Illumina and 454 reads from genomic DNA, respectively, were assembled following different procedures, which produced more than 200,000 differently redundant contigs, with mean length higher than 1,000 nt. Mapping Illumina reads onto the assembled sequences was used to estimate their redundancy. The genome data set was subdivided into highly and medium redundant and nonredundant contigs. By combining identification and mapping of repeated sequences, it was established that tandem repeats represent a very large portion of the olive genome (∼31% of the whole genome), consisting of six main families of different length, two of which were first discovered in these experiments. The other large redundant class in the olive genome is represented by transposable elements (especially long terminal repeat-retrotransposons). On the whole, the results of our analyses show the peculiar landscape of the olive genome, related to the massive amplification of tandem repeats, more than that reported for any other sequenced plant genome. PMID:24671744

The peculiar landscape of repetitive sequences in the olive (Olea europaea L.) genome.

PubMed

Barghini, Elena; Natali, Lucia; Cossu, Rosa Maria; Giordani, Tommaso; Pindo, Massimo; Cattonaro, Federica; Scalabrin, Simone; Velasco, Riccardo; Morgante, Michele; Cavallini, Andrea

2014-04-01

Analyzing genome structure in different species allows to gain an insight into the evolution of plant genome size. Olive (Olea europaea L.) has a medium-sized haploid genome of 1.4 Gb, whose structure is largely uncharacterized, despite the growing importance of this tree as oil crop. Next-generation sequencing technologies and different computational procedures have been used to study the composition of the olive genome and its repetitive fraction. A total of 2.03 and 2.3 genome equivalents of Illumina and 454 reads from genomic DNA, respectively, were assembled following different procedures, which produced more than 200,000 differently redundant contigs, with mean length higher than 1,000 nt. Mapping Illumina reads onto the assembled sequences was used to estimate their redundancy. The genome data set was subdivided into highly and medium redundant and nonredundant contigs. By combining identification and mapping of repeated sequences, it was established that tandem repeats represent a very large portion of the olive genome (∼31% of the whole genome), consisting of six main families of different length, two of which were first discovered in these experiments. The other large redundant class in the olive genome is represented by transposable elements (especially long terminal repeat-retrotransposons). On the whole, the results of our analyses show the peculiar landscape of the olive genome, related to the massive amplification of tandem repeats, more than that reported for any other sequenced plant genome.

BAC-pool 454-sequencing: A rapid and efficient approach to sequence complex tetraploid cotton genomes

USDA-ARS?s Scientific Manuscript database

New and emerging next generation sequencing technologies have been promising in reducing sequencing costs, but not significantly for complex polyploid plant genomes such as cotton. Large and highly repetitive genome of G. hirsutum (~2.5GB) is less amenable and cost-intensive with traditional BAC-by...

Sequencing of 15,622 gene-bearing BACs clarifies the gene-dense regions of the barley genome

USDA-ARS?s Scientific Manuscript database

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework....

Sequencing and annotation of mitochondrial genomes from individual parasitic helminths.

PubMed

Jex, Aaron R; Littlewood, D Timothy; Gasser, Robin B

2015-01-01

Mitochondrial (mt) genomics has significant implications in a range of fundamental areas of parasitology, including evolution, systematics, and population genetics as well as explorations of mt biochemistry, physiology, and function. Mt genomes also provide a rich source of markers to aid molecular epidemiological and ecological studies of key parasites. However, there is still a paucity of information on mt genomes for many metazoan organisms, particularly parasitic helminths, which has often related to challenges linked to sequencing from tiny amounts of material. The advent of next-generation sequencing (NGS) technologies has paved the way for low cost, high-throughput mt genomic research, but there have been obstacles, particularly in relation to post-sequencing assembly and analyses of large datasets. In this chapter, we describe protocols for the efficient amplification and sequencing of mt genomes from small portions of individual helminths, and highlight the utility of NGS platforms to expedite mt genomics. In addition, we recommend approaches for manual or semi-automated bioinformatic annotation and analyses to overcome the bioinformatic "bottleneck" to research in this area. Taken together, these approaches have demonstrated applicability to a range of parasites and provide prospects for using complete mt genomic sequence datasets for large-scale molecular systematic and epidemiological studies. In addition, these methods have broader utility and might be readily adapted to a range of other medium-sized molecular regions (i.e., 10-100 kb), including large genomic operons, and other organellar (e.g., plastid) and viral genomes.

Core-SINE blocks comprise a large fraction of monotreme genomes; implications for vertebrate chromosome evolution.

PubMed

Kirby, Patrick J; Greaves, Ian K; Koina, Edda; Waters, Paul D; Marshall Graves, Jennifer A

2007-01-01

The genomes of the egg-laying platypus and echidna are of particular interest because monotremes are the most basal mammal group. The chromosomal distribution of an ancient family of short interspersed repeats (SINEs), the core-SINEs, was investigated to better understand monotreme genome organization and evolution. Previous studies have identified the core-SINE as the predominant SINE in the platypus genome, and in this study we quantified, characterized and localized subfamilies. Dot blot analysis suggested that a very large fraction (32% of the platypus and 16% of the echidna genome) is composed of Mon core-SINEs. Core-SINE-specific primers were used to amplify PCR products from platypus and echidna genomic DNA. Sequence analysis suggests a common consensus sequence Mon 1-B, shared by platypus and echidna, as well as platypus-specific Mon 1-C and echidna specific Mon 1-D consensus sequences. FISH mapping of the Mon core-SINE products to platypus metaphase spreads demonstrates that the Mon-1C subfamily is responsible for the striking Mon core-SINE accumulation in the distal regions of the six large autosomal pairs and the largest X chromosome. This unusual distribution highlights the dichotomy between the seven large chromosome pairs and the 19 smaller pairs in the monotreme karyotype, which has some similarity to the macro- and micro-chromosomes of birds and reptiles, and suggests that accumulation of repetitive sequences may have enlarged small chromosomes in an ancestral vertebrate. In the forthcoming sequence of the platypus genome there are still large gaps, and the extensive Mon core-SINE accumulation on the distal regions of the six large autosomal pairs may provide one explanation for this missing sequence.

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

CGCI Investigators Reveal Comprehensive Landscape of Diffuse Large B-Cell Lymphoma (DLBCL) Genomes | Office of Cancer Genomics

Cancer.gov

Researchers from British Columbia Cancer Agency used whole genome sequencing to analyze 40 DLBCL cases and 13 cell lines in order to fill in the gaps of the complex landscape of DLBCL genomes. Their analysis, “Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing,” was published online in Blood on May 22. The authors are Ryan Morin, Marco Marra, and colleagues.  

An efficient approach to BAC based assembly of complex genomes.

PubMed

Visendi, Paul; Berkman, Paul J; Hayashi, Satomi; Golicz, Agnieszka A; Bayer, Philipp E; Ruperao, Pradeep; Hurgobin, Bhavna; Montenegro, Juan; Chan, Chon-Kit Kenneth; Staňková, Helena; Batley, Jacqueline; Šimková, Hana; Doležel, Jaroslav; Edwards, David

2016-01-01

There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

Construction of a large collection of small genome variations in French dairy and beef breeds using whole-genome sequences.

PubMed

Boussaha, Mekki; Michot, Pauline; Letaief, Rabia; Hozé, Chris; Fritz, Sébastien; Grohs, Cécile; Esquerré, Diane; Duchesne, Amandine; Philippe, Romain; Blanquet, Véronique; Phocas, Florence; Floriot, Sandrine; Rocha, Dominique; Klopp, Christophe; Capitan, Aurélien; Boichard, Didier

2016-11-15

In recent years, several bovine genome sequencing projects were carried out with the aim of developing genomic tools to improve dairy and beef production efficiency and sustainability. In this study, we describe the first French cattle genome variation dataset obtained by sequencing 274 whole genomes representing several major dairy and beef breeds. This dataset contains over 28 million single nucleotide polymorphisms (SNPs) and small insertions and deletions. Comparisons between sequencing results and SNP array genotypes revealed a very high genotype concordance rate, which indicates the good quality of our data. To our knowledge, this is the first large-scale catalog of small genomic variations in French dairy and beef cattle. This resource will contribute to the study of gene functions and population structure and also help to improve traits through genotype-guided selection.

Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.

PubMed

Wang, Yongjie; Kleespies, Regina G; Ramle, Moslim B; Jehle, Johannes A

2008-09-01

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

UFO: a web server for ultra-fast functional profiling of whole genome protein sequences.

PubMed

Meinicke, Peter

2009-09-02

Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address.

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

Cloud computing for genomic data analysis and collaboration.

PubMed

Langmead, Ben; Nellore, Abhinav

2018-04-01

Next-generation sequencing has made major strides in the past decade. Studies based on large sequencing data sets are growing in number, and public archives for raw sequencing data have been doubling in size every 18 months. Leveraging these data requires researchers to use large-scale computational resources. Cloud computing, a model whereby users rent computers and storage from large data centres, is a solution that is gaining traction in genomics research. Here, we describe how cloud computing is used in genomics for research and large-scale collaborations, and argue that its elasticity, reproducibility and privacy features make it ideally suited for the large-scale reanalysis of publicly available archived data, including privacy-protected data.

Transposable element junctions in marker development and genomic characterization of barley

USDA-ARS?s Scientific Manuscript database

Barley is a model plant in genomic studies of Triticeae species. A complete barley genome sequence will facilitate not only barley breeding programs, but also those for related species. However, the large genome size and high repetitive sequence content complicate the barley genome assembly. The ma...

A whole-genome, radiation hybrid map of wheat

USDA-ARS?s Scientific Manuscript database

Generating a reference sequence of bread wheat (Triticum aestivum L.) is a challenging task because of its large, highly repetitive and allopolyploid genome. Ordering of BAC- and NGS-based contigs in ongoing wheat genome-sequencing projects primarily uses recombination and comparative genomics-base...

Genome resequencing in Populus: Revealing large-scale genome variation and implications on specialized-trait genomics

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

Muchero, Wellington; Labbe, Jessy L; Priya, Ranjan

2014-01-01

To date, Populus ranks among a few plant species with a complete genome sequence and other highly developed genomic resources. With the first genome sequence among all tree species, Populus has been adopted as a suitable model organism for genomic studies in trees. However, far from being just a model species, Populus is a key renewable economic resource that plays a significant role in providing raw materials for the biofuel and pulp and paper industries. Therefore, aside from leading frontiers of basic tree molecular biology and ecological research, Populus leads frontiers in addressing global economic challenges related to fuel andmore » fiber production. The latter fact suggests that research aimed at improving quality and quantity of Populus as a raw material will likely drive the pursuit of more targeted and deeper research in order to unlock the economic potential tied in molecular biology processes that drive this tree species. Advances in genome sequence-driven technologies, such as resequencing individual genotypes, which in turn facilitates large scale SNP discovery and identification of large scale polymorphisms are key determinants of future success in these initiatives. In this treatise we discuss implications of genome sequence-enable technologies on Populus genomic and genetic studies of complex and specialized-traits.« less

BAC-pool sequencing and analysis of large segments of A12 and D12 homoeologous chromosomes in Upland cotton

USDA-ARS?s Scientific Manuscript database

New and emerging next generation sequencing technologies have reduced sequencing costs, but there is room for additional approaches that can be applied to complex polyploid plant genomes. Large (about 2.5GB) and highly repetitive tetraploid genome of G. hirsutum is still cost-intensive with traditi...

mySyntenyPortal: an application package to construct websites for synteny block analysis.

PubMed

Lee, Jongin; Lee, Daehwan; Sim, Mikang; Kwon, Daehong; Kim, Juyeon; Ko, Younhee; Kim, Jaebum

2018-06-05

Advances in sequencing technologies have facilitated large-scale comparative genomics based on whole genome sequencing. Constructing and investigating conserved genomic regions among multiple species (called synteny blocks) are essential in the comparative genomics. However, they require significant amounts of computational resources and time in addition to bioinformatics skills. Many web interfaces have been developed to make such tasks easier. However, these web interfaces cannot be customized for users who want to use their own set of genome sequences or definition of synteny blocks. To resolve this limitation, we present mySyntenyPortal, a stand-alone application package to construct websites for synteny block analyses by using users' own genome data. mySyntenyPortal provides both command line and web-based interfaces to build and manage websites for large-scale comparative genomic analyses. The websites can be also easily published and accessed by other users. To demonstrate the usability of mySyntenyPortal, we present an example study for building websites to compare genomes of three mammalian species (human, mouse, and cow) and show how they can be easily utilized to identify potential genes affected by genome rearrangements. mySyntenyPortal will contribute for extended comparative genomic analyses based on large-scale whole genome sequences by providing unique functionality to support the easy creation of interactive websites for synteny block analyses from user's own genome data.

The Genome Sequence of Avibacterium paragallinarum Strain CL Has a Large Repertoire of Insertion Sequence Elements.

PubMed

Horta-Valerdi, Guillermo; Sanchez-Alonso, Maria Patricia; Perez-Marquez, Victor M; Negrete-Abascal, Erasmo; Vaca-Pacheco, Sergio; Hernandez-Gonzalez, Ismael; Gomez-Lunar, Zulema; Olmedo-Álvarez, Gabriela; Vázquez-Cruz, Candelario

2017-04-13

The draft genome sequence of Avibacterium paragallinarum strain CL serovar C is reported here. The genome comprises 154 contigs corresponding to 2.4 Mb with 41% G+C content and many insertion sequence (IS) elements, a characteristic not previously reported in A. paragallinarum . Copyright © 2017 Horta-Valerdi et al.

Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing | Office of Cancer Genomics

Cancer.gov

Abstract: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer comprising at least two molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease.

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

Using genic sequence capture in combination with a syntenic pseudo genome to map a deletion mutant in a wheat species.

PubMed

Gardiner, Laura-Jayne; Gawroński, Piotr; Olohan, Lisa; Schnurbusch, Thorsten; Hall, Neil; Hall, Anthony

2014-12-01

Mapping-by-sequencing analyses have largely required a complete reference sequence and employed whole genome re-sequencing. In species such as wheat, no finished genome reference sequence is available. Additionally, because of its large genome size (17 Gb), re-sequencing at sufficient depth of coverage is not practical. Here, we extend the utility of mapping by sequencing, developing a bespoke pipeline and algorithm to map an early-flowering locus in einkorn wheat (Triticum monococcum L.) that is closely related to the bread wheat genome A progenitor. We have developed a genomic enrichment approach using the gene-rich regions of hexaploid bread wheat to design a 110-Mbp NimbleGen SeqCap EZ in solution capture probe set, representing the majority of genes in wheat. Here, we use the capture probe set to enrich and sequence an F2 mapping population of the mutant. The mutant locus was identified in T. monococcum, which lacks a complete genome reference sequence, by mapping the enriched data set onto pseudo-chromosomes derived from the capture probe target sequence, with a long-range order of genes based on synteny of wheat with Brachypodium distachyon. Using this approach we are able to map the region and identify a set of deleted genes within the interval. © 2014 The Authors.The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Construction of Pseudomolecule Sequences of the aus Rice Cultivar Kasalath for Comparative Genomics of Asian Cultivated Rice

PubMed Central

Sakai, Hiroaki; Kanamori, Hiroyuki; Arai-Kichise, Yuko; Shibata-Hatta, Mari; Ebana, Kaworu; Oono, Youko; Kurita, Kanako; Fujisawa, Hiroko; Katagiri, Satoshi; Mukai, Yoshiyuki; Hamada, Masao; Itoh, Takeshi; Matsumoto, Takashi; Katayose, Yuichi; Wakasa, Kyo; Yano, Masahiro; Wu, Jianzhong

2014-01-01

Having a deep genetic structure evolved during its domestication and adaptation, the Asian cultivated rice (Oryza sativa) displays considerable physiological and morphological variations. Here, we describe deep whole-genome sequencing of the aus rice cultivar Kasalath by using the advanced next-generation sequencing (NGS) technologies to gain a better understanding of the sequence and structural changes among highly differentiated cultivars. The de novo assembled Kasalath sequences represented 91.1% (330.55 Mb) of the genome and contained 35 139 expressed loci annotated by RNA-Seq analysis. We detected 2 787 250 single-nucleotide polymorphisms (SNPs) and 7393 large insertion/deletion (indel) sites (>100 bp) between Kasalath and Nipponbare, and 2 216 251 SNPs and 3780 large indels between Kasalath and 93-11. Extensive comparison of the gene contents among these cultivars revealed similar rates of gene gain and loss. We detected at least 7.39 Mb of inserted sequences and 40.75 Mb of unmapped sequences in the Kasalath genome in comparison with the Nipponbare reference genome. Mapping of the publicly available NGS short reads from 50 rice accessions proved the necessity and the value of using the Kasalath whole-genome sequence as an additional reference to capture the sequence polymorphisms that cannot be discovered by using the Nipponbare sequence alone. PMID:24578372

BACCardI--a tool for the validation of genomic assemblies, assisting genome finishing and intergenome comparison.

PubMed

Bartels, Daniela; Kespohl, Sebastian; Albaum, Stefan; Drüke, Tanja; Goesmann, Alexander; Herold, Julia; Kaiser, Olaf; Pühler, Alfred; Pfeiffer, Friedhelm; Raddatz, Günter; Stoye, Jens; Meyer, Folker; Schuster, Stephan C

2005-04-01

We provide the graphical tool BACCardI for the construction of virtual clone maps from standard assembler output files or BLAST based sequence comparisons. This new tool has been applied to numerous genome projects to solve various problems including (a) validation of whole genome shotgun assemblies, (b) support for contig ordering in the finishing phase of a genome project, and (c) intergenome comparison between related strains when only one of the strains has been sequenced and a large insert library is available for the other. The BACCardI software can seamlessly interact with various sequence assembly packages. Genomic assemblies generated from sequence information need to be validated by independent methods such as physical maps. The time-consuming task of building physical maps can be circumvented by virtual clone maps derived from read pair information of large insert libraries.

Construction and sequence sampling of deep-coverage, large-insert BAC libraries for three model lepidopteran species

PubMed Central

Wu, Chengcang; Proestou, Dina; Carter, Dorothy; Nicholson, Erica; Santos, Filippe; Zhao, Shaying; Zhang, Hong-Bin; Goldsmith, Marian R

2009-01-01

Background Manduca sexta, Heliothis virescens, and Heliconius erato represent three widely-used insect model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these insects are critical resources for many molecular studies, including physical mapping and genome sequencing, but not available to date. Results We report the construction and characterization of six large-insert BAC libraries for the three species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from 152–175 kb. We estimated that the genome coverage of each library ranged from 6–9 ×, with the two combined libraries of each species being equivalent to 13.0–16.3 × haploid genomes. The genome coverage, quality and utility of the libraries were further confirmed by library screening using 6~8 putative single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC ends of ~200 clones randomly selected from the libraries of each species. The data revealed that the genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the category of low complexity repeats, and are more abundant in retro-elements than DNA transposons. Among the species, the H. erato genome is somewhat more abundant in repeat elements and simple repeats than those of M. sexta and H. virescens. The BLAST analysis of the BAC end sequences suggested that the evolution of the three genomes is widely varied, with the genome of H. virescens being the most conserved as a typical lepidopteran, whereas both genomes of H. erato and M. sexta appear to have evolved significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences. Conclusion The high-quality and large-insert BAC libraries of the insects, together with the identified BACs containing genes of interest, provide valuable information, resources and tools for comprehensive understanding and studies of the insect genomes and for addressing many fundamental questions in Lepidoptera. The sample of the genomic sequences provides the first insight into the constitution and evolution of the insect genomes. PMID:19558662

Whole genome sequencing of a begomovirus-resistant tomato inbred reveals introgressions from wild Solanum species

USDA-ARS?s Scientific Manuscript database

The low cost of next generation sequencing (NGS) technology and the availability of a large number of well annotated plant genomes has made sequencing technology useful to breeding programs. With the published high quality tomato reference genome of the processing cultivar Heinz 1706, we can now uti...

Bioinformatics and genomic analysis of transposable elements in eukaryotic genomes.

PubMed

Janicki, Mateusz; Rooke, Rebecca; Yang, Guojun

2011-08-01

A major portion of most eukaryotic genomes are transposable elements (TEs). During evolution, TEs have introduced profound changes to genome size, structure, and function. As integral parts of genomes, the dynamic presence of TEs will continue to be a major force in reshaping genomes. Early computational analyses of TEs in genome sequences focused on filtering out "junk" sequences to facilitate gene annotation. When the high abundance and diversity of TEs in eukaryotic genomes were recognized, these early efforts transformed into the systematic genome-wide categorization and classification of TEs. The availability of genomic sequence data reversed the classical genetic approaches to discovering new TE families and superfamilies. Curated TE databases and their accurate annotation of genome sequences in turn facilitated the studies on TEs in a number of frontiers including: (1) TE-mediated changes of genome size and structure, (2) the influence of TEs on genome and gene functions, (3) TE regulation by host, (4) the evolution of TEs and their population dynamics, and (5) genomic scale studies of TE activity. Bioinformatics and genomic approaches have become an integral part of large-scale studies on TEs to extract information with pure in silico analyses or to assist wet lab experimental studies. The current revolution in genome sequencing technology facilitates further progress in the existing frontiers of research and emergence of new initiatives. The rapid generation of large-sequence datasets at record low costs on a routine basis is challenging the computing industry on storage capacity and manipulation speed and the bioinformatics community for improvement in algorithms and their implementations.

A Glance at Microsatellite Motifs from 454 Sequencing Reads of Watermelon Genomic DNA

USDA-ARS?s Scientific Manuscript database

A single 454 (Life Sciences Sequencing Technology) run of Charleston Gray watermelon (Citrullus lanatus var. lanatus) genomic DNA was performed and sequence data were assembled. A large scale identification of simple sequence repeat (SSR) was performed and SSR sequence data were used for the develo...

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.

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

Economic importance, taxonomic representation and scientific priority as drivers of genome sequencing projects.

PubMed

Vallée, Geneviève C; Muñoz, Daniella Santos; Sankoff, David

2016-11-11

Of the approximately two hundred sequenced plant genomes, how many and which ones were sequenced motivated by strictly or largely scientific considerations, and how many by chiefly economic, in a wide sense, incentives? And how large a role does publication opportunity play? In an integration of multiple disparate databases and other sources of information, we collect and analyze data on the size (number of species) in the plant orders and families containing sequenced genomes, on the trade value of these species, and of all the same-family or same-order species, and on the publication priority within the family and order. These data are subjected to multiple regression and other statistical analyses. We find that despite the initial importance of model organisms, it is clearly economic considerations that outweigh others in the choice of genome to be sequenced. This has important implications for generalizations about plant genomes, since human choices of plants to harvest (and cultivate) will have incurred many biases with respect to phenotypic characteristics and hence of genomic properties, and recent genomic evolution will also have been affected by human agricultural practices.

Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae).

PubMed

Walker, Joseph F; Zanis, Michael J; Emery, Nancy C

2014-04-01

Complete chloroplast genome studies can help resolve relationships among large, complex plant lineages such as Asteraceae. We present the first whole plastome from the Madieae tribe and compare its sequence variation to other chloroplast genomes in Asteraceae. We used high throughput sequencing to obtain the Lasthenia burkei chloroplast genome. We compared sequence structure and rates of molecular evolution in the small single copy (SSC), large single copy (LSC), and inverted repeat (IR) regions to those for eight Asteraceae accessions and one Solanaceae accession. The chloroplast sequence of L. burkei is 150 746 bp and contains 81 unique protein coding genes and 4 coding ribosomal RNA sequences. We identified three major inversions in the L. burkei chloroplast, all of which have been found in other Asteraceae lineages, and a previously unreported inversion in Lactuca sativa. Regions flanking inversions contained tRNA sequences, but did not have particularly high G + C content. Substitution rates varied among the SSC, LSC, and IR regions, and rates of evolution within each region varied among species. Some observed differences in rates of molecular evolution may be explained by the relative proportion of coding to noncoding sequence within regions. Rates of molecular evolution vary substantially within and among chloroplast genomes, and major inversion events may be promoted by the presence of tRNAs. Collectively, these results provide insight into different mechanisms that may promote intramolecular recombination and the inversion of large genomic regions in the plastome.

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

EGenBio: A Data Management System for Evolutionary Genomics and Biodiversity

PubMed Central

Nahum, Laila A; Reynolds, Matthew T; Wang, Zhengyuan O; Faith, Jeremiah J; Jonna, Rahul; Jiang, Zhi J; Meyer, Thomas J; Pollock, David D

2006-01-01

Background Evolutionary genomics requires management and filtering of large numbers of diverse genomic sequences for accurate analysis and inference on evolutionary processes of genomic and functional change. We developed Evolutionary Genomics and Biodiversity (EGenBio; ) to begin to address this. Description EGenBio is a system for manipulation and filtering of large numbers of sequences, integrating curated sequence alignments and phylogenetic trees, managing evolutionary analyses, and visualizing their output. EGenBio is organized into three conceptual divisions, Evolution, Genomics, and Biodiversity. The Genomics division includes tools for selecting pre-aligned sequences from different genes and species, and for modifying and filtering these alignments for further analysis. Species searches are handled through queries that can be modified based on a tree-based navigation system and saved. The Biodiversity division contains tools for analyzing individual sequences or sequence alignments, whereas the Evolution division contains tools involving phylogenetic trees. Alignments are annotated with analytical results and modification history using our PRAED format. A miscellaneous Tools section and Help framework are also available. EGenBio was developed around our comparative genomic research and a prototype database of mtDNA genomes. It utilizes MySQL-relational databases and dynamic page generation, and calls numerous custom programs. Conclusion EGenBio was designed to serve as a platform for tools and resources to ease combined analysis in evolution, genomics, and biodiversity. PMID:17118150

Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.

PubMed

Li, Qing; Hermanson, Peter J; Springer, Nathan M

2018-01-01

DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. Coupled with next-generation sequencing, whole-genome bisulfite sequencing (WGBS) allows DNA methylation to be evaluated at a genome-wide scale. Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.

A Tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free, and Suitable for Automation.

PubMed

Aubrey, Wayne; Riley, Michael C; Young, Michael; King, Ross D; Oliver, Stephen G; Clare, Amanda

2015-01-01

Many advances in synthetic biology require the removal of a large number of genomic elements from a genome. Most existing deletion methods leave behind markers, and as there are a limited number of markers, such methods can only be applied a fixed number of times. Deletion methods that recycle markers generally are either imprecise (remove untargeted sequences), or leave scar sequences which can cause genome instability and rearrangements. No existing marker recycling method is automation-friendly. We have developed a novel openly available deletion tool that consists of: 1) a method for deleting genomic elements that can be repeatedly used without limit, is precise, scar-free, and suitable for automation; and 2) software to design the method's primers. Our tool is sequence agnostic and could be used to delete large numbers of coding sequences, promoter regions, transcription factor binding sites, terminators, etc in a single genome. We have validated our tool on the deletion of non-essential open reading frames (ORFs) from S. cerevisiae. The tool is applicable to arbitrary genomes, and we provide primer sequences for the deletion of: 90% of the ORFs from the S. cerevisiae genome, 88% of the ORFs from S. pombe genome, and 85% of the ORFs from the L. lactis genome.

A Tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free, and Suitable for Automation

PubMed Central

Aubrey, Wayne; Riley, Michael C.; Young, Michael; King, Ross D.; Oliver, Stephen G.; Clare, Amanda

2015-01-01

Many advances in synthetic biology require the removal of a large number of genomic elements from a genome. Most existing deletion methods leave behind markers, and as there are a limited number of markers, such methods can only be applied a fixed number of times. Deletion methods that recycle markers generally are either imprecise (remove untargeted sequences), or leave scar sequences which can cause genome instability and rearrangements. No existing marker recycling method is automation-friendly. We have developed a novel openly available deletion tool that consists of: 1) a method for deleting genomic elements that can be repeatedly used without limit, is precise, scar-free, and suitable for automation; and 2) software to design the method’s primers. Our tool is sequence agnostic and could be used to delete large numbers of coding sequences, promoter regions, transcription factor binding sites, terminators, etc in a single genome. We have validated our tool on the deletion of non-essential open reading frames (ORFs) from S. cerevisiae. The tool is applicable to arbitrary genomes, and we provide primer sequences for the deletion of: 90% of the ORFs from the S. cerevisiae genome, 88% of the ORFs from S. pombe genome, and 85% of the ORFs from the L. lactis genome. PMID:26630677

The Release 6 reference sequence of the Drosophila melanogaster genome

DOE PAGES

Hoskins, Roger A.; Carlson, Joseph W.; Wan, Kenneth H.; ...

2015-01-14

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy andmore » middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. In conclusion, further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.« less

The Release 6 reference sequence of the Drosophila melanogaster genome

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

Hoskins, Roger A.; Carlson, Joseph W.; Wan, Kenneth H.

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy andmore » middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. In conclusion, further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.« less

Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula

PubMed Central

Macas, Jiří; Neumann, Pavel; Navrátilová, Alice

2007-01-01

Background Extraordinary size variation of higher plant nuclear genomes is in large part caused by differences in accumulation of repetitive DNA. This makes repetitive DNA of great interest for studying the molecular mechanisms shaping architecture and function of complex plant genomes. However, due to methodological constraints of conventional cloning and sequencing, a global description of repeat composition is available for only a very limited number of higher plants. In order to provide further data required for investigating evolutionary patterns of repeated DNA within and between species, we used a novel approach based on massive parallel sequencing which allowed a comprehensive repeat characterization in our model species, garden pea (Pisum sativum). Results Analysis of 33.3 Mb sequence data resulted in quantification and partial sequence reconstruction of major repeat families occurring in the pea genome with at least thousands of copies. Our results showed that the pea genome is dominated by LTR-retrotransposons, estimated at 140,000 copies/1C. Ty3/gypsy elements are less diverse and accumulated to higher copy numbers than Ty1/copia. This is in part due to a large population of Ogre-like retrotransposons which alone make up over 20% of the genome. In addition to numerous types of mobile elements, we have discovered a set of novel satellite repeats and two additional variants of telomeric sequences. Comparative genome analysis revealed that there are only a few repeat sequences conserved between pea and soybean genomes. On the other hand, all major families of pea mobile elements are well represented in M. truncatula. Conclusion We have demonstrated that even in a species with a relatively large genome like pea, where a single 454-sequencing run provided only 0.77% coverage, the generated sequences were sufficient to reconstruct and analyze major repeat families corresponding to a total of 35–48% of the genome. These data provide a starting point for further investigations of legume plant genomes based on their global comparative analysis and for the development of more sophisticated approaches for data mining. PMID:18031571

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.

Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity

PubMed Central

Pope, Welkin H; Bowman, Charles A; Russell, Daniel A; Jacobs-Sera, Deborah; Asai, David J; Cresawn, Steven G; Jacobs, William R; Hendrix, Roger W; Lawrence, Jeffrey G; Hatfull, Graham F; Abbazia, Patrick; Ababio, Amma; Adam, Naazneen

2015-01-01

The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery. DOI: http://dx.doi.org/10.7554/eLife.06416.001 PMID:25919952

Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.

PubMed

Pope, Welkin H; Bowman, Charles A; Russell, Daniel A; Jacobs-Sera, Deborah; Asai, David J; Cresawn, Steven G; Jacobs, William R; Hendrix, Roger W; Lawrence, Jeffrey G; Hatfull, Graham F

2015-04-28

The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.

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/ .

Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies

PubMed Central

2014-01-01

Background The size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination. Results We develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome. Conclusions In addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied. PMID:24647006

Complete Chloroplast Genome Sequences of Important Oilseed Crop Sesamum indicum L

PubMed Central

Yi, Dong-Keun; Kim, Ki-Joong

2012-01-01

Sesamum indicum is an important crop plant species for yielding oil. The complete chloroplast (cp) genome of S. indicum (GenBank acc no. JN637766) is 153,324 bp in length, and has a pair of inverted repeat (IR) regions consisting of 25,141 bp each. The lengths of the large single copy (LSC) and the small single copy (SSC) regions are 85,170 bp and 17,872 bp, respectively. Comparative cp DNA sequence analyses of S. indicum with other cp genomes reveal that the genome structure, gene order, gene and intron contents, AT contents, codon usage, and transcription units are similar to the typical angiosperm cp genomes. Nucleotide diversity of the IR region between Sesamum and three other cp genomes is much lower than that of the LSC and SSC regions in both the coding region and noncoding region. As a summary, the regional constraints strongly affect the sequence evolution of the cp genomes, while the functional constraints weakly affect the sequence evolution of cp genomes. Five short inversions associated with short palindromic sequences that form step-loop structures were observed in the chloroplast genome of S. indicum. Twenty-eight different simple sequence repeat loci have been detected in the chloroplast genome of S. indicum. Almost all of the SSR loci were composed of A or T, so this may also contribute to the A-T richness of the cp genome of S. indicum. Seven large repeated loci in the chloroplast genome of S. indicum were also identified and these loci are useful to developing S. indicum-specific cp genome vectors. The complete cp DNA sequences of S. indicum reported in this paper are prerequisite to modifying this important oilseed crop by cp genetic engineering techniques. PMID:22606240

Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species

PubMed Central

Legault, Boris A; Lopez-Lopez, Arantxa; Alba-Casado, Jose Carlos; Doolittle, W Ford; Bolhuis, Henk; Rodriguez-Valera, Francisco; Papke, R Thane

2006-01-01

Background Mature saturated brine (crystallizers) communities are largely dominated (>80% of cells) by the square halophilic archaeon "Haloquadratum walsbyi". The recent cultivation of the strain HBSQ001 and thesequencing of its genome allows comparison with the metagenome of this taxonomically simplified environment. Similar studies carried out in other extreme environments have revealed very little diversity in gene content among the cell lineages present. Results The metagenome of the microbial community of a crystallizer pond has been analyzed by end sequencing a 2000 clone fosmid library and comparing the sequences obtained with the genome sequence of "Haloquadratum walsbyi". The genome of the sequenced strain was retrieved nearly complete within this environmental DNA library. However, many ORF's that could be ascribed to the "Haloquadratum" metapopulation by common genome characteristics or scaffolding to the strain genome were not present in the specific sequenced isolate. Particularly, three regions of the sequenced genome were associated with multiple rearrangements and the presence of different genes from the metapopulation. Many transposition and phage related genes were found within this pool which, together with the associated atypical GC content in these areas, supports lateral gene transfer mediated by these elements as the most probable genetic cause of this variability. Additionally, these sequences were highly enriched in putative regulatory and signal transduction functions. Conclusion These results point to a large pan-genome (total gene repertoire of the genus/species) even in this highly specialized extremophile and at a single geographic location. The extensive gene repertoire is what might be expected of a population that exploits a diverse nutrient pool, resulting from the degradation of biomass produced at lower salinities. PMID:16820057

Sequencing of a new target genome: the Pediculus humanus humanus (Phthiraptera: Pediculidae) genome project.

PubMed

Pittendrigh, B R; Clark, J M; Johnston, J S; Lee, S H; Romero-Severson, J; Dasch, G A

2006-11-01

The human body louse, Pediculus humanus humanus (L.), and the human head louse, Pediculus humanus capitis, belong to the hemimetabolous order Phthiraptera. The body louse is the primary vector that transmits the bacterial agents of louse-borne relapsing fever, trench fever, and epidemic typhus. The genomes of the bacterial causative agents of several of these aforementioned diseases have been sequenced. Thus, determining the body louse genome will enhance studies of host-vector-pathogen interactions. Although not important as a major disease vector, head lice are of major social concern. Resistance to traditional pesticides used to control head and body lice have developed. It is imperative that new molecular targets be discovered for the development of novel compounds to control these insects. No complete genome sequence exists for a hemimetabolous insect species primarily because hemimetabolous insects often have large (2000 Mb) to very large (up to 16,300 Mb) genomes. Fortuitously, we determined that the human body louse has one of the smallest genome sizes known in insects, suggesting it may be a suitable choice as a minimal hemimetabolous genome in which many genes have been eliminated during its adaptation to human parasitism. Because many louse species infest birds and mammals, the body louse genome-sequencing project will facilitate studies of their comparative genomics. A 6-8X coverage of the body louse genome, plus sequenced expressed sequence tags, should provide the entomological, evolutionary biology, medical, and public health communities with useful genetic information.

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.

Long-range barcode labeling-sequencing

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

Chen, Feng; Zhang, Tao; Singh, Kanwar K.

Methods for sequencing single large DNA molecules by clonal multiple displacement amplification using barcoded primers. Sequences are binned based on barcode sequences and sequenced using a microdroplet-based method for sequencing large polynucleotide templates to enable assembly of haplotype-resolved complex genomes and metagenomes.

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

Complete nucleotide sequence of the Cryptomeria japonica D. Don. chloroplast genome and comparative chloroplast genomics: diversified genomic structure of coniferous species.

PubMed

Hirao, Tomonori; Watanabe, Atsushi; Kurita, Manabu; Kondo, Teiji; Takata, Katsuhiko

2008-06-23

The recent determination of complete chloroplast (cp) genomic sequences of various plant species has enabled numerous comparative analyses as well as advances in plant and genome evolutionary studies. In angiosperms, the complete cp genome sequences of about 70 species have been determined, whereas those of only three gymnosperm species, Cycas taitungensis, Pinus thunbergii, and Pinus koraiensis have been established. The lack of information regarding the gene content and genomic structure of gymnosperm cp genomes may severely hamper further progress of plant and cp genome evolutionary studies. To address this need, we report here the complete nucleotide sequence of the cp genome of Cryptomeria japonica, the first in the Cupressaceae sensu lato of gymnosperms, and provide a comparative analysis of their gene content and genomic structure that illustrates the unique genomic features of gymnosperms. The C. japonica cp genome is 131,810 bp in length, with 112 single copy genes and two duplicated (trnI-CAU, trnQ-UUG) genes that give a total of 116 genes. Compared to other land plant cp genomes, the C. japonica cp has lost one of the relevant large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperms, such as Cycas and Gingko, and additionally has completely lost its trnR-CCG, partially lost its trnT-GGU, and shows diversification of accD. The genomic structure of the C. japonica cp genome also differs significantly from those of other plant species. For example, we estimate that a minimum of 15 inversions would be required to transform the gene organization of the Pinus thunbergii cp genome into that of C. japonica. In the C. japonica cp genome, direct repeat and inverted repeat sequences are observed at the inversion and translocation endpoints, and these sequences may be associated with the genomic rearrangements. The observed differences in genomic structure between C. japonica and other land plants, including pines, strongly support the theory that the large IRs stabilize the cp genome. Furthermore, the deleted large IR and the numerous genomic rearrangements that have occurred in the C. japonica cp genome provide new insights into both the evolutionary lineage of coniferous species in gymnosperm and the evolution of the cp genome.

Draft genome of the living fossil Ginkgo biloba.

PubMed

Guan, Rui; Zhao, Yunpeng; Zhang, He; Fan, Guangyi; Liu, Xin; Zhou, Wenbin; Shi, Chengcheng; Wang, Jiahao; Liu, Weiqing; Liang, Xinming; Fu, Yuanyuan; Ma, Kailong; Zhao, Lijun; Zhang, Fumin; Lu, Zuhong; Lee, Simon Ming-Yuen; Xu, Xun; Wang, Jian; Yang, Huanming; Fu, Chengxin; Ge, Song; Chen, Wenbin

2016-11-21

Ginkgo biloba L. (Ginkgoaceae) is one of the most distinctive plants. It possesses a suite of fascinating characteristics including a large genome, outstanding resistance/tolerance to abiotic and biotic stresses, and dioecious reproduction, making it an ideal model species for biological studies. However, the lack of a high-quality genome sequence has been an impediment to our understanding of its biology and evolution. The 10.61 Gb genome sequence containing 41,840 annotated genes was assembled in the present study. Repetitive sequences account for 76.58% of the assembled sequence, and long terminal repeat retrotransposons (LTR-RTs) are particularly prevalent. The diversity and abundance of LTR-RTs is due to their gradual accumulation and a remarkable amplification between 16 and 24 million years ago, and they contribute to the long introns and large genome. Whole genome duplication (WGD) may have occurred twice, with an ancient WGD consistent with that shown to occur in other seed plants, and a more recent event specific to ginkgo. Abundant gene clusters from tandem duplication were also evident, and enrichment of expanded gene families indicates a remarkable array of chemical and antibacterial defense pathways. The ginkgo genome consists mainly of LTR-RTs resulting from ancient gradual accumulation and two WGD events. The multiple defense mechanisms underlying the characteristic resilience of ginkgo are fostered by a remarkable enrichment in ancient duplicated and ginkgo-specific gene clusters. The present study sheds light on sequencing large genomes, and opens an avenue for further genetic and evolutionary research.

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.

Complete Genome Sequence of Bradyrhizobium sp. Strain CCGE-LA001, Isolated from Field Nodules of the Enigmatic Wild Bean Phaseolus microcarpus

PubMed Central

Servín-Garcidueñas, Luis E.; Rogel, Marco A.; Ormeño-Orrillo, Ernesto; Zayas-del Moral, Alejandra; Sánchez, Federico

2016-01-01

We present the complete genome sequence of Bradyrhizobium sp. strain CCGE-LA001, a nitrogen-fixing bacterium isolated from nodules of Phaseolus microcarpus. Strain CCGE-LA001 represents the first sequenced bradyrhizobial strain obtained from a wild Phaseolus sp. Its genome revealed a large and novel symbiotic island. PMID:26988045

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

Comparative analysis of the complete sequence of the plastid genome of Parthenium argentatum and identification of DNA barcodes to differentiate Parthenium species and lines

PubMed Central

2009-01-01

Background Parthenium argentatum (guayule) is an industrial crop that produces latex, which was recently commercialized as a source of latex rubber safe for people with Type I latex allergy. The complete plastid genome of P. argentatum was sequenced. The sequence provides important information useful for genetic engineering strategies. Comparison to the sequences of plastid genomes from three other members of the Asteraceae, Lactuca sativa, Guitozia abyssinica and Helianthus annuus revealed details of the evolution of the four genomes. Chloroplast-specific DNA barcodes were developed for identification of Parthenium species and lines. Results The complete plastid genome of P. argentatum is 152,803 bp. Based on the overall comparison of individual protein coding genes with those in L. sativa, G. abyssinica and H. annuus, we demonstrate that the P. argentatum chloroplast genome sequence is most closely related to that of H. annuus. Similar to chloroplast genomes in G. abyssinica, L. sativa and H. annuus, the plastid genome of P. argentatum has a large 23 kb inversion with a smaller 3.4 kb inversion, within the large inversion. Using the matK and psbA-trnH spacer chloroplast DNA barcodes, three of the four Parthenium species tested, P. tomentosum, P. hysterophorus and P. schottii, can be differentiated from P. argentatum. In addition, we identified lines within P. argentatum. Conclusion The genome sequence of the P. argentatum chloroplast will enrich the sequence resources of plastid genomes in commercial crops. The availability of the complete plastid genome sequence may facilitate transformation efficiency by using the precise sequence of endogenous flanking sequences and regulatory elements in chloroplast transformation vectors. The DNA barcoding study forms the foundation for genetic identification of commercially significant lines of P. argentatum that are important for producing latex. PMID:19917140

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.

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.

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.

Structure and evolution of cereal genomes.

PubMed

Paterson, Andrew H; Bowers, John E; Peterson, Daniel G; Estill, James C; Chapman, Brad A

2003-12-01

The cereal species, of central importance to our diet, began to diverge 50-70 million years ago. For the past few thousand years, these species have undergone largely parallel selection regimes associated with domestication and improvement. The rice genome sequence provides a platform for organizing information about diverse cereals, and together with genetic maps and sequence samples from other cereals is yielding new insights into both the shared and the independent dimensions of cereal evolution. New data and population-based approaches are identifying genes that have been involved in cereal improvement. Reduced-representation sequencing promises to accelerate gene discovery in many large-genome cereals, and to better link the under-explored genomes of 'orphan' cereals with state-of-the-art knowledge.

Correcting Inconsistencies and Errors in Bacterial Genome Metadata Using an Automated Curation Tool in Excel (AutoCurE).

PubMed

Schmedes, Sarah E; King, Jonathan L; Budowle, Bruce

2015-01-01

Whole-genome data are invaluable for large-scale comparative genomic studies. Current sequencing technologies have made it feasible to sequence entire bacterial genomes with relative ease and time with a substantially reduced cost per nucleotide, hence cost per genome. More than 3,000 bacterial genomes have been sequenced and are available at the finished status. Publically available genomes can be readily downloaded; however, there are challenges to verify the specific supporting data contained within the download and to identify errors and inconsistencies that may be present within the organizational data content and metadata. AutoCurE, an automated tool for bacterial genome database curation in Excel, was developed to facilitate local database curation of supporting data that accompany downloaded genomes from the National Center for Biotechnology Information. AutoCurE provides an automated approach to curate local genomic databases by flagging inconsistencies or errors by comparing the downloaded supporting data to the genome reports to verify genome name, RefSeq accession numbers, the presence of archaea, BioProject/UIDs, and sequence file descriptions. Flags are generated for nine metadata fields if there are inconsistencies between the downloaded genomes and genomes reports and if erroneous or missing data are evident. AutoCurE is an easy-to-use tool for local database curation for large-scale genome data prior to downstream analyses.

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

Efficient high-throughput sequencing of a laser microdissected chromosome arm

PubMed Central

2013-01-01

Background Genomic sequence assemblies are key tools for a broad range of gene function and evolutionary studies. The diploid amphibian Xenopus tropicalis plays a pivotal role in these fields due to its combination of experimental flexibility, diploid genome, and early-branching tetrapod taxonomic position, having diverged from the amniote lineage ~360 million years ago. A genome assembly and a genetic linkage map have recently been made available. Unfortunately, large gaps in the linkage map attenuate long-range integrity of the genome assembly. Results We laser dissected the short arm of X. tropicalis chromosome 7 for next generation sequencing and computational mapping to the reference genome. This arm is of particular interest as it encodes the sex determination locus, but its genetic map contains large gaps which undermine available genome assemblies. Whole genome amplification of 15 laser-microdissected 7p arms followed by next generation sequencing yielded ~35 million reads, over four million of which uniquely mapped to the X. tropicalis genome. Our analysis placed more than 200 previously unmapped scaffolds on the analyzed chromosome arm, providing valuable low-resolution physical map information for de novo genome assembly. Conclusion We present a new approach for improving and validating genetic maps and sequence assemblies. Whole genome amplification of 15 microdissected chromosome arms provided sufficient high-quality material for localizing previously unmapped scaffolds and genes as well as recognizing mislocalized scaffolds. PMID:23714049

Targeted enrichment strategies for next-generation plant biology

Treesearch

Richard Cronn; Brian J. Knaus; Aaron Liston; Peter J. Maughan; Matthew Parks; John V. Syring; Joshua Udall

2012-01-01

The dramatic advances offered by modem DNA sequencers continue to redefine the limits of what can be accomplished in comparative plant biology. Even with recent achievements, however, plant genomes present obstacles that can make it difficult to execute large-scale population and phylogenetic studies on next-generation sequencing platforms. Factors like large genome...

DArT Markers Effectively Target Gene Space in the Rye Genome

PubMed Central

Gawroński, Piotr; Pawełkowicz, Magdalena; Tofil, Katarzyna; Uszyński, Grzegorz; Sharifova, Saida; Ahluwalia, Shivaksh; Tyrka, Mirosław; Wędzony, Maria; Kilian, Andrzej; Bolibok-Brągoszewska, Hanna

2016-01-01

Large genome size and complexity hamper considerably the genomics research in relevant species. Rye (Secale cereale L.) has one of the largest genomes among cereal crops and repetitive sequences account for over 90% of its length. Diversity Arrays Technology is a high-throughput genotyping method, in which a preferential sampling of gene-rich regions is achieved through the use of methylation sensitive restriction enzymes. We obtained sequences of 6,177 rye DArT markers and following a redundancy analysis assembled them into 3,737 non-redundant sequences, which were then used in homology searches against five Pooideae sequence sets. In total 515 DArT sequences could be incorporated into publicly available rye genome zippers providing a starting point for the integration of DArT- and transcript-based genomics resources in rye. Using Blast2Go pipeline we attributed putative gene functions to 1101 (29.4%) of the non-redundant DArT marker sequences, including 132 sequences with putative disease resistance-related functions, which were found to be preferentially located in the 4RL and 6RL chromosomes. Comparative analysis based on the DArT sequences revealed obvious inconsistencies between two recently published high density consensus maps of rye. Furthermore we demonstrated that DArT marker sequences can be a source of SSR polymorphisms. Obtained data demonstrate that DArT markers effectively target gene space in the large, complex, and repetitive rye genome. Through the annotation of putative gene functions and the alignment of DArT sequences relative to reference genomes we obtained information, that will complement the results of the studies, where DArT genotyping was deployed, by simplifying the gene ontology and microcolinearity based identification of candidate genes. PMID:27833625

DArT Markers Effectively Target Gene Space in the Rye Genome.

PubMed

Gawroński, Piotr; Pawełkowicz, Magdalena; Tofil, Katarzyna; Uszyński, Grzegorz; Sharifova, Saida; Ahluwalia, Shivaksh; Tyrka, Mirosław; Wędzony, Maria; Kilian, Andrzej; Bolibok-Brągoszewska, Hanna

2016-01-01

Large genome size and complexity hamper considerably the genomics research in relevant species. Rye ( Secale cereale L.) has one of the largest genomes among cereal crops and repetitive sequences account for over 90% of its length. Diversity Arrays Technology is a high-throughput genotyping method, in which a preferential sampling of gene-rich regions is achieved through the use of methylation sensitive restriction enzymes. We obtained sequences of 6,177 rye DArT markers and following a redundancy analysis assembled them into 3,737 non-redundant sequences, which were then used in homology searches against five Pooideae sequence sets. In total 515 DArT sequences could be incorporated into publicly available rye genome zippers providing a starting point for the integration of DArT- and transcript-based genomics resources in rye. Using Blast2Go pipeline we attributed putative gene functions to 1101 (29.4%) of the non-redundant DArT marker sequences, including 132 sequences with putative disease resistance-related functions, which were found to be preferentially located in the 4RL and 6RL chromosomes. Comparative analysis based on the DArT sequences revealed obvious inconsistencies between two recently published high density consensus maps of rye. Furthermore we demonstrated that DArT marker sequences can be a source of SSR polymorphisms. Obtained data demonstrate that DArT markers effectively target gene space in the large, complex, and repetitive rye genome. Through the annotation of putative gene functions and the alignment of DArT sequences relative to reference genomes we obtained information, that will complement the results of the studies, where DArT genotyping was deployed, by simplifying the gene ontology and microcolinearity based identification of candidate genes.

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

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

PubMed

Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

2016-01-01

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

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...

Deep whole-genome sequencing of 90 Han Chinese genomes.

PubMed

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

2017-09-01

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

Complete Chloroplast Genome Sequences of Mongolia Medicine Artemisia frigida and Phylogenetic Relationships with Other Plants

PubMed Central

Liu, Yue; Huo, Naxin; Dong, Lingli; Wang, Yi; Zhang, Shuixian; Young, Hugh A.; Feng, Xiaoxiao; Gu, Yong Qiang

2013-01-01

Background Artemisia frigida Willd. is an important Mongolian traditional medicinal plant with pharmacological functions of stanch and detumescence. However, there is little sequence and genomic information available for Artemisia frigida, which makes phylogenetic identification, evolutionary studies, and genetic improvement of its value very difficult. We report the complete chloroplast genome sequence of Artemisia frigida based on 454 pyrosequencing. Methodology/Principal Findings The complete chloroplast genome of Artemisia frigida is 151,076 bp including a large single copy (LSC) region of 82,740 bp, a small single copy (SSC) region of 18,394 bp and a pair of inverted repeats (IRs) of 24,971 bp. The genome contains 114 unique genes and 18 duplicated genes. The chloroplast genome of Artemisia frigida contains a small 3.4 kb inversion within a large 23 kb inversion in the LSC region, a unique feature in Asteraceae. The gene order in the SSC region of Artemisia frigida is inverted compared with the other 6 Asteraceae species with the chloroplast genomes sequenced. This inversion is likely caused by an intramolecular recombination event only occurred in Artemisia frigida. The existence of rich SSR loci in the Artemisia frigida chloroplast genome provides a rare opportunity to study population genetics of this Mongolian medicinal plant. Phylogenetic analysis demonstrates a sister relationship between Artemisia frigida and four other species in Asteraceae, including Ageratina adenophora, Helianthus annuus, Guizotia abyssinica and Lactuca sativa, based on 61 protein-coding sequences. Furthermore, Artemisia frigida was placed in the tribe Anthemideae in the subfamily Asteroideae (Asteraceae) based on ndhF and trnL-F sequence comparisons. Conclusion The chloroplast genome sequence of Artemisia frigida was assembled and analyzed in this study, representing the first plastid genome sequenced in the Anthemideae tribe. This complete chloroplast genome sequence will be useful for molecular ecology and molecular phylogeny studies within Artemisia species and also within the Asteraceae family. PMID:23460871

Major soybean maturity gene haplotypes revealed by SNPViz analysis of 72 sequenced soybean genomes

USDA-ARS?s Scientific Manuscript database

In this Genomics Era, vast amounts of next generation sequencing data have become publicly-available for multiple genomes across hundreds of species. Analysis of these large-scale datasets can become cumbersome, especially when comparing nucleotide polymorphisms across many samples within a dataset...

CattleTickBase: An integrated Internet-based bioinformatics resource for Rhipicephalus (Boophilus) microplus

USDA-ARS?s Scientific Manuscript database

The Rhipicephalus microplus genome is large and complex in structure, making a genome sequence difficult to assemble and costly to resource the required bioinformatics. In light of this, a consortium of international collaborators was formed to pool resources to begin sequencing this genome. We have...

Recombination-dependent replication and gene conversion homogenize repeat sequences and diversify plastid genome structure.

PubMed

Ruhlman, Tracey A; Zhang, Jin; Blazier, John C; Sabir, Jamal S M; Jansen, Robert K

2017-04-01

There is a misinterpretation in the literature regarding the variable orientation of the small single copy region of plastid genomes (plastomes). The common phenomenon of small and large single copy inversion, hypothesized to occur through intramolecular recombination between inverted repeats (IR) in a circular, single unit-genome, in fact, more likely occurs through recombination-dependent replication (RDR) of linear plastome templates. If RDR can be primed through both intra- and intermolecular recombination, then this mechanism could not only create inversion isomers of so-called single copy regions, but also an array of alternative sequence arrangements. We used Illumina paired-end and PacBio single-molecule real-time (SMRT) sequences to characterize repeat structure in the plastome of Monsonia emarginata (Geraniaceae). We used OrgConv and inspected nucleotide alignments to infer ancestral nucleotides and identify gene conversion among repeats and mapped long (>1 kb) SMRT reads against the unit-genome assembly to identify alternative sequence arrangements. Although M. emarginata lacks the canonical IR, we found that large repeats (>1 kilobase; kb) represent ∼22% of the plastome nucleotide content. Among the largest repeats (>2 kb), we identified GC-biased gene conversion and mapping filtered, long SMRT reads to the M. emarginata unit-genome assembly revealed alternative, substoichiometric sequence arrangements. We offer a model based on RDR and gene conversion between long repeated sequences in the M. emarginata plastome and provide support that both intra-and intermolecular recombination between large repeats, particularly in repeat-rich plastomes, varies unit-genome structure while homogenizing the nucleotide sequence of repeats. © 2017 Botanical Society of America.

De novo assembly of human genomes with massively parallel short read sequencing.

PubMed

Li, Ruiqiang; Zhu, Hongmei; Ruan, Jue; Qian, Wubin; Fang, Xiaodong; Shi, Zhongbin; Li, Yingrui; Li, Shengting; Shan, Gao; Kristiansen, Karsten; Li, Songgang; Yang, Huanming; Wang, Jian; Wang, Jun

2010-02-01

Next-generation massively parallel DNA sequencing technologies provide ultrahigh throughput at a substantially lower unit data cost; however, the data are very short read length sequences, making de novo assembly extremely challenging. Here, we describe a novel method for de novo assembly of large genomes from short read sequences. We successfully assembled both the Asian and African human genome sequences, achieving an N50 contig size of 7.4 and 5.9 kilobases (kb) and scaffold of 446.3 and 61.9 kb, respectively. The development of this de novo short read assembly method creates new opportunities for building reference sequences and carrying out accurate analyses of unexplored genomes in a cost-effective way.

Legume genome evolution viewed through the Medicago truncatula and Lotus japonicus genomes

PubMed Central

Cannon, Steven B.; Sterck, Lieven; Rombauts, Stephane; Sato, Shusei; Cheung, Foo; Gouzy, Jérôme; Wang, Xiaohong; Mudge, Joann; Vasdewani, Jayprakash; Schiex, Thomas; Spannagl, Manuel; Monaghan, Erin; Nicholson, Christine; Humphray, Sean J.; Schoof, Heiko; Mayer, Klaus F. X.; Rogers, Jane; Quétier, Francis; Oldroyd, Giles E.; Debellé, Frédéric; Cook, Douglas R.; Retzel, Ernest F.; Roe, Bruce A.; Town, Christopher D.; Tabata, Satoshi; Van de Peer, Yves; Young, Nevin D.

2006-01-01

Genome sequencing of the model legumes, Medicago truncatula and Lotus japonicus, provides an opportunity for large-scale sequence-based comparison of two genomes in the same plant family. Here we report synteny comparisons between these species, including details about chromosome relationships, large-scale synteny blocks, microsynteny within blocks, and genome regions lacking clear correspondence. The Lotus and Medicago genomes share a minimum of 10 large-scale synteny blocks, each with substantial collinearity and frequently extending the length of whole chromosome arms. The proportion of genes syntenic and collinear within each synteny block is relatively homogeneous. Medicago–Lotus comparisons also indicate similar and largely homogeneous gene densities, although gene-containing regions in Mt occupy 20–30% more space than Lj counterparts, primarily because of larger numbers of Mt retrotransposons. Because the interpretation of genome comparisons is complicated by large-scale genome duplications, we describe synteny, synonymous substitutions and phylogenetic analyses to identify and date a probable whole-genome duplication event. There is no direct evidence for any recent large-scale genome duplication in either Medicago or Lotus but instead a duplication predating speciation. Phylogenetic comparisons place this duplication within the Rosid I clade, clearly after the split between legumes and Salicaceae (poplar). PMID:17003129

Imputation of unordered markers and the impact on genomic selection accuracy

USDA-ARS?s Scientific Manuscript database

Genomic selection, a breeding method that promises to accelerate rates of genetic gain, requires dense, genome-wide marker data. Genotyping-by-sequencing can generate a large number of de novo markers. However, without a reference genome, these markers are unordered and typically have a large propo...

Evaluation of Targeted Sequencing for Transcriptional Analysis of Archival Formalin-Fixed Paraffin-Embedded (FFPE) Samples

EPA Science Inventory

Next-generation sequencing provides unprecedented access to genomic information in archival FFPE tissue samples. However, costs and technical challenges related to RNA isolation and enrichment limit use of whole-genome RNA-sequencing for large-scale studies of FFPE specimens. Rec...

A new strategy for genome assembly using short sequence reads and reduced representation libraries.

PubMed

Young, Andrew L; Abaan, Hatice Ozel; Zerbino, Daniel; Mullikin, James C; Birney, Ewan; Margulies, Elliott H

2010-02-01

We have developed a novel approach for using massively parallel short-read sequencing to generate fast and inexpensive de novo genomic assemblies comparable to those generated by capillary-based methods. The ultrashort (<100 base) sequences generated by this technology pose specific biological and computational challenges for de novo assembly of large genomes. To account for this, we devised a method for experimentally partitioning the genome using reduced representation (RR) libraries prior to assembly. We use two restriction enzymes independently to create a series of overlapping fragment libraries, each containing a tractable subset of the genome. Together, these libraries allow us to reassemble the entire genome without the need of a reference sequence. As proof of concept, we applied this approach to sequence and assembled the majority of the 125-Mb Drosophila melanogaster genome. We subsequently demonstrate the accuracy of our assembly method with meaningful comparisons against the current available D. melanogaster reference genome (dm3). The ease of assembly and accuracy for comparative genomics suggest that our approach will scale to future mammalian genome-sequencing efforts, saving both time and money without sacrificing quality.

Characterization of the complete chloroplast genome of Platycarya strobilacea (Juglandaceae)

Treesearch

Jing Yan; Kai Han; Shuyun Zeng; Peng Zhao; Keith Woeste; Jianfang Li; Zhan-Lin Liu

2017-01-01

The whole chloroplast genome (cp genome) sequence of Platycarya strobilacea was characterized from Illumina pair-end sequencing data. The complete cp genome was 160,994 bp in length and contained a large single copy region (LSC) of 90,225 bp and a small single copy region (SSC) of 18,371 bp, which were separated by a pair of inverted repeat regions...

Home - The Cancer Genome Atlas - Cancer Genome - TCGA

Cancer.gov

The Cancer Genome Atlas (TCGA) is a comprehensive and coordinated effort to accelerate our understanding of the molecular basis of cancer through the application of genome analysis technologies, including large-scale genome sequencing.

Genome empowerment for the Puerto Rican parrot – Amazona vittata

PubMed Central

2012-01-01

A unique community-funded project in Puerto Rico has launched whole-genome sequencing of the critically endangered Puerto Rican Parrot (Amazona vittata), with interpretation by genome bioinformaticians and students, and deposition into public online databases. This is the first article that focuses on the whole genome of a parrot species, one endemic to the USA and recently threatened with extinction. It provides invaluable conservation tools and a vivid example of hopeful prospects for future genome assessment of so many new species. It also demonstrates inventive ways for smaller institutions to contribute to a field largely considered the domain of large sequencing centers. PMID:23587407

Exploring the feasibility of using copy number variants as genetic markers through large-scale whole genome sequencing experiments

USDA-ARS?s Scientific Manuscript database

Copy number variants (CNV) are large scale duplications or deletions of genomic sequence that are caused by a diverse set of molecular phenomena that are distinct from single nucleotide polymorphism (SNP) formation. Due to their different mechanisms of formation, CNVs are often difficult to track us...

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.

Selected Insights from Application of Whole Genome Sequencing for Outbreak Investigations

PubMed Central

Le, Vien Thi Minh; Diep, Binh An

2014-01-01

Purpose of review The advent of high-throughput whole genome sequencing has the potential to revolutionize the conduct of outbreak investigation. Because of its ultimate pathogen strain resolution, whole genome sequencing could augment traditional epidemiologic investigations of infectious disease outbreaks. Recent findings The combination of whole genome sequencing and intensive epidemiologic analysis provided new insights on the sources and transmission dynamics of large-scale epidemics caused by Escherichia coli and Vibrio cholerae, nosocomial outbreaks caused by methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, and Mycobacterium abscessus, community-centered outbreaks caused by Mycobacterium tuberculosis, and natural disaster-associated outbreak caused by environmentally acquired molds. Summary When combined with traditional epidemiologic investigation, whole genome sequencing has proven useful for elucidating sources and transmission dynamics of disease outbreaks. Development of a fully automated bioinformatics pipeline for analysis of whole genome sequence data is much needed to make this powerful tool more widely accessible. PMID:23856896

Genomic diversity of the human intestinal parasite Entamoeba histolytica

PubMed Central

2012-01-01

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

Draft Genome Sequences of 510 Listeria monocytogenes Strains from Food Isolates and Human Listeriosis Cases from Northern Italy.

PubMed

Lomonaco, Sara; Gallina, Silvia; Filipello, Virginia; Sanchez Leon, Maria; Kastanis, George John; Allard, Marc; Brown, Eric; Amato, Ettore; Pontello, Mirella; Decastelli, Lucia

2018-01-18

Listeriosis outbreaks are frequently multistate/multicountry outbreaks, underlining the importance of molecular typing data for several diverse and well-characterized isolates. Large-scale whole-genome sequencing studies on Listeria monocytogenes isolates from non-U.S. locations have been limited. Herein, we describe the draft genome sequences of 510 L. monocytogenes isolates from northern Italy from different sources.

Complete genome sequence of Aminobacterium colombiense type strain (ALA-1T)

PubMed Central

Chertkov, Olga; Sikorski, Johannes; Brambilla, Evelyne; Lapidus, Alla; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Lucas, Susan; Tice, Hope; Cheng, Jan-Fang; Han, Cliff; Detter, John C.; Bruce, David; Tapia, Roxanne; Goodwin, Lynne; Pitluck, Sam; Liolios, Konstantinos; Ivanova, Natalia; Mavromatis, Konstantinos; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jeffries, Cynthia D.; Spring, Stefan; Rohde, Manfred; Göker, Markus; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C.; Klenk, Hans-Peter

2010-01-01

Aminobacterium colombiense Baena et al. 1999 is the type species of the genus Aminobacterium. This genus is of large interest because of its isolated phylogenetic location in the family Synergistaceae, its strictly anaerobic lifestyle, and its ability to grow by fermentation of a limited range of amino acids but not carbohydrates. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the second completed genome sequence of a member of the family Synergistaceae and the first genome sequence of a member of the genus Aminobacterium. The 1,980,592 bp long genome with its 1,914 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project. PMID:21304712

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

PubMed

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

Pooled-DNA Sequencing for Elucidating New Genomic Risk Factors, Rare Variants Underlying Alzheimer's Disease.

PubMed

Jin, Sheng Chih; Benitez, Bruno A; Deming, Yuetiva; Cruchaga, Carlos

2016-01-01

Analyses of genome-wide association studies (GWAS) for complex disorders usually identify common variants with a relatively small effect size that only explain a small proportion of phenotypic heritability. Several studies have suggested that a significant fraction of heritability may be explained by low-frequency (minor allele frequency (MAF) of 1-5 %) and rare-variants that are not contained in the commercial GWAS genotyping arrays (Schork et al., Curr Opin Genet Dev 19:212, 2009). Rare variants can also have relatively large effects on risk for developing human diseases or disease phenotype (Cruchaga et al., PLoS One 7:e31039, 2012). However, it is necessary to perform next-generation sequencing (NGS) studies in a large population (>4,000 samples) to detect a significant rare-variant association. Several NGS methods, such as custom capture sequencing and amplicon-based sequencing, are designed to screen a small proportion of the genome, but most of these methods are limited in the number of samples that can be multiplexed (i.e. most sequencing kits only provide 96 distinct index). Additionally, the sequencing library preparation for 4,000 samples remains expensive and thus conducting NGS studies with the aforementioned methods are not feasible for most research laboratories.The need for low-cost large scale rare-variant detection makes pooled-DNA sequencing an ideally efficient and cost-effective technique to identify rare variants in target regions by sequencing hundreds to thousands of samples. Our recent work has demonstrated that pooled-DNA sequencing can accurately detect rare variants in targeted regions in multiple DNA samples with high sensitivity and specificity (Jin et al., Alzheimers Res Ther 4:34, 2012). In these studies we used a well-established pooled-DNA sequencing approach and a computational package, SPLINTER (short indel prediction by large deviation inference and nonlinear true frequency estimation by recursion) (Vallania et al., Genome Res 20:1711, 2010), for accurate identification of rare variants in large DNA pools. Given an average sequencing coverage of 30× per haploid genome, SPLINTER can detect rare variants and short indels up to 4 base pairs (bp) with high sensitivity and specificity (up to 1 haploid allele in a pool as large as 500 individuals). Step-by-step instructions on how to conduct pooled-DNA sequencing experiments and data analyses are described in this chapter.

Living laboratory: whole-genome sequencing as a learning healthcare enterprise.

PubMed

Angrist, M; Jamal, L

2015-04-01

With the proliferation of affordable large-scale human genomic data come profound and vexing questions about management of such data and their clinical uncertainty. These issues challenge the view that genomic research on human beings can (or should) be fully segregated from clinical genomics, either conceptually or practically. Here, we argue that the sharp distinction between clinical care and research is especially problematic in the context of large-scale genomic sequencing of people with suspected genetic conditions. Core goals of both enterprises (e.g. understanding genotype-phenotype relationships; generating an evidence base for genomic medicine) are more likely to be realized at a population scale if both those ordering and those undergoing sequencing for diagnostic reasons are routinely and longitudinally studied. Rather than relying on expensive and lengthy randomized clinical trials and meta-analyses, we propose leveraging nascent clinical-research hybrid frameworks into a broader, more permanent instantiation of exploratory medical sequencing. Such an investment could enlighten stakeholders about the real-life challenges posed by whole-genome sequencing, such as establishing the clinical actionability of genetic variants, returning 'off-target' results to families, developing effective service delivery models and monitoring long-term outcomes. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Minimal Absent Words in Four Human Genome Assemblies

PubMed Central

Garcia, Sara P.; Pinho, Armando J.

2011-01-01

Minimal absent words have been computed in genomes of organisms from all domains of life. Here, we aim to contribute to the catalogue of human genomic variation by investigating the variation in number and content of minimal absent words within a species, using four human genome assemblies. We compare the reference human genome GRCh37 assembly, the HuRef assembly of the genome of Craig Venter, the NA12878 assembly from cell line GM12878, and the YH assembly of the genome of a Han Chinese individual. We find the variation in number and content of minimal absent words between assemblies more significant for large and very large minimal absent words, where the biases of sequencing and assembly methodologies become more pronounced. Moreover, we find generally greater similarity between the human genome assemblies sequenced with capillary-based technologies (GRCh37 and HuRef) than between the human genome assemblies sequenced with massively parallel technologies (NA12878 and YH). Finally, as expected, we find the overall variation in number and content of minimal absent words within a species to be generally smaller than the variation between species. PMID:22220210

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/.

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 yam chlorotic necrosis virus, a novel macluravirus infecting yam

USDA-ARS?s Scientific Manuscript database

Complete genomic sequence of a novel member of the genus Macluravirus was determined from yam plants with chlorotic and necrotic symptoms in China. The genomic RNA consists of 8,261 nucleotides (nt) excluding the 3’-terminal poly (A) tail, containing one long open reading frame (ORF) encoding a larg...

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.

Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis.

PubMed

Jakupciak, John P; Wells, Jeffrey M; Karalus, Richard J; Pawlowski, David R; Lin, Jeffrey S; Feldman, Andrew B

2013-01-01

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.

Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis

PubMed Central

Jakupciak, John P.; Wells, Jeffrey M.; Karalus, Richard J.; Pawlowski, David R.; Lin, Jeffrey S.; Feldman, Andrew B.

2013-01-01

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations. PMID:24455204

Genomic sequencing of Pleistocene cave bears

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

Noonan, James P.; Hofreiter, Michael; Smith, Doug

2005-04-01

Despite the information content of genomic DNA, ancient DNA studies to date have largely been limited to amplification of mitochondrial DNA due to technical hurdles such as contamination and degradation of ancient DNAs. In this study, we describe two metagenomic libraries constructed using unamplified DNA extracted from the bones of two 40,000-year-old extinct cave bears. Analysis of {approx}1 Mb of sequence from each library showed that, despite significant microbial contamination, 5.8 percent and 1.1 percent of clones in the libraries contain cave bear inserts, yielding 26,861 bp of cave bear genome sequence. Alignment of this sequence to the dog genome,more » the closest sequenced genome to cave bear in terms of evolutionary distance, revealed roughly the expected ratio of cave bear exons, repeats and conserved noncoding sequences. Only 0.04 percent of all clones sequenced were derived from contamination with modern human DNA. Comparison of cave bear with orthologous sequences from several modern bear species revealed the evolutionary relationship of these lineages. Using the metagenomic approach described here, we have recovered substantial quantities of mammalian genomic sequence more than twice as old as any previously reported, establishing the feasibility of ancient DNA genomic sequencing programs.« less

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

Viral Genome DataBase: storing and analyzing genes and proteins from complete viral genomes.

PubMed

Hiscock, D; Upton, C

2000-05-01

The Viral Genome DataBase (VGDB) contains detailed information of the genes and predicted protein sequences from 15 completely sequenced genomes of large (&100 kb) viruses (2847 genes). The data that is stored includes DNA sequence, protein sequence, GenBank and user-entered notes, molecular weight (MW), isoelectric point (pI), amino acid content, A + T%, nucleotide frequency, dinucleotide frequency and codon use. The VGDB is a mySQL database with a user-friendly JAVA GUI. Results of queries can be easily sorted by any of the individual parameters. The software and additional figures and information are available at http://athena.bioc.uvic.ca/genomes/index.html .

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

PubMed

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

2017-11-01

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

The first complete chloroplast genome sequence of a lycophyte,Huperzia lucidula (Lycopodiaceae)

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

Wolf, Paul G.; Karol, Kenneth G.; Mandoli, Dina F.

2005-02-01

We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8x depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,671 bp. Gene order is more similarmore » to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperziachloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophytechloroplast genome data also enable a better reconstruction of the basaltracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.« less

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives.

PubMed

Zhao, Min; Wang, Qingguo; Wang, Quan; Jia, Peilin; Zhao, Zhongming

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development.

Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives

PubMed Central

2013-01-01

Copy number variation (CNV) is a prevalent form of critical genetic variation that leads to an abnormal number of copies of large genomic regions in a cell. Microarray-based comparative genome hybridization (arrayCGH) or genotyping arrays have been standard technologies to detect large regions subject to copy number changes in genomes until most recently high-resolution sequence data can be analyzed by next-generation sequencing (NGS). During the last several years, NGS-based analysis has been widely applied to identify CNVs in both healthy and diseased individuals. Correspondingly, the strong demand for NGS-based CNV analyses has fuelled development of numerous computational methods and tools for CNV detection. In this article, we review the recent advances in computational methods pertaining to CNV detection using whole genome and whole exome sequencing data. Additionally, we discuss their strengths and weaknesses and suggest directions for future development. PMID:24564169

Beyond Linear Sequence Comparisons: The use of genome-levelcharacters for phylogenetic reconstruction

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

Boore, Jeffrey L.

2004-11-27

Although the phylogenetic relationships of many organisms have been convincingly resolved by the comparisons of nucleotide or amino acid sequences, others have remained equivocal despite great effort. Now that large-scale genome sequencing projects are sampling many lineages, it is becoming feasible to compare large data sets of genome-level features and to develop this as a tool for phylogenetic reconstruction that has advantages over conventional sequence comparisons. Although it is unlikely that these will address a large number of evolutionary branch points across the broad tree of life due to the infeasibility of such sampling, they have great potential for convincinglymore » resolving many critical, contested relationships for which no other data seems promising. However, it is important that we recognize potential pitfalls, establish reasonable standards for acceptance, and employ rigorous methodology to guard against a return to earlier days of scenario-driven evolutionary reconstructions.« less

The Use of Weighted Graphs for Large-Scale Genome Analysis

PubMed Central

Zhou, Fang; Toivonen, Hannu; King, Ross D.

2014-01-01

There is an acute need for better tools to extract knowledge from the growing flood of sequence data. For example, thousands of complete genomes have been sequenced, and their metabolic networks inferred. Such data should enable a better understanding of evolution. However, most existing network analysis methods are based on pair-wise comparisons, and these do not scale to thousands of genomes. Here we propose the use of weighted graphs as a data structure to enable large-scale phylogenetic analysis of networks. We have developed three types of weighted graph for enzymes: taxonomic (these summarize phylogenetic importance), isoenzymatic (these summarize enzymatic variety/redundancy), and sequence-similarity (these summarize sequence conservation); and we applied these types of weighted graph to survey prokaryotic metabolism. To demonstrate the utility of this approach we have compared and contrasted the large-scale evolution of metabolism in Archaea and Eubacteria. Our results provide evidence for limits to the contingency of evolution. PMID:24619061

Fast selection of miRNA candidates based on large-scale pre-computed MFE sets of randomized sequences.

PubMed

Warris, Sven; Boymans, Sander; Muiser, Iwe; Noback, Michiel; Krijnen, Wim; Nap, Jan-Peter

2014-01-13

Small RNAs are important regulators of genome function, yet their prediction in genomes is still a major computational challenge. Statistical analyses of pre-miRNA sequences indicated that their 2D structure tends to have a minimal free energy (MFE) significantly lower than MFE values of equivalently randomized sequences with the same nucleotide composition, in contrast to other classes of non-coding RNA. The computation of many MFEs is, however, too intensive to allow for genome-wide screenings. Using a local grid infrastructure, MFE distributions of random sequences were pre-calculated on a large scale. These distributions follow a normal distribution and can be used to determine the MFE distribution for any given sequence composition by interpolation. It allows on-the-fly calculation of the normal distribution for any candidate sequence composition. The speedup achieved makes genome-wide screening with this characteristic of a pre-miRNA sequence practical. Although this particular property alone will not be able to distinguish miRNAs from other sequences sufficiently discriminative, the MFE-based P-value should be added to the parameters of choice to be included in the selection of potential miRNA candidates for experimental verification.

Large-scale chromosome folding versus genomic DNA sequences: A discrete double Fourier transform technique.

PubMed

Chechetkin, V R; Lobzin, V V

2017-08-07

Using state-of-the-art techniques combining imaging methods and high-throughput genomic mapping tools leaded to the significant progress in detailing chromosome architecture of various organisms. However, a gap still remains between the rapidly growing structural data on the chromosome folding and the large-scale genome organization. Could a part of information on the chromosome folding be obtained directly from underlying genomic DNA sequences abundantly stored in the databanks? To answer this question, we developed an original discrete double Fourier transform (DDFT). DDFT serves for the detection of large-scale genome regularities associated with domains/units at the different levels of hierarchical chromosome folding. The method is versatile and can be applied to both genomic DNA sequences and corresponding physico-chemical parameters such as base-pairing free energy. The latter characteristic is closely related to the replication and transcription and can also be used for the assessment of temperature or supercoiling effects on the chromosome folding. We tested the method on the genome of E. coli K-12 and found good correspondence with the annotated domains/units established experimentally. As a brief illustration of further abilities of DDFT, the study of large-scale genome organization for bacteriophage PHIX174 and bacterium Caulobacter crescentus was also added. The combined experimental, modeling, and bioinformatic DDFT analysis should yield more complete knowledge on the chromosome architecture and genome organization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reference-guided assembly of four diverse Arabidopsis thaliana genomes

PubMed Central

Schneeberger, Korbinian; Ossowski, Stephan; Ott, Felix; Klein, Juliane D.; Wang, Xi; Lanz, Christa; Smith, Lisa M.; Cao, Jun; Fitz, Joffrey; Warthmann, Norman; Henz, Stefan R.; Huson, Daniel H.; Weigel, Detlef

2011-01-01

We present whole-genome assemblies of four divergent Arabidopsis thaliana strains that complement the 125-Mb reference genome sequence released a decade ago. Using a newly developed reference-guided approach, we assembled large contigs from 9 to 42 Gb of Illumina short-read data from the Landsberg erecta (Ler-1), C24, Bur-0, and Kro-0 strains, which have been sequenced as part of the 1,001 Genomes Project for this species. Using alignments against the reference sequence, we first reduced the complexity of the de novo assembly and later integrated reads without similarity to the reference sequence. As an example, half of the noncentromeric C24 genome was covered by scaffolds that are longer than 260 kb, with a maximum of 2.2 Mb. Moreover, over 96% of the reference genome was covered by the reference-guided assembly, compared with only 87% with a complete de novo assembly. Comparisons with 2 Mb of dideoxy sequence reveal that the per-base error rate of the reference-guided assemblies was below 1 in 10,000. Our assemblies provide a detailed, genomewide picture of large-scale differences between A. thaliana individuals, most of which are difficult to access with alignment-consensus methods only. We demonstrate their practical relevance in studying the expression differences of polymorphic genes and show how the analysis of sRNA sequencing data can lead to erroneous conclusions if aligned against the reference genome alone. Genome assemblies, raw reads, and further information are accessible through http://1001genomes.org/projects/assemblies.html. PMID:21646520

The Human Genome Project: big science transforms biology and medicine.

PubMed

Hood, Leroy; Rowen, Lee

2013-01-01

The Human Genome Project has transformed biology through its integrated big science approach to deciphering a reference human genome sequence along with the complete sequences of key model organisms. The project exemplifies the power, necessity and success of large, integrated, cross-disciplinary efforts - so-called 'big science' - directed towards complex major objectives. In this article, we discuss the ways in which this ambitious endeavor led to the development of novel technologies and analytical tools, and how it brought the expertise of engineers, computer scientists and mathematicians together with biologists. It established an open approach to data sharing and open-source software, thereby making the data resulting from the project accessible to all. The genome sequences of microbes, plants and animals have revolutionized many fields of science, including microbiology, virology, infectious disease and plant biology. Moreover, deeper knowledge of human sequence variation has begun to alter the practice of medicine. The Human Genome Project has inspired subsequent large-scale data acquisition initiatives such as the International HapMap Project, 1000 Genomes, and The Cancer Genome Atlas, as well as the recently announced Human Brain Project and the emerging Human Proteome Project.

The Human Genome Project: big science transforms biology and medicine

PubMed Central

2013-01-01

The Human Genome Project has transformed biology through its integrated big science approach to deciphering a reference human genome sequence along with the complete sequences of key model organisms. The project exemplifies the power, necessity and success of large, integrated, cross-disciplinary efforts - so-called ‘big science’ - directed towards complex major objectives. In this article, we discuss the ways in which this ambitious endeavor led to the development of novel technologies and analytical tools, and how it brought the expertise of engineers, computer scientists and mathematicians together with biologists. It established an open approach to data sharing and open-source software, thereby making the data resulting from the project accessible to all. The genome sequences of microbes, plants and animals have revolutionized many fields of science, including microbiology, virology, infectious disease and plant biology. Moreover, deeper knowledge of human sequence variation has begun to alter the practice of medicine. The Human Genome Project has inspired subsequent large-scale data acquisition initiatives such as the International HapMap Project, 1000 Genomes, and The Cancer Genome Atlas, as well as the recently announced Human Brain Project and the emerging Human Proteome Project. PMID:24040834

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

A draft sequence of the rice genome (Oryza sativa L. ssp. indica).

PubMed

Yu, Jun; Hu, Songnian; Wang, Jun; Wong, Gane Ka-Shu; Li, Songgang; Liu, Bin; Deng, Yajun; Dai, Li; Zhou, Yan; Zhang, Xiuqing; Cao, Mengliang; Liu, Jing; Sun, Jiandong; Tang, Jiabin; Chen, Yanjiong; Huang, Xiaobing; Lin, Wei; Ye, Chen; Tong, Wei; Cong, Lijuan; Geng, Jianing; Han, Yujun; Li, Lin; Li, Wei; Hu, Guangqiang; Huang, Xiangang; Li, Wenjie; Li, Jian; Liu, Zhanwei; Li, Long; Liu, Jianping; Qi, Qiuhui; Liu, Jinsong; Li, Li; Li, Tao; Wang, Xuegang; Lu, Hong; Wu, Tingting; Zhu, Miao; Ni, Peixiang; Han, Hua; Dong, Wei; Ren, Xiaoyu; Feng, Xiaoli; Cui, Peng; Li, Xianran; Wang, Hao; Xu, Xin; Zhai, Wenxue; Xu, Zhao; Zhang, Jinsong; He, Sijie; Zhang, Jianguo; Xu, Jichen; Zhang, Kunlin; Zheng, Xianwu; Dong, Jianhai; Zeng, Wanyong; Tao, Lin; Ye, Jia; Tan, Jun; Ren, Xide; Chen, Xuewei; He, Jun; Liu, Daofeng; Tian, Wei; Tian, Chaoguang; Xia, Hongai; Bao, Qiyu; Li, Gang; Gao, Hui; Cao, Ting; Wang, Juan; Zhao, Wenming; Li, Ping; Chen, Wei; Wang, Xudong; Zhang, Yong; Hu, Jianfei; Wang, Jing; Liu, Song; Yang, Jian; Zhang, Guangyu; Xiong, Yuqing; Li, Zhijie; Mao, Long; Zhou, Chengshu; Zhu, Zhen; Chen, Runsheng; Hao, Bailin; Zheng, Weimou; Chen, Shouyi; Guo, Wei; Li, Guojie; Liu, Siqi; Tao, Ming; Wang, Jian; Zhu, Lihuang; Yuan, Longping; Yang, Huanming

2002-04-05

We have produced a draft sequence of the rice genome for the most widely cultivated subspecies in China, Oryza sativa L. ssp. indica, by whole-genome shotgun sequencing. The genome was 466 megabases in size, with an estimated 46,022 to 55,615 genes. Functional coverage in the assembled sequences was 92.0%. About 42.2% of the genome was in exact 20-nucleotide oligomer repeats, and most of the transposons were in the intergenic regions between genes. Although 80.6% of predicted Arabidopsis thaliana genes had a homolog in rice, only 49.4% of predicted rice genes had a homolog in A. thaliana. The large proportion of rice genes with no recognizable homologs is due to a gradient in the GC content of rice coding sequences.

Enabling large-scale next-generation sequence assembly with Blacklight

PubMed Central

Couger, M. Brian; Pipes, Lenore; Squina, Fabio; Prade, Rolf; Siepel, Adam; Palermo, Robert; Katze, Michael G.; Mason, Christopher E.; Blood, Philip D.

2014-01-01

Summary A variety of extremely challenging biological sequence analyses were conducted on the XSEDE large shared memory resource Blacklight, using current bioinformatics tools and encompassing a wide range of scientific applications. These include genomic sequence assembly, very large metagenomic sequence assembly, transcriptome assembly, and sequencing error correction. The data sets used in these analyses included uncategorized fungal species, reference microbial data, very large soil and human gut microbiome sequence data, and primate transcriptomes, composed of both short-read and long-read sequence data. A new parallel command execution program was developed on the Blacklight resource to handle some of these analyses. These results, initially reported previously at XSEDE13 and expanded here, represent significant advances for their respective scientific communities. The breadth and depth of the results achieved demonstrate the ease of use, versatility, and unique capabilities of the Blacklight XSEDE resource for scientific analysis of genomic and transcriptomic sequence data, and the power of these resources, together with XSEDE support, in meeting the most challenging scientific problems. PMID:25294974

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

Phased genotyping-by-sequencing enhances analysis of genetic diversity and reveals divergent copy number variants in maize

USDA-ARS?s Scientific Manuscript database

High-throughput sequencing of reduced representation genomic libraries has ushered in an era of genotyping-by-sequencing (GBS), where genome-wide genotype data can be obtained for nearly any species. However, there remains a need for imputation-free GBS methods for genotyping large samples taken fr...

Protein Sequence Annotation Tool (PSAT): A centralized web-based meta-server for high-throughput sequence annotations

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

Leung, Elo; Huang, Amy; Cadag, Eithon

In this study, we introduce the Protein Sequence Annotation Tool (PSAT), a web-based, sequence annotation meta-server for performing integrated, high-throughput, genome-wide sequence analyses. Our goals in building PSAT were to (1) create an extensible platform for integration of multiple sequence-based bioinformatics tools, (2) enable functional annotations and enzyme predictions over large input protein fasta data sets, and (3) provide a web interface for convenient execution of the tools. In this paper, we demonstrate the utility of PSAT by annotating the predicted peptide gene products of Herbaspirillum sp. strain RV1423, importing the results of PSAT into EC2KEGG, and using the resultingmore » functional comparisons to identify a putative catabolic pathway, thereby distinguishing RV1423 from a well annotated Herbaspirillum species. This analysis demonstrates that high-throughput enzyme predictions, provided by PSAT processing, can be used to identify metabolic potential in an otherwise poorly annotated genome. Lastly, PSAT is a meta server that combines the results from several sequence-based annotation and function prediction codes, and is available at http://psat.llnl.gov/psat/. PSAT stands apart from other sequencebased genome annotation systems in providing a high-throughput platform for rapid de novo enzyme predictions and sequence annotations over large input protein sequence data sets in FASTA. PSAT is most appropriately applied in annotation of large protein FASTA sets that may or may not be associated with a single genome.« less

Protein Sequence Annotation Tool (PSAT): A centralized web-based meta-server for high-throughput sequence annotations

DOE PAGES

Leung, Elo; Huang, Amy; Cadag, Eithon; ...

2016-01-20

In this study, we introduce the Protein Sequence Annotation Tool (PSAT), a web-based, sequence annotation meta-server for performing integrated, high-throughput, genome-wide sequence analyses. Our goals in building PSAT were to (1) create an extensible platform for integration of multiple sequence-based bioinformatics tools, (2) enable functional annotations and enzyme predictions over large input protein fasta data sets, and (3) provide a web interface for convenient execution of the tools. In this paper, we demonstrate the utility of PSAT by annotating the predicted peptide gene products of Herbaspirillum sp. strain RV1423, importing the results of PSAT into EC2KEGG, and using the resultingmore » functional comparisons to identify a putative catabolic pathway, thereby distinguishing RV1423 from a well annotated Herbaspirillum species. This analysis demonstrates that high-throughput enzyme predictions, provided by PSAT processing, can be used to identify metabolic potential in an otherwise poorly annotated genome. Lastly, PSAT is a meta server that combines the results from several sequence-based annotation and function prediction codes, and is available at http://psat.llnl.gov/psat/. PSAT stands apart from other sequencebased genome annotation systems in providing a high-throughput platform for rapid de novo enzyme predictions and sequence annotations over large input protein sequence data sets in FASTA. PSAT is most appropriately applied in annotation of large protein FASTA sets that may or may not be associated with a single genome.« less

The structure and evolution of angiosperm nuclear genomes.

PubMed

Bennetzen, J L

1998-04-01

Despite several decades of investigation, the organization of angiosperm genomes remained largely unknown until very recently. Data describing the sequence composition of large segments of genomes, covering hundreds of kilobases of contiguous sequence, have only become available in the past two years. Recent results indicate commonalities in the characteristics of many plant genomes, including in the structure of chromosomal components like telomeres and centromeres, and in the order and content of genes. Major differences between angiosperms have been associated mainly with repetitive DNAs, both gene families and mobile elements. Intriguing new studies have begun to characterize the dynamic three-dimensional structures of chromosomes and chromatin, and the relationship between genome structure and co-ordinated gene function.

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.

Enriching public descriptions of marine phages using the Genomic Standards Consortium MIGS standard

PubMed Central

Duhaime, Melissa Beth; Kottmann, Renzo; Field, Dawn; Glöckner, Frank Oliver

2011-01-01

In any sequencing project, the possible depth of comparative analysis is determined largely by the amount and quality of the accompanying contextual data. The structure, content, and storage of this contextual data should be standardized to ensure consistent coverage of all sequenced entities and facilitate comparisons. The Genomic Standards Consortium (GSC) has developed the “Minimum Information about Genome/Metagenome Sequences (MIGS/MIMS)” checklist for the description of genomes and here we annotate all 30 publicly available marine bacteriophage sequences to the MIGS standard. These annotations build on existing International Nucleotide Sequence Database Collaboration (INSDC) records, and confirm, as expected that current submissions lack most MIGS fields. MIGS fields were manually curated from the literature and placed in XML format as specified by the Genomic Contextual Data Markup Language (GCDML). These “machine-readable” reports were then analyzed to highlight patterns describing this collection of genomes. Completed reports are provided in GCDML. This work represents one step towards the annotation of our complete collection of genome sequences and shows the utility of capturing richer metadata along with raw sequences. PMID:21677864

Efficient privacy-preserving string search and an application in genomics.

PubMed

Shimizu, Kana; Nuida, Koji; Rätsch, Gunnar

2016-06-01

Personal genomes carry inherent privacy risks and protecting privacy poses major social and technological challenges. We consider the case where a user searches for genetic information (e.g. an allele) on a server that stores a large genomic database and aims to receive allele-associated information. The user would like to keep the query and result private and the server the database. We propose a novel approach that combines efficient string data structures such as the Burrows-Wheeler transform with cryptographic techniques based on additive homomorphic encryption. We assume that the sequence data is searchable in efficient iterative query operations over a large indexed dictionary, for instance, from large genome collections and employing the (positional) Burrows-Wheeler transform. We use a technique called oblivious transfer that is based on additive homomorphic encryption to conceal the sequence query and the genomic region of interest in positional queries. We designed and implemented an efficient algorithm for searching sequences of SNPs in large genome databases. During search, the user can only identify the longest match while the server does not learn which sequence of SNPs the user queried. In an experiment based on 2184 aligned haploid genomes from the 1000 Genomes Project, our algorithm was able to perform typical queries within [Formula: see text] 4.6 s and [Formula: see text] 10.8 s for client and server side, respectively, on laptop computers. The presented algorithm is at least one order of magnitude faster than an exhaustive baseline algorithm. https://github.com/iskana/PBWT-sec and https://github.com/ratschlab/PBWT-sec shimizu-kana@aist.go.jp or Gunnar.Ratsch@ratschlab.org Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Efficient privacy-preserving string search and an application in genomics

PubMed Central

Shimizu, Kana; Nuida, Koji; Rätsch, Gunnar

2016-01-01

Motivation: Personal genomes carry inherent privacy risks and protecting privacy poses major social and technological challenges. We consider the case where a user searches for genetic information (e.g. an allele) on a server that stores a large genomic database and aims to receive allele-associated information. The user would like to keep the query and result private and the server the database. Approach: We propose a novel approach that combines efficient string data structures such as the Burrows–Wheeler transform with cryptographic techniques based on additive homomorphic encryption. We assume that the sequence data is searchable in efficient iterative query operations over a large indexed dictionary, for instance, from large genome collections and employing the (positional) Burrows–Wheeler transform. We use a technique called oblivious transfer that is based on additive homomorphic encryption to conceal the sequence query and the genomic region of interest in positional queries. Results: We designed and implemented an efficient algorithm for searching sequences of SNPs in large genome databases. During search, the user can only identify the longest match while the server does not learn which sequence of SNPs the user queried. In an experiment based on 2184 aligned haploid genomes from the 1000 Genomes Project, our algorithm was able to perform typical queries within ≈ 4.6 s and ≈ 10.8 s for client and server side, respectively, on laptop computers. The presented algorithm is at least one order of magnitude faster than an exhaustive baseline algorithm. Availability and implementation: https://github.com/iskana/PBWT-sec and https://github.com/ratschlab/PBWT-sec. Contacts: shimizu-kana@aist.go.jp or Gunnar.Ratsch@ratschlab.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153731

Dramatic improvement in genome assembly achieved using doubled-haploid genomes.

PubMed

Zhang, Hong; Tan, Engkong; Suzuki, Yutaka; Hirose, Yusuke; Kinoshita, Shigeharu; Okano, Hideyuki; Kudoh, Jun; Shimizu, Atsushi; Saito, Kazuyoshi; Watabe, Shugo; Asakawa, Shuichi

2014-10-27

Improvement in de novo assembly of large genomes is still to be desired. Here, we improved draft genome sequence quality by employing doubled-haploid individuals. We sequenced wildtype and doubled-haploid Takifugu rubripes genomes, under the same conditions, using the Illumina platform and assembled contigs with SOAPdenovo2. We observed 5.4-fold and 2.6-fold improvement in the sizes of the N50 contig and scaffold of doubled-haploid individuals, respectively, compared to the wildtype, indicating that the use of a doubled-haploid genome aids in accurate genome analysis.

Using SQL Databases for Sequence Similarity Searching and Analysis.

PubMed

Pearson, William R; Mackey, Aaron J

2017-09-13

Relational databases can integrate diverse types of information and manage large sets of similarity search results, greatly simplifying genome-scale analyses. By focusing on taxonomic subsets of sequences, relational databases can reduce the size and redundancy of sequence libraries and improve the statistical significance of homologs. In addition, by loading similarity search results into a relational database, it becomes possible to explore and summarize the relationships between all of the proteins in an organism and those in other biological kingdoms. This unit describes how to use relational databases to improve the efficiency of sequence similarity searching and demonstrates various large-scale genomic analyses of homology-related data. It also describes the installation and use of a simple protein sequence database, seqdb_demo, which is used as a basis for the other protocols. The unit also introduces search_demo, a database that stores sequence similarity search results. The search_demo database is then used to explore the evolutionary relationships between E. coli proteins and proteins in other organisms in a large-scale comparative genomic analysis. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Comparison of the genomic sequence of the microminipig, a novel breed of swine, with the genomic database for conventional pig.

PubMed

Miura, Naoki; Kucho, Ken-Ichi; Noguchi, Michiko; Miyoshi, Noriaki; Uchiumi, Toshiki; Kawaguchi, Hiroaki; Tanimoto, Akihide

2014-01-01

The microminipig, which weighs less than 10 kg at an early stage of maturity, has been reported as a potential experimental model animal. Its extremely small size and other distinct characteristics suggest the possibility of a number of differences between the genome of the microminipig and that of conventional pigs. In this study, we analyzed the genomes of two healthy microminipigs using a next-generation sequencer SOLiD™ system. We then compared the obtained genomic sequences with a genomic database for the domestic pig (Sus scrofa). The mapping coverage of sequenced tag from the microminipig to conventional pig genomic sequences was greater than 96% and we detected no clear, substantial genomic variance from these data. The results may indicate that the distinct characteristics of the microminipig derive from small-scale alterations in the genome, such as Single Nucleotide Polymorphisms or translational modifications, rather than large-scale deletion or insertion polymorphisms. Further investigation of the entire genomic sequence of the microminipig with methods enabling deeper coverage is required to elucidate the genetic basis of its distinct phenotypic traits. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Complete genome sequence of Hirschia baltica type strain (IFAM 1418T)

PubMed Central

Chertkov, Olga; Brown, Pamela J.B.; Kysela, David T.; de Pedro, Miguel A.; Lucas, Susan; Copeland, Alex; Lapidus, Alla; Del Rio, Tijana Glavina; Tice, Hope; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Detter, John C.; Han, Cliff; Larimer, Frank; Chang, Yun-juan; Jeffries, Cynthia D.; Land, Miriam; Hauser, Loren; Kyrpides, Nikos C.; Ivanova, Natalia; Ovchinnikova, Galina; Tindall, Brian J.; Göker, Markus; Klenk, Hans-Peter; Brun, Yves V.

2011-01-01

The family Hyphomonadaceae within the Alphaproteobacteria is largely comprised of bacteria isolated from marine environments with striking morphologies and an unusual mode of cell growth. Here, we report the complete genome sequence Hirschia baltica, which is only the second a member of the Hyphomonadaceae with a published genome sequence. H. baltica is of special interest because it has a dimorphic life cycle and is a stalked, budding bacterium. The 3,455,622 bp long chromosome and 84,492 bp plasmid with a total of 3,222 protein-coding and 44 RNA genes were sequenced as part of the DOE Joint Genome Institute Program CSP 2008. PMID:22675580

Molecular characterization of a novel rhabdovirus infecting blackcurrant identified by high-throughput sequencing.

PubMed

Wu, L-P; Yang, T; Liu, H-W; Postman, J; Li, R

2018-05-01

A large contig with sequence similarities to several nucleorhabdoviruses was identified by high-throughput sequencing analysis from a black currant (Ribes nigrum L.) cultivar. The complete genome sequence of this new nucleorhabdovirus is 14,432 nucleotides long. Its genomic organization is very similar to those of unsegmented plant rhabdoviruses, containing six open reading frames in the order 3'-N-P-P3-M-G-L-5. The virus, which is provisionally named "black currant-associated rhabdovirus", is 41-52% identical in its genome nucleotide sequence to other nucleorhabdoviruses and may represent a new species in the genus Nucleorhabdovirus.

Complete genome sequence of Paris mosaic necrosis virus, a distinct member of the genus Potyvirus

USDA-ARS?s Scientific Manuscript database

The complete genomic sequence of a novel potyvirus was determined from Paris polyphylla var. yunnanensis. Its genomic RNA consists of 9,660 nucleotides (nt) excluding the 3’-terminal poly (A) tail, containing a single open reading frame (ORF) encoding a large polyprotein. The virus shares 52.1-69.7%...

Genome-wide association study based on multiple imputation with low-depth sequencing data: application to biofuel traits in reed canarygrass

USDA-ARS?s Scientific Manuscript database

Genotyping by sequencing allows for large-scale genetic analyses in plant species with no reference genome, but sets the challenge of sound inference in presence of uncertain genotypes. We report an imputation-based genome-wide association study (GWAS) in reed canarygrass (Phalaris arundinacea L., P...

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

USDA-ARS?s Scientific Manuscript database

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

WheatGenome.info: an integrated database and portal for wheat genome information.

PubMed

Lai, Kaitao; Berkman, Paul J; Lorenc, Michal Tadeusz; Duran, Chris; Smits, Lars; Manoli, Sahana; Stiller, Jiri; Edwards, David

2012-02-01

Bread wheat (Triticum aestivum) is one of the most important crop plants, globally providing staple food for a large proportion of the human population. However, improvement of this crop has been limited due to its large and complex genome. Advances in genomics are supporting wheat crop improvement. We provide a variety of web-based systems hosting wheat genome and genomic data to support wheat research and crop improvement. WheatGenome.info is an integrated database resource which includes multiple web-based applications. These include a GBrowse2-based wheat genome viewer with BLAST search portal, TAGdb for searching wheat second-generation genome sequence data, wheat autoSNPdb, links to wheat genetic maps using CMap and CMap3D, and a wheat genome Wiki to allow interaction between diverse wheat genome sequencing activities. This system includes links to a variety of wheat genome resources hosted at other research organizations. This integrated database aims to accelerate wheat genome research and is freely accessible via the web interface at http://www.wheatgenome.info/.

Targeted sequencing for high-resolution evolutionary analyses following genome duplication in salmonid fish: Proof of concept for key components of the insulin-like growth factor axis.

PubMed

Lappin, Fiona M; Shaw, Rebecca L; Macqueen, Daniel J

2016-12-01

High-throughput sequencing has revolutionised comparative and evolutionary genome biology. It has now become relatively commonplace to generate multiple genomes and/or transcriptomes to characterize the evolution of large taxonomic groups of interest. Nevertheless, such efforts may be unsuited to some research questions or remain beyond the scope of some research groups. Here we show that targeted high-throughput sequencing offers a viable alternative to study genome evolution across a vertebrate family of great scientific interest. Specifically, we exploited sequence capture and Illumina sequencing to characterize the evolution of key components from the insulin-like growth (IGF) signalling axis of salmonid fish at unprecedented phylogenetic resolution. The IGF axis represents a central governor of vertebrate growth and its core components were expanded by whole genome duplication in the salmonid ancestor ~95Ma. Using RNA baits synthesised to genes encoding the complete family of IGF binding proteins (IGFBP) and an IGF hormone (IGF2), we captured, sequenced and assembled orthologous and paralogous exons from species representing all ten salmonid genera. This approach generated 299 novel sequences, most as complete or near-complete protein-coding sequences. Phylogenetic analyses confirmed congruent evolutionary histories for all nineteen recognized salmonid IGFBP family members and identified novel salmonid-specific IGF2 paralogues. Moreover, we reconstructed the evolution of duplicated IGF axis paralogues across a replete salmonid phylogeny, revealing complex historic selection regimes - both ancestral to salmonids and lineage-restricted - that frequently involved asymmetric paralogue divergence under positive and/or relaxed purifying selection. Our findings add to an emerging literature highlighting diverse applications for targeted sequencing in comparative-evolutionary genomics. We also set out a viable approach to obtain large sets of nuclear genes for any member of the salmonid family, which should enable insights into the evolutionary role of whole genome duplication before additional nuclear genome sequences become available. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Complete genome sequencing and analysis of Saprospira grandis str. Lewin, a predatory marine bacterium

PubMed Central

Saw, Jimmy H. W.; Yuryev, Anton; Kanbe, Masaomi; Hou, Shaobin; Young, Aaron G.; Aizawa, Shin-Ichi

2012-01-01

Saprospira grandis is a coastal marine bacterium that can capture and prey upon other marine bacteria using a mechanism known as ‘ixotrophy’. Here, we present the complete genome sequence of Saprospira grandis str. Lewin isolated from La Jolla beach in San Diego, California. The complete genome sequence comprises a chromosome of 4.35 Mbp and a plasmid of 54.9 Kbp. Genome analysis revealed incomplete pathways for the biosynthesis of nine essential amino acids but presence of a large number of peptidases. The genome encodes multiple copies of sensor globin-coupled rsbR genes thought to be essential for stress response and the presence of such sensor globins in Bacteroidetes is unprecedented. A total of 429 spacer sequences within the three CRISPR repeat regions were identified in the genome and this number is the largest among all the Bacteroidetes sequenced to date. PMID:22675601

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.

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.

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

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.

Between Two Fern Genomes

PubMed Central

2014-01-01

Ferns are the only major lineage of vascular plants not represented by a sequenced nuclear genome. This lack of genome sequence information significantly impedes our ability to understand and reconstruct genome evolution not only in ferns, but across all land plants. Azolla and Ceratopteris are ideal and complementary candidates to be the first ferns to have their nuclear genomes sequenced. They differ dramatically in genome size, life history, and habit, and thus represent the immense diversity of extant ferns. Together, this pair of genomes will facilitate myriad large-scale comparative analyses across ferns and all land plants. Here we review the unique biological characteristics of ferns and describe a number of outstanding questions in plant biology that will benefit from the addition of ferns to the set of taxa with sequenced nuclear genomes. We explain why the fern clade is pivotal for understanding genome evolution across land plants, and we provide a rationale for how knowledge of fern genomes will enable progress in research beyond the ferns themselves. PMID:25324969

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

PubMed

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

2017-01-01

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

D-GENIES: dot plot large genomes in an interactive, efficient and simple way.

PubMed

Cabanettes, Floréal; Klopp, Christophe

2018-01-01

Dot plots are widely used to quickly compare sequence sets. They provide a synthetic similarity overview, highlighting repetitions, breaks and inversions. Different tools have been developed to easily generated genomic alignment dot plots, but they are often limited in the input sequence size. D-GENIES is a standalone and web application performing large genome alignments using minimap2 software package and generating interactive dot plots. It enables users to sort query sequences along the reference, zoom in the plot and download several image, alignment or sequence files. D-GENIES is an easy-to-install, open-source software package (GPL) developed in Python and JavaScript. The source code is available at https://github.com/genotoul-bioinfo/dgenies and it can be tested at http://dgenies.toulouse.inra.fr/.

From the Battlefield to the Bedside: Supporting Warfighter and Civilian Health With the "ART" of Whole Genome Sequencing for Antibiotic Resistance and Outbreak Investigations.

PubMed

Lesho, Emil; Lin, Xiaoxu; Clifford, Robert; Snesrud, Erik; Onmus-Leone, Fatma; Appalla, Lakshmi; Ong, Ana; Maybank, Rosslyn; Nielsen, Lindsey; Kwak, Yoon; Hinkle, Mary; Turco, John; Marin, Juan A; Hooks, Sally; Matthews, Stacy; Hyland, Stephen; Little, Jered; Waterman, Paige; McGann, Patrick

2016-07-01

Awareness, responsiveness, and throughput characterize an approach for enhancing the clinical impact of whole genome sequencing for austere environments and for large geographically dispersed health systems. This Department of Defense approach is informing interagency efforts linking antibiograms of multidrug-resistant organisms to their genome sequences in a public database. Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.

Human centromere genomics: now it's personal.

PubMed

Hayden, Karen E

2012-07-01

Advances in human genomics have accelerated studies in evolution, disease, and cellular regulation. However, centromere sequences, defining the chromosomal interface with spindle microtubules, remain largely absent from ongoing genomic studies and disconnected from functional, genome-wide analyses. This disparity results from the challenge of predicting the linear order of multi-megabase-sized regions that are composed almost entirely of near-identical satellite DNA. Acknowledging these challenges, the field of human centromere genomics possesses the potential to rapidly advance given the availability of individual, or personalized, genome projects matched with the promise of long-read sequencing technologies. Here I review the current genomic model of human centromeres in consideration of those studies involving functional datasets that examine the role of sequence in centromere identity.

PET-Tool: a software suite for comprehensive processing and managing of Paired-End diTag (PET) sequence data.

PubMed

Chiu, Kuo Ping; Wong, Chee-Hong; Chen, Qiongyu; Ariyaratne, Pramila; Ooi, Hong Sain; Wei, Chia-Lin; Sung, Wing-Kin Ken; Ruan, Yijun

2006-08-25

We recently developed the Paired End diTag (PET) strategy for efficient characterization of mammalian transcriptomes and genomes. The paired end nature of short PET sequences derived from long DNA fragments raised a new set of bioinformatics challenges, including how to extract PETs from raw sequence reads, and correctly yet efficiently map PETs to reference genome sequences. To accommodate and streamline data analysis of the large volume PET sequences generated from each PET experiment, an automated PET data process pipeline is desirable. We designed an integrated computation program package, PET-Tool, to automatically process PET sequences and map them to the genome sequences. The Tool was implemented as a web-based application composed of four modules: the Extractor module for PET extraction; the Examiner module for analytic evaluation of PET sequence quality; the Mapper module for locating PET sequences in the genome sequences; and the Project Manager module for data organization. The performance of PET-Tool was evaluated through the analyses of 2.7 million PET sequences. It was demonstrated that PET-Tool is accurate and efficient in extracting PET sequences and removing artifacts from large volume dataset. Using optimized mapping criteria, over 70% of quality PET sequences were mapped specifically to the genome sequences. With a 2.4 GHz LINUX machine, it takes approximately six hours to process one million PETs from extraction to mapping. The speed, accuracy, and comprehensiveness have proved that PET-Tool is an important and useful component in PET experiments, and can be extended to accommodate other related analyses of paired-end sequences. The Tool also provides user-friendly functions for data quality check and system for multi-layer data management.

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  .

The global catalogue of microorganisms 10K type strain sequencing project: closing the genomic gaps for the validly published prokaryotic and fungi species.

PubMed

Wu, Linhuan; McCluskey, Kevin; Desmeth, Philippe; Liu, Shuangjiang; Hideaki, Sugawara; Yin, Ye; Moriya, Ohkuma; Itoh, Takashi; Kim, Cha Young; Lee, Jung-Sook; Zhou, Yuguang; Kawasaki, Hiroko; Hazbón, Manzour Hernando; Robert, Vincent; Boekhout, Teun; Lima, Nelson; Evtushenko, Lyudmila; Boundy-Mills, Kyria; Bunk, Boyke; Moore, Edward R B; Eurwilaichitr, Lily; Ingsriswang, Supawadee; Shah, Heena; Yao, Su; Jin, Tao; Huang, Jinqun; Shi, Wenyu; Sun, Qinglan; Fan, Guomei; Li, Wei; Li, Xian; Kurtböke, Ipek; Ma, Juncai

2018-05-01

Genomic information is essential for taxonomic, phylogenetic, and functional studies to comprehensively decipher the characteristics of microorganisms, to explore microbiomes through metagenomics, and to answer fundamental questions of nature and human life. However, large gaps remain in the available genomic sequencing information published for bacterial and archaeal species, and the gaps are even larger for fungal type strains. The Global Catalogue of Microorganisms (GCM) leads an internationally coordinated effort to sequence type strains and close gaps in the genomic maps of microorganisms. Hence, the GCM aims to promote research by deep-mining genomic data.

Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing

PubMed Central

Eastman, Alexander W.; Yuan, Ze-Chun

2015-01-01

Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects. PMID:25653642

Phylogenomics from Whole Genome Sequences Using aTRAM.

PubMed

Allen, Julie M; Boyd, Bret; Nguyen, Nam-Phuong; Vachaspati, Pranjal; Warnow, Tandy; Huang, Daisie I; Grady, Patrick G S; Bell, Kayce C; Cronk, Quentin C B; Mugisha, Lawrence; Pittendrigh, Barry R; Leonardi, M Soledad; Reed, David L; Johnson, Kevin P

2017-09-01

Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes ($<$1000 Mbp) it is feasible to sequence the entire genome at modest coverage ($10-30\\times$). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single-copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out-groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close- to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above $5-10\\times$, aTRAM was successful at assembling 80-90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.]. © The Author(s) 2017. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Draft genome sequence of Staphylococcus aureus KT/312045, an ST1-MSSA PVL positive isolated from pus sample in East Coast Malaysia.

PubMed

Suhaili, Zarizal; Lean, Soo-Sum; Mohamad, Noor Muzamil; Rachman, Abdul R Abdul; Desa, Mohd Nasir Mohd; Yeo, Chew Chieng

2016-09-01

Most of the efforts in elucidating the molecular relatedness and epidemiology of Staphylococcus aureus in Malaysia have been largely focused on methicillin-resistant S. aureus (MRSA). Therefore, here we report the draft genome sequence of the methicillin-susceptible Staphylococcus aureus (MSSA) with sequence type 1 (ST1), spa type t127 with Panton-Valentine Leukocidin (pvl) pathogenic determinant isolated from pus sample designated as KT/314250 strain. The size of the draft genome is 2.86 Mbp with 32.7% of G + C content consisting 2673 coding sequences. The draft genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession number AOCP00000000.

Complete genome sequence of the sulfate-reducing firmicute Desulfotomaculum ruminis type strain (DLT)

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

Spring, Stefan; Visser, Michael; Lu, Megan

2012-12-11

Desulfotomaculum ruminis Campbell and Postgate 1965 is a member of the large genus Desulfotomaculum which contains 30 species and is contained in the family Peptococcaceae. This species is of interest because it represents one of the few sulfate- reducing bacteria that have been isolated from the rumen. Here we describe the features of D. ruminis together with the complete genome sequence and annotation. The 3,969,014 bp long chromosome with a total of 3,901 protein-coding and 85 RNA genes is the second completed genome sequence of a type strain of the genus Desulfotomaculum to be pub- lished, and was sequenced asmore » part of the DOE Joint Genome Institute Community Sequencing Program 2009.« less

One chromosome, one contig: complete microbial genomes from long-read sequencing and assembly.

PubMed

Koren, Sergey; Phillippy, Adam M

2015-02-01

Like a jigsaw puzzle with large pieces, a genome sequenced with long reads is easier to assemble. However, recent sequencing technologies have favored lowering per-base cost at the expense of read length. This has dramatically reduced sequencing cost, but resulted in fragmented assemblies, which negatively affect downstream analyses and hinder the creation of finished (gapless, high-quality) genomes. In contrast, emerging long-read sequencing technologies can now produce reads tens of kilobases in length, enabling the automated finishing of microbial genomes for under $1000. This promises to improve the quality of reference databases and facilitate new studies of chromosomal structure and variation. We present an overview of these new technologies and the methods used to assemble long reads into complete genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evolution and Diversity of the Human Hepatitis D Virus Genome

PubMed Central

Huang, Chi-Ruei; Lo, Szecheng J.

2010-01-01

Human hepatitis delta virus (HDV) is the smallest RNA virus in genome. HDV genome is divided into a viroid-like sequence and a protein-coding sequence which could have originated from different resources and the HDV genome was eventually constituted through RNA recombination. The genome subsequently diversified through accumulation of mutations selected by interactions between the mutated RNA and proteins with host factors to successfully form the infectious virions. Therefore, we propose that the conservation of HDV nucleotide sequence is highly related with its functionality. Genome analysis of known HDV isolates shows that the C-terminal coding sequences of large delta antigen (LDAg) are the highest diversity than other regions of protein-coding sequences but they still retain biological functionality to interact with the heavy chain of clathrin can be selected and maintained. Since viruses interact with many host factors, including escaping the host immune response, how to design a program to predict RNA genome evolution is a great challenging work. PMID:20204073

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

Five Complete Chloroplast Genome Sequences from Diospyros: Genome Organization and Comparative Analysis.

PubMed

Fu, Jianmin; Liu, Huimin; Hu, Jingjing; Liang, Yuqin; Liang, Jinjun; Wuyun, Tana; Tan, Xiaofeng

2016-01-01

Diospyros is the largest genus in Ebenaceae, comprising more than 500 species with remarkable economic value, especially Diospyros kaki Thunb., which has traditionally been an important food resource in China, Korea, and Japan. Complete chloroplast (cp) genomes from D. kaki, D. lotus L., D. oleifera Cheng., D. glaucifolia Metc., and Diospyros 'Jinzaoshi' were sequenced using Illumina sequencing technology. This is the first cp genome reported in Ebenaceae. The cp genome sequences of Diospyros ranged from 157,300 to 157,784 bp in length, presenting a typical quadripartite structure with two inverted repeats each separated by one large and one small single-copy region. For each cp genome, 134 genes were annotated, including 80 protein-coding, 31 tRNA, and 4 rRNA unique genes. In all, 179 repeats and 283 single sequence repeats were identified. Four hypervariable regions, namely, intergenic region of trnQ_rps16, trnV_ndhC, and psbD_trnT, and intron of ndhA, were identified in the Diospyros genomes. Phylogenetic analyses based on the whole cp genome, protein-coding, and intergenic and intron sequences indicated that D. oleifera is closely related to D. kaki and could be used as a model plant for future research on D. kaki; to our knowledge, this is proposed for the first time. Further, these analyses together with two large deletions (301 and 140 bp) in the cp genome of D. 'Jinzaoshi', support its placement as a new species in Diospyros. Both maximum parsimony and likelihood analyses for 19 taxa indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales.

Five Complete Chloroplast Genome Sequences from Diospyros: Genome Organization and Comparative Analysis

PubMed Central

Hu, Jingjing; Liang, Yuqin; Liang, Jinjun; Wuyun, Tana; Tan, Xiaofeng

2016-01-01

Diospyros is the largest genus in Ebenaceae, comprising more than 500 species with remarkable economic value, especially Diospyros kaki Thunb., which has traditionally been an important food resource in China, Korea, and Japan. Complete chloroplast (cp) genomes from D. kaki, D. lotus L., D. oleifera Cheng., D. glaucifolia Metc., and Diospyros ‘Jinzaoshi’ were sequenced using Illumina sequencing technology. This is the first cp genome reported in Ebenaceae. The cp genome sequences of Diospyros ranged from 157,300 to 157,784 bp in length, presenting a typical quadripartite structure with two inverted repeats each separated by one large and one small single-copy region. For each cp genome, 134 genes were annotated, including 80 protein-coding, 31 tRNA, and 4 rRNA unique genes. In all, 179 repeats and 283 single sequence repeats were identified. Four hypervariable regions, namely, intergenic region of trnQ_rps16, trnV_ndhC, and psbD_trnT, and intron of ndhA, were identified in the Diospyros genomes. Phylogenetic analyses based on the whole cp genome, protein-coding, and intergenic and intron sequences indicated that D. oleifera is closely related to D. kaki and could be used as a model plant for future research on D. kaki; to our knowledge, this is proposed for the first time. Further, these analyses together with two large deletions (301 and 140 bp) in the cp genome of D. ‘Jinzaoshi’, support its placement as a new species in Diospyros. Both maximum parsimony and likelihood analyses for 19 taxa indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales. PMID:27442423

Assembly of the Genome of the Disease Vector Aedes aegypti onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission

PubMed Central

Juneja, Punita; Osei-Poku, Jewelna; Ho, Yung S.; Ariani, Cristina V.; Palmer, William J.; Pain, Arnab; Jiggins, Francis M.

2014-01-01

The mosquito Aedes aegypti transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled — there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of Ae. aegypti, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes Ae. aegypti resistant to the human parasite Brugia malayi, and generated a list of candidate genes that could be affecting the trait. PMID:24498447

Sequence-specific epigenetic effects of the maternal somatic genome on developmental rearrangements of the zygotic genome in Paramecium primaurelia.

PubMed Central

Meyer, E; Butler, A; Dubrana, K; Duharcourt, S; Caron, F

1997-01-01

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions; the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences. PMID:9199294

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

The number of genomes from uncultivated microbes will soon surpass the number of isolate genomes in public databases (Hugenholtz, Skarshewski, & Parks, 2016). Technological advancements in high-throughput sequencing and assembly, including single-cell genomics and the computational extraction of genomes from metagenomes (GFMs), are largely responsible. Here we propose community standards for reporting the Minimum Information about a Single-Cell Genome (MIxS-SCG) and Minimum Information about Genomes extracted From Metagenomes (MIxS-GFM) specific for Bacteria and Archaea. The standards have been developed in the context of the International Genomics Standards Consortium (GSC) community (Field et al., 2014) and can be viewed as amore » supplement to other GSC checklists including the Minimum Information about a Genome Sequence (MIGS), Minimum information about a Metagenomic Sequence(s) (MIMS) (Field et al., 2008) and Minimum Information about a Marker Gene Sequence (MIMARKS) (P. Yilmaz et al., 2011). Community-wide acceptance of MIxS-SCG and MIxS-GFM for Bacteria and Archaea will enable broad comparative analyses of genomes from the majority of taxa that remain uncultivated, improving our understanding of microbial function, ecology, and evolution.« less

Kullback Leibler divergence in complete bacterial and phage genomes

PubMed Central

Akhter, Sajia; Kashef, Mona T.; Ibrahim, Eslam S.; Bailey, Barbara

2017-01-01

The amino acid content of the proteins encoded by a genome may predict the coding potential of that genome and may reflect lifestyle restrictions of the organism. Here, we calculated the Kullback–Leibler divergence from the mean amino acid content as a metric to compare the amino acid composition for a large set of bacterial and phage genome sequences. Using these data, we demonstrate that (i) there is a significant difference between amino acid utilization in different phylogenetic groups of bacteria and phages; (ii) many of the bacteria with the most skewed amino acid utilization profiles, or the bacteria that host phages with the most skewed profiles, are endosymbionts or parasites; (iii) the skews in the distribution are not restricted to certain metabolic processes but are common across all bacterial genomic subsystems; (iv) amino acid utilization profiles strongly correlate with GC content in bacterial genomes but very weakly correlate with the G+C percent in phage genomes. These findings might be exploited to distinguish coding from non-coding sequences in large data sets, such as metagenomic sequence libraries, to help in prioritizing subsequent analyses. PMID:29204318

Kullback Leibler divergence in complete bacterial and phage genomes.

PubMed

Akhter, Sajia; Aziz, Ramy K; Kashef, Mona T; Ibrahim, Eslam S; Bailey, Barbara; Edwards, Robert A

2017-01-01

The amino acid content of the proteins encoded by a genome may predict the coding potential of that genome and may reflect lifestyle restrictions of the organism. Here, we calculated the Kullback-Leibler divergence from the mean amino acid content as a metric to compare the amino acid composition for a large set of bacterial and phage genome sequences. Using these data, we demonstrate that (i) there is a significant difference between amino acid utilization in different phylogenetic groups of bacteria and phages; (ii) many of the bacteria with the most skewed amino acid utilization profiles, or the bacteria that host phages with the most skewed profiles, are endosymbionts or parasites; (iii) the skews in the distribution are not restricted to certain metabolic processes but are common across all bacterial genomic subsystems; (iv) amino acid utilization profiles strongly correlate with GC content in bacterial genomes but very weakly correlate with the G+C percent in phage genomes. These findings might be exploited to distinguish coding from non-coding sequences in large data sets, such as metagenomic sequence libraries, to help in prioritizing subsequent analyses.

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-...

ALLPATHS: Assembling Large Genomes with Short Illumina Reads

ScienceCinema

Gnerre, Sante

2018-02-06

Sante Gnerre from the Broad Institute speaks on the challenge of developing high quality assemblies of large genomes using short reads at the "Sequencing, Finishing, Analysis in the Future" meeting in Santa Fe, NM.

An Exploration into Fern Genome Space.

PubMed

Wolf, Paul G; Sessa, Emily B; Marchant, Daniel Blaine; Li, Fay-Wei; Rothfels, Carl J; Sigel, Erin M; Gitzendanner, Matthew A; Visger, Clayton J; Banks, Jo Ann; Soltis, Douglas E; Soltis, Pamela S; Pryer, Kathleen M; Der, Joshua P

2015-08-26

Ferns are one of the few remaining major clades of land plants for which a complete genome sequence is lacking. Knowledge of genome space in ferns will enable broad-scale comparative analyses of land plant genes and genomes, provide insights into genome evolution across green plants, and shed light on genetic and genomic features that characterize ferns, such as their high chromosome numbers and large genome sizes. As part of an initial exploration into fern genome space, we used a whole genome shotgun sequencing approach to obtain low-density coverage (∼0.4X to 2X) for six fern species from the Polypodiales (Ceratopteris, Pteridium, Polypodium, Cystopteris), Cyatheales (Plagiogyria), and Gleicheniales (Dipteris). We explore these data to characterize the proportion of the nuclear genome represented by repetitive sequences (including DNA transposons, retrotransposons, ribosomal DNA, and simple repeats) and protein-coding genes, and to extract chloroplast and mitochondrial genome sequences. Such initial sweeps of fern genomes can provide information useful for selecting a promising candidate fern species for whole genome sequencing. We also describe variation of genomic traits across our sample and highlight some differences and similarities in repeat structure between ferns and seed plants. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genome sequencing and analysis of the model grass Brachypodium distachyon

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

Yang, Xiaohan; Kalluri, Udaya C; Tuskan, Gerald A

Three subfamilies of grasses, the Ehrhartoideae, Panicoideae and Pooideae, provide the bulk of human nutrition and are poised to become major sources of renewable energy. Here we describe the genome sequence of the wild grass Brachypodium distachyon (Brachypodium), which is, to our knowledge, the first member of the Pooideae subfamily to be sequenced. Comparison of the Brachypodium, rice and sorghum genomes shows a precise history of genome evolution across a broad diversity of the grasses, and establishes a template for analysis of the large genomes of economically important pooid grasses such as wheat. The high-quality genome sequence, coupled with easemore » of cultivation and transformation, small size and rapid life cycle, will help Brachypodium reach its potential as an important model system for developing new energy and food crops.« less

Complete genome sequence of Leptotrichia buccalis type strain (C-1013-bT)

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

Ivanova, Natalia; Gronow, Sabine; Lapidus, Alla

2009-05-20

Leptotrichia buccalis (Robin 1853) Trevisan 1879 is the type species of the genus, and is of phylogenetic interest because of its isolated location in the sparsely populated and neither taxonomically nor genomically adequately accessed family 'Leptotrichiaceae' within the phylum 'Fusobacteria'. Species of Leptotrichia are large fusiform non-motile, non-sporulating rods, which often populate the human oral flora. L. buccalis is anaerobic to aerotolerant, and saccharolytic. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of the order 'Fusobacteriales' and no more than the second sequence from themore » phylum 'Fusobacteria'. The 2,465,610 bp long single replicon genome with its 2306 protein-coding and 61 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.« less

Complete genome sequence of Leptotrichia buccalis type strain (C-1013-bT)

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

Ivanova, N; Gronow, Sabine; Lapidus, Alla L.

2009-01-01

Leptotrichia buccalis (Robin 1853) Trevisan 1879 is the type species of the genus, and is of phylogenetic interest because of its isolated location in the sparsely populated and neither taxonomically nor genomically adequately accessed family 'Leptotrichiaceae' within the phylum 'Fusobacteria'. Species of Leptotrichia are large, fusiform, non-motile, non-sporulating rods, which often populate the human oral flora. L. buccalis is anaerobic to aerotolerant, and saccharolytic. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of the order 'Fusobacteriales' and no more than the second sequence from themore » phylum 'Fusobacteria'. The 2,465,610 bp long single replicon genome with its 2306 protein-coding and 61 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.« less

Optical mapping and its potential for large-scale sequencing projects.

PubMed

Aston, C; Mishra, B; Schwartz, D C

1999-07-01

Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.

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.

Reefgenomics.Org - a repository for marine genomics data.

PubMed

Liew, Yi Jin; Aranda, Manuel; Voolstra, Christian R

2016-01-01

Over the last decade, technological advancements have substantially decreased the cost and time of obtaining large amounts of sequencing data. Paired with the exponentially increased computing power, individual labs are now able to sequence genomes or transcriptomes to investigate biological questions of interest. This has led to a significant increase in available sequence data. Although the bulk of data published in articles are stored in public sequence databases, very often, only raw sequencing data are available; miscellaneous data such as assembled transcriptomes, genome annotations etc. are not easily obtainable through the same means. Here, we introduce our website (http://reefgenomics.org) that aims to centralize genomic and transcriptomic data from marine organisms. Besides providing convenient means to download sequences, we provide (where applicable) a genome browser to explore available genomic features, and a BLAST interface to search through the hosted sequences. Through the interface, multiple datasets can be queried simultaneously, allowing for the retrieval of matching sequences from organisms of interest. The minimalistic, no-frills interface reduces visual clutter, making it convenient for end-users to search and explore processed sequence data. DATABASE URL: http://reefgenomics.org. © The Author(s) 2016. Published by Oxford University Press.

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.

Fast selection of miRNA candidates based on large-scale pre-computed MFE sets of randomized sequences

PubMed Central

2014-01-01

Background Small RNAs are important regulators of genome function, yet their prediction in genomes is still a major computational challenge. Statistical analyses of pre-miRNA sequences indicated that their 2D structure tends to have a minimal free energy (MFE) significantly lower than MFE values of equivalently randomized sequences with the same nucleotide composition, in contrast to other classes of non-coding RNA. The computation of many MFEs is, however, too intensive to allow for genome-wide screenings. Results Using a local grid infrastructure, MFE distributions of random sequences were pre-calculated on a large scale. These distributions follow a normal distribution and can be used to determine the MFE distribution for any given sequence composition by interpolation. It allows on-the-fly calculation of the normal distribution for any candidate sequence composition. Conclusion The speedup achieved makes genome-wide screening with this characteristic of a pre-miRNA sequence practical. Although this particular property alone will not be able to distinguish miRNAs from other sequences sufficiently discriminative, the MFE-based P-value should be added to the parameters of choice to be included in the selection of potential miRNA candidates for experimental verification. PMID:24418292

Impact of Lateral Transfers on the Genomes of Lepidoptera

PubMed Central

Drezen, Jean-Michel; Josse, Thibaut; Bézier, Annie; Gauthier, Jérémy; Huguet, Elisabeth

2017-01-01

Transfer of DNA sequences between species regardless of their evolutionary distance is very common in bacteria, but evidence that horizontal gene transfer (HGT) also occurs in multicellular organisms has been accumulating in the past few years. The actual extent of this phenomenon is underestimated due to frequent sequence filtering of “alien” DNA before genome assembly. However, recent studies based on genome sequencing have revealed, and experimentally verified, the presence of foreign DNA sequences in the genetic material of several species of Lepidoptera. Large DNA viruses, such as baculoviruses and the symbiotic viruses of parasitic wasps (bracoviruses), have the potential to mediate these transfers in Lepidoptera. In particular, using ultra-deep sequencing, newly integrated transposons have been identified within baculovirus genomes. Bacterial genes have also been acquired by genomes of Lepidoptera, as in other insects and nematodes. In addition, insertions of bracovirus sequences were present in the genomes of certain moth and butterfly lineages, that were likely corresponding to rearrangements of ancient integrations. The viral genes present in these sequences, sometimes of hymenopteran origin, have been co-opted by lepidopteran species to confer some protection against pathogens. PMID:29120392

Genome sequencing elucidates Sardinian genetic architecture and augments association analyses for lipid and blood inflammatory markers

PubMed Central

Zoledziewska, Magdalena; Mulas, Antonella; Pistis, Giorgio; Steri, Maristella; Danjou, Fabrice; Kwong, Alan; Ortega del Vecchyo, Vicente Diego; Chiang, Charleston W. K.; Bragg-Gresham, Jennifer; Pitzalis, Maristella; Nagaraja, Ramaiah; Tarrier, Brendan; Brennan, Christine; Uzzau, Sergio; Fuchsberger, Christian; Atzeni, Rossano; Reinier, Frederic; Berutti, Riccardo; Huang, Jie; Timpson, Nicholas J; Toniolo, Daniela; Gasparini, Paolo; Malerba, Giovanni; Dedoussis, George; Zeggini, Eleftheria; Soranzo, Nicole; Jones, Chris; Lyons, Robert; Angius, Andrea; Kang, Hyun M.; Novembre, John; Sanna, Serena; Schlessinger, David; Cucca, Francesco; Abecasis, Gonçalo R

2015-01-01

We report ~17.6M genetic variants from whole-genome sequencing of 2,120 Sardinians; 22% are absent from prior sequencing-based compilations and enriched for predicted functional consequence. Furthermore, ~76K variants common in our sample (frequency >5%) are rare elsewhere (<0.5% in the 1000 Genomes Project). We assessed the impact of these variants on circulating lipid levels and five inflammatory biomarkers. Fourteen signals, including two major new loci, were observed for lipid levels, and 19, including two novel loci, for inflammatory markers. New associations would be missed in analyses based on 1000 Genomes data, underlining the advantages of large-scale sequencing in this founder population. PMID:26366554

Complete Chloroplast Genome of the Multifunctional Crop Globe Artichoke and Comparison with Other Asteraceae

PubMed Central

Curci, Pasquale L.; De Paola, Domenico; Danzi, Donatella; Vendramin, Giovanni G.; Sonnante, Gabriella

2015-01-01

With over 20,000 species, Asteraceae is the second largest plant family. High-throughput sequencing of nuclear and chloroplast genomes has allowed for a better understanding of the evolutionary relationships within large plant families. Here, the globe artichoke chloroplast (cp) genome was obtained by a combination of whole-genome and BAC clone high-throughput sequencing. The artichoke cp genome is 152,529 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 25,155 bp, representing the longest IRs found in the Asteraceae family so far. The large (LSC) and the small (SSC) single-copy regions span 83,578 bp and 18,641 bp, respectively. The artichoke cp sequence was compared to the other eight Asteraceae complete cp genomes available, revealing an IR expansion at the SSC/IR boundary. This expansion consists of 17 bp of the ndhF gene generating an overlap between the ndhF and ycf1 genes. A total of 127 cp simple sequence repeats (cpSSRs) were identified in the artichoke cp genome, potentially suitable for future population studies in the Cynara genus. Parsimony-informative regions were evaluated and allowed to place a Cynara species within the Asteraceae family tree. The eight most informative coding regions were also considered and tested for “specific barcode” purpose in the Asteraceae family. Our results highlight the usefulness of cp genome sequencing in exploring plant genome diversity and retrieving reliable molecular resources for phylogenetic and evolutionary studies, as well as for specific barcodes in plants. PMID:25774672

Complete chloroplast genome of the multifunctional crop globe artichoke and comparison with other Asteraceae.

PubMed

Curci, Pasquale L; De Paola, Domenico; Danzi, Donatella; Vendramin, Giovanni G; Sonnante, Gabriella

2015-01-01

With over 20,000 species, Asteraceae is the second largest plant family. High-throughput sequencing of nuclear and chloroplast genomes has allowed for a better understanding of the evolutionary relationships within large plant families. Here, the globe artichoke chloroplast (cp) genome was obtained by a combination of whole-genome and BAC clone high-throughput sequencing. The artichoke cp genome is 152,529 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 25,155 bp, representing the longest IRs found in the Asteraceae family so far. The large (LSC) and the small (SSC) single-copy regions span 83,578 bp and 18,641 bp, respectively. The artichoke cp sequence was compared to the other eight Asteraceae complete cp genomes available, revealing an IR expansion at the SSC/IR boundary. This expansion consists of 17 bp of the ndhF gene generating an overlap between the ndhF and ycf1 genes. A total of 127 cp simple sequence repeats (cpSSRs) were identified in the artichoke cp genome, potentially suitable for future population studies in the Cynara genus. Parsimony-informative regions were evaluated and allowed to place a Cynara species within the Asteraceae family tree. The eight most informative coding regions were also considered and tested for "specific barcode" purpose in the Asteraceae family. Our results highlight the usefulness of cp genome sequencing in exploring plant genome diversity and retrieving reliable molecular resources for phylogenetic and evolutionary studies, as well as for specific barcodes in plants.

A high resolution radiation hybrid map of wheat chromosome 4A

USDA-ARS?s Scientific Manuscript database

Bread wheat has a large and complex allohexaploid genome with low recombination level at chromosome centromeric and peri-centromeric regions. This significantly hampers ordering of markers, contigs of physical maps and sequence scaffolds and impedes obtaining of high-quality reference genome sequenc...

Atlas2 Cloud: a framework for personal genome analysis in the cloud

PubMed Central

2012-01-01

Background Until recently, sequencing has primarily been carried out in large genome centers which have invested heavily in developing the computational infrastructure that enables genomic sequence analysis. The recent advancements in next generation sequencing (NGS) have led to a wide dissemination of sequencing technologies and data, to highly diverse research groups. It is expected that clinical sequencing will become part of diagnostic routines shortly. However, limited accessibility to computational infrastructure and high quality bioinformatic tools, and the demand for personnel skilled in data analysis and interpretation remains a serious bottleneck. To this end, the cloud computing and Software-as-a-Service (SaaS) technologies can help address these issues. Results We successfully enabled the Atlas2 Cloud pipeline for personal genome analysis on two different cloud service platforms: a community cloud via the Genboree Workbench, and a commercial cloud via the Amazon Web Services using Software-as-a-Service model. We report a case study of personal genome analysis using our Atlas2 Genboree pipeline. We also outline a detailed cost structure for running Atlas2 Amazon on whole exome capture data, providing cost projections in terms of storage, compute and I/O when running Atlas2 Amazon on a large data set. Conclusions We find that providing a web interface and an optimized pipeline clearly facilitates usage of cloud computing for personal genome analysis, but for it to be routinely used for large scale projects there needs to be a paradigm shift in the way we develop tools, in standard operating procedures, and in funding mechanisms. PMID:23134663

Atlas2 Cloud: a framework for personal genome analysis in the cloud.

PubMed

Evani, Uday S; Challis, Danny; Yu, Jin; Jackson, Andrew R; Paithankar, Sameer; Bainbridge, Matthew N; Jakkamsetti, Adinarayana; Pham, Peter; Coarfa, Cristian; Milosavljevic, Aleksandar; Yu, Fuli

2012-01-01

Until recently, sequencing has primarily been carried out in large genome centers which have invested heavily in developing the computational infrastructure that enables genomic sequence analysis. The recent advancements in next generation sequencing (NGS) have led to a wide dissemination of sequencing technologies and data, to highly diverse research groups. It is expected that clinical sequencing will become part of diagnostic routines shortly. However, limited accessibility to computational infrastructure and high quality bioinformatic tools, and the demand for personnel skilled in data analysis and interpretation remains a serious bottleneck. To this end, the cloud computing and Software-as-a-Service (SaaS) technologies can help address these issues. We successfully enabled the Atlas2 Cloud pipeline for personal genome analysis on two different cloud service platforms: a community cloud via the Genboree Workbench, and a commercial cloud via the Amazon Web Services using Software-as-a-Service model. We report a case study of personal genome analysis using our Atlas2 Genboree pipeline. We also outline a detailed cost structure for running Atlas2 Amazon on whole exome capture data, providing cost projections in terms of storage, compute and I/O when running Atlas2 Amazon on a large data set. We find that providing a web interface and an optimized pipeline clearly facilitates usage of cloud computing for personal genome analysis, but for it to be routinely used for large scale projects there needs to be a paradigm shift in the way we develop tools, in standard operating procedures, and in funding mechanisms.

The mitochondrial genomes of the acoelomorph worms Paratomella rubra, Isodiametra pulchra and Archaphanostoma ylvae.

PubMed

Robertson, Helen E; Lapraz, François; Egger, Bernhard; Telford, Maximilian J; Schiffer, Philipp H

2017-05-12

Acoels are small, ubiquitous - but understudied - marine worms with a very simple body plan. Their internal phylogeny is still not fully resolved, and the position of their proposed phylum Xenacoelomorpha remains debated. Here we describe mitochondrial genome sequences from the acoels Paratomella rubra and Isodiametra pulchra, and the complete mitochondrial genome of the acoel Archaphanostoma ylvae. The P. rubra and A. ylvae sequences are typical for metazoans in size and gene content. The larger I. pulchra  mitochondrial genome contains both ribosomal genes, 21 tRNAs, but only 11 protein-coding genes. We find evidence suggesting a duplicated sequence in the I. pulchra mitochondrial genome. The P. rubra, I. pulchra and A. ylvae mitochondria have a unique genome organisation in comparison to other metazoan mitochondrial genomes. We found a large degree of protein-coding gene and tRNA overlap with little non-coding sequence in the compact P. rubra genome. Conversely, the A. ylvae and I. pulchra genomes have many long non-coding sequences between genes, likely driving genome size expansion in the latter. Phylogenetic trees inferred from mitochondrial genes retrieve Xenacoelomorpha as an early branching taxon in the deuterostomes. Sequence divergence analysis between P. rubra sampled in England and Spain indicates cryptic diversity.

An efficient and scalable analysis framework for variant extraction and refinement from population-scale DNA sequence data.

PubMed

Jun, Goo; Wing, Mary Kate; Abecasis, Gonçalo R; Kang, Hyun Min

2015-06-01

The analysis of next-generation sequencing data is computationally and statistically challenging because of the massive volume of data and imperfect data quality. We present GotCloud, a pipeline for efficiently detecting and genotyping high-quality variants from large-scale sequencing data. GotCloud automates sequence alignment, sample-level quality control, variant calling, filtering of likely artifacts using machine-learning techniques, and genotype refinement using haplotype information. The pipeline can process thousands of samples in parallel and requires less computational resources than current alternatives. Experiments with whole-genome and exome-targeted sequence data generated by the 1000 Genomes Project show that the pipeline provides effective filtering against false positive variants and high power to detect true variants. Our pipeline has already contributed to variant detection and genotyping in several large-scale sequencing projects, including the 1000 Genomes Project and the NHLBI Exome Sequencing Project. We hope it will now prove useful to many medical sequencing studies. © 2015 Jun et al.; Published by Cold Spring Harbor Laboratory Press.

COMPUTATIONAL RESOURCES FOR BIOFUEL FEEDSTOCK SPECIES

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

Buell, Carol Robin; Childs, Kevin L

2013-05-07

While current production of ethanol as a biofuel relies on starch and sugar inputs, it is anticipated that sustainable production of ethanol for biofuel use will utilize lignocellulosic feedstocks. Candidate plant species to be used for lignocellulosic ethanol production include a large number of species within the Grass, Pine and Birch plant families. For these biofuel feedstock species, there are variable amounts of genome sequence resources available, ranging from complete genome sequences (e.g. sorghum, poplar) to transcriptome data sets (e.g. switchgrass, pine). These data sets are not only dispersed in location but also disparate in content. It will be essentialmore » to leverage and improve these genomic data sets for the improvement of biofuel feedstock production. The objectives of this project were to provide computational tools and resources for data-mining genome sequence/annotation and large-scale functional genomic datasets available for biofuel feedstock species. We have created a Bioenergy Feedstock Genomics Resource that provides a web-based portal or clearing house for genomic data for plant species relevant to biofuel feedstock production. Sequence data from a total of 54 plant species are included in the Bioenergy Feedstock Genomics Resource including model plant species that permit leveraging of knowledge across taxa to biofuel feedstock species.We have generated additional computational analyses of these data, including uniform annotation, to facilitate genomic approaches to improved biofuel feedstock production. These data have been centralized in the publicly available Bioenergy Feedstock Genomics Resource (http://bfgr.plantbiology.msu.edu/).« less

Extensive sequencing of seven human genomes to characterize benchmark reference materials

PubMed Central

Zook, Justin M.; Catoe, David; McDaniel, Jennifer; Vang, Lindsay; Spies, Noah; Sidow, Arend; Weng, Ziming; Liu, Yuling; Mason, Christopher E.; Alexander, Noah; Henaff, Elizabeth; McIntyre, Alexa B.R.; Chandramohan, Dhruva; Chen, Feng; Jaeger, Erich; Moshrefi, Ali; Pham, Khoa; Stedman, William; Liang, Tiffany; Saghbini, Michael; Dzakula, Zeljko; Hastie, Alex; Cao, Han; Deikus, Gintaras; Schadt, Eric; Sebra, Robert; Bashir, Ali; Truty, Rebecca M.; Chang, Christopher C.; Gulbahce, Natali; Zhao, Keyan; Ghosh, Srinka; Hyland, Fiona; Fu, Yutao; Chaisson, Mark; Xiao, Chunlin; Trow, Jonathan; Sherry, Stephen T.; Zaranek, Alexander W.; Ball, Madeleine; Bobe, Jason; Estep, Preston; Church, George M.; Marks, Patrick; Kyriazopoulou-Panagiotopoulou, Sofia; Zheng, Grace X.Y.; Schnall-Levin, Michael; Ordonez, Heather S.; Mudivarti, Patrice A.; Giorda, Kristina; Sheng, Ying; Rypdal, Karoline Bjarnesdatter; Salit, Marc

2016-01-01

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly. PMID:27271295

Non-contiguous finished genome sequence of the opportunistic oral pathogen Prevotella multisaccharivorax type strain (PPPA20T)

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

Pati, Amrita; Gronow, Sabine; Lu, Megan

2011-01-01

Prevotella multisaccharivorax Sakamoto et al. 2005 is a species of the large genus Prevotella, which belongs to the family Prevotellaceae. The species is of medical interest because its members are able to cause diseases in the human oral cavity such as periodontitis, root caries and others. Although 77 Prevotella genomes have already been sequenced or are targeted for sequencing, this is only the second completed genome sequence of a type strain of a species within the genus Prevotella to be published. The 3,388,644 bp long genome is assembled in three non-contiguous contigs, harbors 2,876 protein-coding and 75 RNA genes andmore » is a part of the Genomic Encyclopedia of Bacteria and Archaea project.« less

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

PubMed

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

2013-01-01

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

Discovery, genotyping and characterization of structural variation and novel sequence at single nucleotide resolution from de novo genome assemblies on a population scale.

PubMed

Liu, Siyang; Huang, Shujia; Rao, Junhua; Ye, Weijian; Krogh, Anders; Wang, Jun

2015-01-01

Comprehensive recognition of genomic variation in one individual is important for understanding disease and developing personalized medication and treatment. Many tools based on DNA re-sequencing exist for identification of single nucleotide polymorphisms, small insertions and deletions (indels) as well as large deletions. However, these approaches consistently display a substantial bias against the recovery of complex structural variants and novel sequence in individual genomes and do not provide interpretation information such as the annotation of ancestral state and formation mechanism. We present a novel approach implemented in a single software package, AsmVar, to discover, genotype and characterize different forms of structural variation and novel sequence from population-scale de novo genome assemblies up to nucleotide resolution. Application of AsmVar to several human de novo genome assemblies captures a wide spectrum of structural variants and novel sequences present in the human population in high sensitivity and specificity. Our method provides a direct solution for investigating structural variants and novel sequences from de novo genome assemblies, facilitating the construction of population-scale pan-genomes. Our study also highlights the usefulness of the de novo assembly strategy for definition of genome structure.

The mitochondrial genome of the legume Vigna radiata and the analysis of recombination across short mitochondrial repeats.

PubMed

Alverson, Andrew J; Zhuo, Shi; Rice, Danny W; Sloan, Daniel B; Palmer, Jeffrey D

2011-01-20

The mitochondrial genomes of seed plants are exceptionally fluid in size, structure, and sequence content, with the accumulation and activity of repetitive sequences underlying much of this variation. We report the first fully sequenced mitochondrial genome of a legume, Vigna radiata (mung bean), and show that despite its unexceptional size (401,262 nt), the genome is unusually depauperate in repetitive DNA and "promiscuous" sequences from the chloroplast and nuclear genomes. Although Vigna lacks the large, recombinationally active repeats typical of most other seed plants, a PCR survey of its modest repertoire of short (38-297 nt) repeats nevertheless revealed evidence for recombination across all of them. A set of novel control assays showed, however, that these results could instead reflect, in part or entirely, artifacts of PCR-mediated recombination. Consequently, we recommend that other methods, especially high-depth genome sequencing, be used instead of PCR to infer patterns of plant mitochondrial recombination. The average-sized but repeat- and feature-poor mitochondrial genome of Vigna makes it ever more difficult to generalize about the factors shaping the size and sequence content of plant mitochondrial genomes.

Genomic and Genetic Diversity within the Pseudomonas fluorescens Complex

PubMed Central

Garrido-Sanz, Daniel; Meier-Kolthoff, Jan P.; Göker, Markus; Martín, Marta; Rivilla, Rafael; Redondo-Nieto, Miguel

2016-01-01

The Pseudomonas fluorescens complex includes Pseudomonas strains that have been taxonomically assigned to more than fifty different species, many of which have been described as plant growth-promoting rhizobacteria (PGPR) with potential applications in biocontrol and biofertilization. So far the phylogeny of this complex has been analyzed according to phenotypic traits, 16S rDNA, MLSA and inferred by whole-genome analysis. However, since most of the type strains have not been fully sequenced and new species are frequently described, correlation between taxonomy and phylogenomic analysis is missing. In recent years, the genomes of a large number of strains have been sequenced, showing important genomic heterogeneity and providing information suitable for genomic studies that are important to understand the genomic and genetic diversity shown by strains of this complex. Based on MLSA and several whole-genome sequence-based analyses of 93 sequenced strains, we have divided the P. fluorescens complex into eight phylogenomic groups that agree with previous works based on type strains. Digital DDH (dDDH) identified 69 species and 75 subspecies within the 93 genomes. The eight groups corresponded to clustering with a threshold of 31.8% dDDH, in full agreement with our MLSA. The Average Nucleotide Identity (ANI) approach showed inconsistencies regarding the assignment to species and to the eight groups. The small core genome of 1,334 CDSs and the large pan-genome of 30,848 CDSs, show the large diversity and genetic heterogeneity of the P. fluorescens complex. However, a low number of strains were enough to explain most of the CDSs diversity at core and strain-specific genomic fractions. Finally, the identification and analysis of group-specific genome and the screening for distinctive characters revealed a phylogenomic distribution of traits among the groups that provided insights into biocontrol and bioremediation applications as well as their role as PGPR. PMID:26915094

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

PubMed Central

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

2015-01-01

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

Evolution of the Australian lungfish (Neoceratodus forsteri) genome: a major role for CR1 and L2 LINE elements.

PubMed

Metcalfe, Cushla J; Filée, Jonathan; Germon, Isabelle; Joss, Jean; Casane, Didier

2012-11-01

Haploid genomes greater than 25,000 Mb are rare, within the animals only the lungfish and some of the salamanders and crustaceans are known to have genomes this large. There is very little data on the structure of genomes this size. It is known, however, that for animal genomes up to 3,000 Mb, there is in general a good correlation between genome size and the percent of the genome composed of repetitive sequence and that this repetitive component is highly dynamic. In this study, we sampled the Australian lungfish genome using three mini-genomic libraries and found that with very little sequence, the results converged on an estimate of 40% of the genome being composed of recognizable transposable elements (TEs), chiefly from the CR1 and L2 long interspersed nuclear element clades. We further characterized the CR1 and L2 elements in the lungfish genome and show that although most CR1 elements probably represent recent amplifications, the L2 elements are more diverse and are more likely the result of a series of amplifications. We suggest that our sampling method has probably underestimated the recognizable TE content. However, on the basis of the most likely sources of error, we suggest that this very large genome is not largely composed of recently amplified, undetected TEs but may instead include a large component of older degenerate TEs. Based on these estimates, and on Thomson's (Thomson K. 1972. An attempt to reconstruct evolutionary changes in the cellular DNA content of lungfish. J Exp Zool. 180:363-372) inference that in the lineage leading to the extant Australian lungfish, there was massive increase in genome size between 350 and 200 mya, after which the size of the genome changed little, we speculate that the very large Australian lungfish genome may be the result of a massive amplification of TEs followed by a long period with a very low rate of sequence removal and some ongoing TE activity.

About TCGA - TCGA

Cancer.gov

Find out about The Cancer Genome Atlas (TCGA) is a comprehensive and coordinated effort to accelerate our understanding of the molecular basis of cancer through the application of genome analysis technologies, including large-scale genome sequencing.

Radiation hybrid maps of the D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes.

PubMed

Kumar, Ajay; Seetan, Raed; Mergoum, Mohamed; Tiwari, Vijay K; Iqbal, Muhammad J; Wang, Yi; Al-Azzam, Omar; Šimková, Hana; Luo, Ming-Cheng; Dvorak, Jan; Gu, Yong Q; Denton, Anne; Kilian, Andrzej; Lazo, Gerard R; Kianian, Shahryar F

2015-10-16

The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high resolution genome maps with saturated marker scaffolds to anchor and orient BAC contigs/ sequence scaffolds for whole genome assembly. Radiation hybrid (RH) mapping has proven to be an excellent tool for the development of such maps for it offers much higher and more uniform marker resolution across the length of the chromosome compared to genetic mapping and does not require marker polymorphism per se, as it is based on presence (retention) vs. absence (deletion) marker assay. In this study, a 178 line RH panel was genotyped with SSRs and DArT markers to develop the first high resolution RH maps of the entire D-genome of Ae. tauschii accession AL8/78. To confirm map order accuracy, the AL8/78-RH maps were compared with:1) a DArT consensus genetic map constructed using more than 100 bi-parental populations, 2) a RH map of the D-genome of reference hexaploid wheat 'Chinese Spring', and 3) two SNP-based genetic maps, one with anchored D-genome BAC contigs and another with anchored D-genome sequence scaffolds. Using marker sequences, the RH maps were also anchored with a BAC contig based physical map and draft sequence of the D-genome of Ae. tauschii. A total of 609 markers were mapped to 503 unique positions on the seven D-genome chromosomes, with a total map length of 14,706.7 cR. The average distance between any two marker loci was 29.2 cR which corresponds to 2.1 cM or 9.8 Mb. The average mapping resolution across the D-genome was estimated to be 0.34 Mb (Mb/cR) or 0.07 cM (cM/cR). The RH maps showed almost perfect agreement with several published maps with regard to chromosome assignments of markers. The mean rank correlations between the position of markers on AL8/78 maps and the four published maps, ranged from 0.75 to 0.92, suggesting a good agreement in marker order. With 609 mapped markers, a total of 2481 deletions for the whole D-genome were detected with an average deletion size of 42.0 Mb. A total of 520 markers were anchored to 216 Ae. tauschii sequence scaffolds, 116 of which were not anchored earlier to the D-genome. This study reports the development of first high resolution RH maps for the D-genome of Ae. tauschii accession AL8/78, which were then used for the anchoring of unassigned sequence scaffolds. This study demonstrates how RH mapping, which offered high and uniform resolution across the length of the chromosome, can facilitate the complete sequence assembly of the large and complex plant genomes.

Genome of the Actinomycete Plant Pathogen Clavibacter michiganensis subsp. sepedonicus Suggests Recent Niche Adaptation▿ †

PubMed Central

Bentley, Stephen D.; Corton, Craig; Brown, Susan E.; Barron, Andrew; Clark, Louise; Doggett, Jon; Harris, Barbara; Ormond, Doug; Quail, Michael A.; May, Georgiana; Francis, David; Knudson, Dennis; Parkhill, Julian; Ishimaru, Carol A.

2008-01-01

Clavibacter michiganensis subsp. sepedonicus is a plant-pathogenic bacterium and the causative agent of bacterial ring rot, a devastating agricultural disease under strict quarantine control and zero tolerance in the seed potato industry. This organism appears to be largely restricted to an endophytic lifestyle, proliferating within plant tissues and unable to persist in the absence of plant material. Analysis of the genome sequence of C. michiganensis subsp. sepedonicus and comparison with the genome sequences of related plant pathogens revealed a dramatic recent evolutionary history. The genome contains 106 insertion sequence elements, which appear to have been active in extensive rearrangement of the chromosome compared to that of Clavibacter michiganensis subsp. michiganensis. There are 110 pseudogenes with overrepresentation in functions associated with carbohydrate metabolism, transcriptional regulation, and pathogenicity. Genome comparisons also indicated that there is substantial gene content diversity within the species, probably due to differential gene acquisition and loss. These genomic features and evolutionary dating suggest that there was recent adaptation for life in a restricted niche where nutrient diversity and perhaps competition are low, correlated with a reduced ability to exploit previously occupied complex niches outside the plant. Toleration of factors such as multiplication and integration of insertion sequence elements, genome rearrangements, and functional disruption of many genes and operons seems to indicate that there has been general relaxation of selective pressure on a large proportion of the genome. PMID:18192393

Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ)

PubMed Central

Mascher, Martin; Muehlbauer, Gary J; Rokhsar, Daniel S; Chapman, Jarrod; Schmutz, Jeremy; Barry, Kerrie; Muñoz-Amatriaín, María; Close, Timothy J; Wise, Roger P; Schulman, Alan H; Himmelbach, Axel; Mayer, Klaus FX; Scholz, Uwe; Poland, Jesse A; Stein, Nils; Waugh, Robbie

2013-01-01

Next-generation whole-genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence-based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost-efficient establishment of powerful genomic information for many species. PMID:23998490

Revealing the Genomic Landscape of Pediatric T-ALL | Office of Cancer Genomics

Cancer.gov

T-lineage acute lymphoblastic leukemia (T-ALL) comprises 15-20% of childhood ALL and has historically been associated with inferior outcome to B-cell  ALL (B-ALL). Recent studies have used genome-wide sequencing approaches to identify new subtypes and targets of mutation in B-ALL, but comprehensive sequencing studies of large cohorts of T-ALL have not been performed.

Complete genome sequence of Nakamurella multipartita type strain (Y-104).

PubMed

Tice, Hope; Mayilraj, Shanmugam; Sims, David; Lapidus, Alla; Nolan, Matt; Lucas, Susan; Glavina Del Rio, Tijana; Copeland, Alex; Cheng, Jan-Fang; Meincke, Linda; Bruce, David; Goodwin, Lynne; Pitluck, Sam; Ivanova, Natalia; Mavromatis, Konstantinos; Ovchinnikova, Galina; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jeffries, Cynthia D; Detter, John C; Brettin, Thomas; Rohde, Manfred; Göker, Markus; Bristow, Jim; Eisen, Jonathan A; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C; Klenk, Hans-Peter; Chen, Feng

2010-03-30

Nakamurella multipartita (Yoshimi et al. 1996) Tao et al. 2004 is the type species of the monospecific genus Nakamurella in the actinobacterial suborder Frankineae. The nonmotile, coccus-shaped strain was isolated from activated sludge acclimated with sugar-containing synthetic wastewater, and is capable of accumulating large amounts of polysaccharides in its cells. Here we describe the features of the organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the family Nakamurellaceae. The 6,060,298 bp long single replicon genome with its 5415 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Extensive structural variations between mitochondrial genomes of CMS and normal peppers (Capsicum annuum L.) revealed by complete nucleotide sequencing.

PubMed

Jo, Yeong Deuk; Choi, Yoomi; Kim, Dong-Hwan; Kim, Byung-Dong; Kang, Byoung-Cheorl

2014-07-04

Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp. We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (ψatp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and ψatp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes. Although large portion of sequence context was shared by mitochondrial genomes of CMS and male-fertile pepper lines, extensive genome rearrangements were detected. CMS candidate genes located on the edges of highly-rearranged CMS-specific DNA regions and near to repeat sequences. These characteristics were detected among CMS-associated genes in other species, implying a common mechanism might be involved in the evolution of CMS-associated genes.

Integrative Genomics Viewer (IGV) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

The Integrative Genomics Viewer (IGV) is a high-performance visualization tool for interactive exploration of large, integrated genomic datasets. It supports a wide variety of data types, including array-based and next-generation sequence data, and genomic annotations.

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

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

Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Jr., Richard A.

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

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

DOE PAGES

Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Jr., Richard A.; ...

2017-07-18

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

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

PubMed Central

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

2017-01-01

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences. PMID:28769883

A High Quality Draft Consensus Sequence of the Genome of a Heterozygous Grapevine Variety

PubMed Central

Cartwright, Dustin A.; Cestaro, Alessandro; Pruss, Dmitry; Pindo, Massimo; FitzGerald, Lisa M.; Vezzulli, Silvia; Reid, Julia; Malacarne, Giulia; Iliev, Diana; Coppola, Giuseppina; Wardell, Bryan; Micheletti, Diego; Macalma, Teresita; Facci, Marco; Mitchell, Jeff T.; Perazzolli, Michele; Eldredge, Glenn; Gatto, Pamela; Oyzerski, Rozan; Moretto, Marco; Gutin, Natalia; Stefanini, Marco; Chen, Yang; Segala, Cinzia; Davenport, Christine; Demattè, Lorenzo; Mraz, Amy; Battilana, Juri; Stormo, Keith; Costa, Fabrizio; Tao, Quanzhou; Si-Ammour, Azeddine; Harkins, Tim; Lackey, Angie; Perbost, Clotilde; Taillon, Bruce; Stella, Alessandra; Solovyev, Victor; Fawcett, Jeffrey A.; Sterck, Lieven; Vandepoele, Klaas; Grando, Stella M.; Toppo, Stefano; Moser, Claudio; Lanchbury, Jerry; Bogden, Robert; Skolnick, Mark; Sgaramella, Vittorio; Bhatnagar, Satish K.; Fontana, Paolo; Gutin, Alexander; Van de Peer, Yves; Salamini, Francesco; Viola, Roberto

2007-01-01

Background Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented. Principal Findings We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before). Conclusions Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape. PMID:18094749

Group-based variant calling leveraging next-generation supercomputing for large-scale whole-genome sequencing studies.

PubMed

Standish, Kristopher A; Carland, Tristan M; Lockwood, Glenn K; Pfeiffer, Wayne; Tatineni, Mahidhar; Huang, C Chris; Lamberth, Sarah; Cherkas, Yauheniya; Brodmerkel, Carrie; Jaeger, Ed; Smith, Lance; Rajagopal, Gunaretnam; Curran, Mark E; Schork, Nicholas J

2015-09-22

Next-generation sequencing (NGS) technologies have become much more efficient, allowing whole human genomes to be sequenced faster and cheaper than ever before. However, processing the raw sequence reads associated with NGS technologies requires care and sophistication in order to draw compelling inferences about phenotypic consequences of variation in human genomes. It has been shown that different approaches to variant calling from NGS data can lead to different conclusions. Ensuring appropriate accuracy and quality in variant calling can come at a computational cost. We describe our experience implementing and evaluating a group-based approach to calling variants on large numbers of whole human genomes. We explore the influence of many factors that may impact the accuracy and efficiency of group-based variant calling, including group size, the biogeographical backgrounds of the individuals who have been sequenced, and the computing environment used. We make efficient use of the Gordon supercomputer cluster at the San Diego Supercomputer Center by incorporating job-packing and parallelization considerations into our workflow while calling variants on 437 whole human genomes generated as part of large association study. We ultimately find that our workflow resulted in high-quality variant calls in a computationally efficient manner. We argue that studies like ours should motivate further investigations combining hardware-oriented advances in computing systems with algorithmic developments to tackle emerging 'big data' problems in biomedical research brought on by the expansion of NGS technologies.

The complete chloroplast genome sequence of the medicinal plant Salvia miltiorrhiza.

PubMed

Qian, Jun; Song, Jingyuan; Gao, Huanhuan; Zhu, Yingjie; Xu, Jiang; Pang, Xiaohui; Yao, Hui; Sun, Chao; Li, Xian'en; Li, Chuyuan; Liu, Juyan; Xu, Haibin; Chen, Shilin

2013-01-01

Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp) genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp) and small (SSC, 17,555 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp). It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.

Comparative Genomic and Transcriptomic Characterization of the Toxigenic Marine Dinoflagellate Alexandrium ostenfeldii

PubMed Central

Jaeckisch, Nina; Yang, Ines; Wohlrab, Sylke; Glöckner, Gernot; Kroymann, Juergen; Vogel, Heiko; Cembella, Allan; John, Uwe

2011-01-01

Many dinoflagellate species are notorious for the toxins they produce and ecological and human health consequences associated with harmful algal blooms (HABs). Dinoflagellates are particularly refractory to genomic analysis due to the enormous genome size, lack of knowledge about their DNA composition and structure, and peculiarities of gene regulation, such as spliced leader (SL) trans-splicing and mRNA transposition mechanisms. Alexandrium ostenfeldii is known to produce macrocyclic imine toxins, described as spirolides. We characterized the genome of A. ostenfeldii using a combination of transcriptomic data and random genomic clones for comparison with other dinoflagellates, particularly Alexandrium species. Examination of SL sequences revealed similar features as in other dinoflagellates, including Alexandrium species. SL sequences in decay indicate frequent retro-transposition of mRNA species. This probably contributes to overall genome complexity by generating additional gene copies. Sequencing of several thousand fosmid and bacterial artificial chromosome (BAC) ends yielded a wealth of simple repeats and tandemly repeated longer sequence stretches which we estimated to comprise more than half of the whole genome. Surprisingly, the repeats comprise a very limited set of 79–97 bp sequences; in part the genome is thus a relatively uniform sequence space interrupted by coding sequences. Our genomic sequence survey (GSS) represents the largest genomic data set of a dinoflagellate to date. Alexandrium ostenfeldii is a typical dinoflagellate with respect to its transcriptome and mRNA transposition but demonstrates Alexandrium-like stop codon usage. The large portion of repetitive sequences and the organization within the genome is in agreement with several other studies on dinoflagellates using different approaches. It remains to be determined whether this unusual composition is directly correlated to the exceptionally genome organization of dinoflagellates with a low amount of histones and histone-like proteins. PMID:22164224

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

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.

Molecular analysis of the anaerobic rumen fungus Orpinomyces - insights into an AT-rich genome.

PubMed

Nicholson, Matthew J; Theodorou, Michael K; Brookman, Jayne L

2005-01-01

The anaerobic gut fungi occupy a unique niche in the intestinal tract of large herbivorous animals and are thought to act as primary colonizers of plant material during digestion. They are the only known obligately anaerobic fungi but molecular analysis of this group has been hampered by difficulties in their culture and manipulation, and by their extremely high A+T nucleotide content. This study begins to answer some of the fundamental questions about the structure and organization of the anaerobic gut fungal genome. Directed plasmid libraries using genomic DNA digested with highly or moderately rich AT-specific restriction enzymes (VspI and EcoRI) were prepared from a polycentric Orpinomyces isolate. Clones were sequenced from these libraries and the breadth of genomic inserts, both genic and intergenic, was characterized. Genes encoding numerous functions not previously characterized for these fungi were identified, including cytoskeletal, secretory pathway and transporter genes. A peptidase gene with no introns and having sequence similarity to a gene encoding a bacterial peptidase was also identified, extending the range of metabolic enzymes resulting from apparent trans-kingdom transfer from bacteria to fungi, as previously characterized largely for genes encoding plant-degrading enzymes. This paper presents the first thorough analysis of the genic, intergenic and rDNA regions of a variety of genomic segments from an anaerobic gut fungus and provides observations on rules governing intron boundaries, the codon biases observed with different types of genes, and the sequence of only the second anaerobic gut fungal promoter reported. Large numbers of retrotransposon sequences of different types were found and the authors speculate on the possible consequences of any such transposon activity in the genome. The coding sequences identified included several orphan gene sequences, including one with regions strongly suggestive of structural proteins such as collagens and lampirin. This gene was present as a single copy in Orpinomyces, was expressed during vegetative growth and was also detected in genomes from another gut fungal genus, Neocallimastix.

Conserved intergenic sequences revealed by CTAG-profiling in Salmonella: thermodynamic modeling for function prediction

NASA Astrophysics Data System (ADS)

Tang, Le; Zhu, Songling; Mastriani, Emilio; Fang, Xin; Zhou, Yu-Jie; Li, Yong-Guo; Johnston, Randal N.; Guo, Zheng; Liu, Gui-Rong; Liu, Shu-Lin

2017-03-01

Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

The sequence and de novo assembly of the giant panda genome

PubMed Central

Li, Ruiqiang; Fan, Wei; Tian, Geng; Zhu, Hongmei; He, Lin; Cai, Jing; Huang, Quanfei; Cai, Qingle; Li, Bo; Bai, Yinqi; Zhang, Zhihe; Zhang, Yaping; Wang, Wen; Li, Jun; Wei, Fuwen; Li, Heng; Jian, Min; Li, Jianwen; Zhang, Zhaolei; Nielsen, Rasmus; Li, Dawei; Gu, Wanjun; Yang, Zhentao; Xuan, Zhaoling; Ryder, Oliver A.; Leung, Frederick Chi-Ching; Zhou, Yan; Cao, Jianjun; Sun, Xiao; Fu, Yonggui; Fang, Xiaodong; Guo, Xiaosen; Wang, Bo; Hou, Rong; Shen, Fujun; Mu, Bo; Ni, Peixiang; Lin, Runmao; Qian, Wubin; Wang, Guodong; Yu, Chang; Nie, Wenhui; Wang, Jinhuan; Wu, Zhigang; Liang, Huiqing; Min, Jiumeng; Wu, Qi; Cheng, Shifeng; Ruan, Jue; Wang, Mingwei; Shi, Zhongbin; Wen, Ming; Liu, Binghang; Ren, Xiaoli; Zheng, Huisong; Dong, Dong; Cook, Kathleen; Shan, Gao; Zhang, Hao; Kosiol, Carolin; Xie, Xueying; Lu, Zuhong; Zheng, Hancheng; Li, Yingrui; Steiner, Cynthia C.; Lam, Tommy Tsan-Yuk; Lin, Siyuan; Zhang, Qinghui; Li, Guoqing; Tian, Jing; Gong, Timing; Liu, Hongde; Zhang, Dejin; Fang, Lin; Ye, Chen; Zhang, Juanbin; Hu, Wenbo; Xu, Anlong; Ren, Yuanyuan; Zhang, Guojie; Bruford, Michael W.; Li, Qibin; Ma, Lijia; Guo, Yiran; An, Na; Hu, Yujie; Zheng, Yang; Shi, Yongyong; Li, Zhiqiang; Liu, Qing; Chen, Yanling; Zhao, Jing; Qu, Ning; Zhao, Shancen; Tian, Feng; Wang, Xiaoling; Wang, Haiyin; Xu, Lizhi; Liu, Xiao; Vinar, Tomas; Wang, Yajun; Lam, Tak-Wah; Yiu, Siu-Ming; Liu, Shiping; Zhang, Hemin; Li, Desheng; Huang, Yan; Wang, Xia; Yang, Guohua; Jiang, Zhi; Wang, Junyi; Qin, Nan; Li, Li; Li, Jingxiang; Bolund, Lars; Kristiansen, Karsten; Wong, Gane Ka-Shu; Olson, Maynard; Zhang, Xiuqing; Li, Songgang; Yang, Huanming; Wang, Jian; Wang, Jun

2013-01-01

Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes. PMID:20010809

Whole genome sequences of a male and female supercentenarian, ages greater than 114 years.

PubMed

Sebastiani, Paola; Riva, Alberto; Montano, Monty; Pham, Phillip; Torkamani, Ali; Scherba, Eugene; Benson, Gary; Milton, Jacqueline N; Baldwin, Clinton T; Andersen, Stacy; Schork, Nicholas J; Steinberg, Martin H; Perls, Thomas T

2011-01-01

Supercentenarians (age 110+ years old) generally delay or escape age-related diseases and disability well beyond the age of 100 and this exceptional survival is likely to be influenced by a genetic predisposition that includes both common and rare genetic variants. In this report, we describe the complete genomic sequences of male and female supercentenarians, both age >114 years old. We show that: (1) the sequence variant spectrum of these two individuals' DNA sequences is largely comparable to existing non-supercentenarian genomes; (2) the two individuals do not appear to carry most of the well-established human longevity enabling variants already reported in the literature; (3) they have a comparable number of known disease-associated variants relative to most human genomes sequenced to-date; (4) approximately 1% of the variants these individuals possess are novel and may point to new genes involved in exceptional longevity; and (5) both individuals are enriched for coding variants near longevity-associated variants that we discovered through a large genome-wide association study. These analyses suggest that there are both common and rare longevity-associated variants that may counter the effects of disease-predisposing variants and extend lifespan. The continued analysis of the genomes of these and other rare individuals who have survived to extremely old ages should provide insight into the processes that contribute to the maintenance of health during extreme aging.

Whole Genome Sequences of a Male and Female Supercentenarian, Ages Greater than 114 Years

PubMed Central

Sebastiani, Paola; Riva, Alberto; Montano, Monty; Pham, Phillip; Torkamani, Ali; Scherba, Eugene; Benson, Gary; Milton, Jacqueline N.; Baldwin, Clinton T.; Andersen, Stacy; Schork, Nicholas J.; Steinberg, Martin H.; Perls, Thomas T.

2012-01-01

Supercentenarians (age 110+ years old) generally delay or escape age-related diseases and disability well beyond the age of 100 and this exceptional survival is likely to be influenced by a genetic predisposition that includes both common and rare genetic variants. In this report, we describe the complete genomic sequences of male and female supercentenarians, both age >114 years old. We show that: (1) the sequence variant spectrum of these two individuals’ DNA sequences is largely comparable to existing non-supercentenarian genomes; (2) the two individuals do not appear to carry most of the well-established human longevity enabling variants already reported in the literature; (3) they have a comparable number of known disease-associated variants relative to most human genomes sequenced to-date; (4) approximately 1% of the variants these individuals possess are novel and may point to new genes involved in exceptional longevity; and (5) both individuals are enriched for coding variants near longevity-associated variants that we discovered through a large genome-wide association study. These analyses suggest that there are both common and rare longevity-associated variants that may counter the effects of disease-predisposing variants and extend lifespan. The continued analysis of the genomes of these and other rare individuals who have survived to extremely old ages should provide insight into the processes that contribute to the maintenance of health during extreme aging. PMID:22303384

Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level.

PubMed

Schmid, Michael; Muri, Jonathan; Melidis, Damianos; Varadarajan, Adithi R; Somerville, Vincent; Wicki, Adrian; Moser, Aline; Bourqui, Marc; Wenzel, Claudia; Eugster-Meier, Elisabeth; Frey, Juerg E; Irmler, Stefan; Ahrens, Christian H

2018-01-01

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus -to our knowledge-identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus . Notably, the functional Clusters of Orthologous Groups of proteins categories "cell wall/membrane biogenesis" and "defense mechanisms" were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.

Comparative Genomics of Completely Sequenced Lactobacillus helveticus Genomes Provides Insights into Strain-Specific Genes and Resolves Metagenomics Data Down to the Strain Level

PubMed Central

Schmid, Michael; Muri, Jonathan; Melidis, Damianos; Varadarajan, Adithi R.; Somerville, Vincent; Wicki, Adrian; Moser, Aline; Bourqui, Marc; Wenzel, Claudia; Eugster-Meier, Elisabeth; Frey, Juerg E.; Irmler, Stefan; Ahrens, Christian H.

2018-01-01

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus—to our knowledge—identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories “cell wall/membrane biogenesis” and “defense mechanisms” were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level. PMID:29441050

The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation.

PubMed

Mavromatis, Konstantinos; Land, Miriam L; Brettin, Thomas S; Quest, Daniel J; Copeland, Alex; Clum, Alicia; Goodwin, Lynne; Woyke, Tanja; Lapidus, Alla; Klenk, Hans Peter; Cottingham, Robert W; Kyrpides, Nikos C

2012-01-01

The emergence of next generation sequencing (NGS) has provided the means for rapid and high throughput sequencing and data generation at low cost, while concomitantly creating a new set of challenges. The number of available assembled microbial genomes continues to grow rapidly and their quality reflects the quality of the sequencing technology used, but also of the analysis software employed for assembly and annotation. In this work, we have explored the quality of the microbial draft genomes across various sequencing technologies. We have compared the draft and finished assemblies of 133 microbial genomes sequenced at the Department of Energy-Joint Genome Institute and finished at the Los Alamos National Laboratory using a variety of combinations of sequencing technologies, reflecting the transition of the institute from Sanger-based sequencing platforms to NGS platforms. The quality of the public assemblies and of the associated gene annotations was evaluated using various metrics. Results obtained with the different sequencing technologies, as well as their effects on downstream processes, were analyzed. Our results demonstrate that the Illumina HiSeq 2000 sequencing system, the primary sequencing technology currently used for de novo genome sequencing and assembly at JGI, has various advantages in terms of total sequence throughput and cost, but it also introduces challenges for the downstream analyses. In all cases assembly results although on average are of high quality, need to be viewed critically and consider sources of errors in them prior to analysis. These data follow the evolution of microbial sequencing and downstream processing at the JGI from draft genome sequences with large gaps corresponding to missing genes of significant biological role to assemblies with multiple small gaps (Illumina) and finally to assemblies that generate almost complete genomes (Illumina+PacBio).

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates.

PubMed

Leung, Kaston; Klaus, Anders; Lin, Bill K; Laks, Emma; Biele, Justina; Lai, Daniel; Bashashati, Ali; Huang, Yi-Fei; Aniba, Radhouane; Moksa, Michelle; Steif, Adi; Mes-Masson, Anne-Marie; Hirst, Martin; Shah, Sohrab P; Aparicio, Samuel; Hansen, Carl L

2016-07-26

The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in high-throughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform high-throughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10(-6) and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells.

Identification and characterization of dinucleotide repeat (CA)[sub n] markers for genetic mapping in dog

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

Ostrander, E.A.; Sprague, G.F. Jr.; Rine, J.

1993-04-01

A large block of simple sequence repeat (SSR) polymorphisms for the dog genome has been isolated and characterized. Screening of primary libraries by conventional hybridization methods as well as by screening of enriched marker-selected libraries led to the isolation of a large number of genomic clones that contained (CA)[sub n] repeats. The sequences of 101 clones showed that the size and complexity of (CA)[sub n] repeats in the dog genome were similar to those reported for these markers in the human genome. Detailed analysis of a representative subset of these markers revealed that most markers were moderately to highly polymorphic,more » with PIC values exceeding 0.70 for 33% of the markers tested. An association between higher PIC values and markers containing longer (CA)[sub n] repeats was observed in these studies, as previously noted for similar markers in the human genome. A list of primer sequences that tag each characterized marker is provided, and a comprehensive system of nomenclature for the dog genome is suggested. 28 refs., 4 figs., 2 tabs.« less

Mapping and phasing of structural variation in patient genomes using nanopore sequencing.

PubMed

Cretu Stancu, Mircea; van Roosmalen, Markus J; Renkens, Ivo; Nieboer, Marleen M; Middelkamp, Sjors; de Ligt, Joep; Pregno, Giulia; Giachino, Daniela; Mandrile, Giorgia; Espejo Valle-Inclan, Jose; Korzelius, Jerome; de Bruijn, Ewart; Cuppen, Edwin; Talkowski, Michael E; Marschall, Tobias; de Ridder, Jeroen; Kloosterman, Wigard P

2017-11-06

Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline-NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications.

Low levels of LTR retrotransposon deletion by ectopic recombination in the gigantic genomes of salamanders.

PubMed

Frahry, Matthew Blake; Sun, Cheng; Chong, Rebecca A; Mueller, Rachel Lockridge

2015-02-01

Across the tree of life, species vary dramatically in nuclear genome size. Mutations that add or remove sequences from genomes-insertions or deletions, or indels-are the ultimate source of this variation. Differences in the tempo and mode of insertion and deletion across taxa have been proposed to contribute to evolutionary diversity in genome size. Among vertebrates, most of the largest genomes are found within the salamanders, an amphibian clade with genome sizes ranging from ~14 to ~120 Gb. Salamander genomes have been shown to experience slower rates of DNA loss through small (i.e., <30 bp) deletions than do other vertebrate genomes. However, no studies have addressed DNA loss from salamander genomes resulting from larger deletions. Here, we focus on one type of large deletion-ectopic-recombination-mediated removal of LTR retrotransposon sequences. In ectopic recombination, double-strand breaks are repaired using a "wrong" (i.e., ectopic, or non-allelic) template sequence-typically another locus of similar sequence. When breaks occur within the LTR portions of LTR retrotransposons, ectopic-recombination-mediated repair can produce deletions that remove the internal transposon sequence and the equivalent of one of the two LTR sequences. These deletions leave a signature in the genome-a solo LTR sequence. We compared levels of solo LTRs in the genomes of four salamander species with levels present in five vertebrates with smaller genomes. Our results demonstrate that salamanders have low levels of solo LTRs, suggesting that ectopic-recombination-mediated deletion of LTR retrotransposons occurs more slowly than in other vertebrates with smaller genomes.

Genome-Wide Search Identifies 1.9 Mb from the Polar Bear Y Chromosome for Evolutionary Analyses

PubMed Central

Bidon, Tobias; Schreck, Nancy; Hailer, Frank; Nilsson, Maria A.; Janke, Axel

2015-01-01

The male-inherited Y chromosome is the major haploid fraction of the mammalian genome, rendering Y-linked sequences an indispensable resource for evolutionary research. However, despite recent large-scale genome sequencing approaches, only a handful of Y chromosome sequences have been characterized to date, mainly in model organisms. Using polar bear (Ursus maritimus) genomes, we compare two different in silico approaches to identify Y-linked sequences: 1) Similarity to known Y-linked genes and 2) difference in the average read depth of autosomal versus sex chromosomal scaffolds. Specifically, we mapped available genomic sequencing short reads from a male and a female polar bear against the reference genome and identify 112 Y-chromosomal scaffolds with a combined length of 1.9 Mb. We verified the in silico findings for the longer polar bear scaffolds by male-specific in vitro amplification, demonstrating the reliability of the average read depth approach. The obtained Y chromosome sequences contain protein-coding sequences, single nucleotide polymorphisms, microsatellites, and transposable elements that are useful for evolutionary studies. A high-resolution phylogeny of the polar bear patriline shows two highly divergent Y chromosome lineages, obtained from analysis of the identified Y scaffolds in 12 previously published male polar bear genomes. Moreover, we find evidence of gene conversion among ZFX and ZFY sequences in the giant panda lineage and in the ancestor of ursine and tremarctine bears. Thus, the identification of Y-linked scaffold sequences from unordered genome sequences yields valuable data to infer phylogenomic and population-genomic patterns in bears. PMID:26019166

Virtual Genome Walking across the 32 Gb Ambystoma mexicanum genome; assembling gene models and intronic sequence.

PubMed

Evans, Teri; Johnson, Andrew D; Loose, Matthew

2018-01-12

Large repeat rich genomes present challenges for assembly using short read technologies. The 32 Gb axolotl genome is estimated to contain ~19 Gb of repetitive DNA making an assembly from short reads alone effectively impossible. Indeed, this model species has been sequenced to 20× coverage but the reads could not be conventionally assembled. Using an alternative strategy, we have assembled subsets of these reads into scaffolds describing over 19,000 gene models. We call this method Virtual Genome Walking as it locally assembles whole genome reads based on a reference transcriptome, identifying exons and iteratively extending them into surrounding genomic sequence. These assemblies are then linked and refined to generate gene models including upstream and downstream genomic, and intronic, sequence. Our assemblies are validated by comparison with previously published axolotl bacterial artificial chromosome (BAC) sequences. Our analyses of axolotl intron length, intron-exon structure, repeat content and synteny provide novel insights into the genic structure of this model species. This resource will enable new experimental approaches in axolotl, such as ChIP-Seq and CRISPR and aid in future whole genome sequencing efforts. The assembled sequences and annotations presented here are freely available for download from https://tinyurl.com/y8gydc6n . The software pipeline is available from https://github.com/LooseLab/iterassemble .

Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102.

PubMed

Brown, Nathan M; Mueller, Ryan S; Shepardson, Jonathan W; Landry, Zachary C; Morré, Jeffrey T; Maier, Claudia S; Hardy, F Joan; Dreher, Theo W

2016-06-13

Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture. The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90. Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.

The Large Mitochondrial Genome of Symbiodinium minutum Reveals Conserved Noncoding Sequences between Dinoflagellates and Apicomplexans

PubMed Central

Shoguchi, Eiichi; Shinzato, Chuya; Hisata, Kanako; Satoh, Nori; Mungpakdee, Sutada

2015-01-01

Even though mitochondrial genomes, which characterize eukaryotic cells, were first discovered more than 50 years ago, mitochondrial genomics remains an important topic in molecular biology and genome sciences. The Phylum Alveolata comprises three major groups (ciliates, apicomplexans, and dinoflagellates), the mitochondrial genomes of which have diverged widely. Even though the gene content of dinoflagellate mitochondrial genomes is reportedly comparable to that of apicomplexans, the highly fragmented and rearranged genome structures of dinoflagellates have frustrated whole genomic analysis. Consequently, noncoding sequences and gene arrangements of dinoflagellate mitochondrial genomes have not been well characterized. Here we report that the continuous assembled genome (∼326 kb) of the dinoflagellate, Symbiodinium minutum, is AT-rich (∼64.3%) and that it contains three protein-coding genes. Based upon in silico analysis, the remaining 99% of the genome comprises transcriptomic noncoding sequences. RNA edited sites and unique, possible start and stop codons clarify conserved regions among dinoflagellates. Our massive transcriptome analysis shows that almost all regions of the genome are transcribed, including 27 possible fragmented ribosomal RNA genes and 12 uncharacterized small RNAs that are similar to mitochondrial RNA genes of the malarial parasite, Plasmodium falciparum. Gene map comparisons show that gene order is only slightly conserved between S. minutum and P. falciparum. However, small RNAs and intergenic sequences share sequence similarities with P. falciparum, suggesting that the function of noncoding sequences has been preserved despite development of very different genome structures. PMID:26199191

The genome sequence of the model ascomycete fungus Podospora anserina.

PubMed

Espagne, Eric; Lespinet, Olivier; Malagnac, Fabienne; Da Silva, Corinne; Jaillon, Olivier; Porcel, Betina M; Couloux, Arnaud; Aury, Jean-Marc; Ségurens, Béatrice; Poulain, Julie; Anthouard, Véronique; Grossetete, Sandrine; Khalili, Hamid; Coppin, Evelyne; Déquard-Chablat, Michelle; Picard, Marguerite; Contamine, Véronique; Arnaise, Sylvie; Bourdais, Anne; Berteaux-Lecellier, Véronique; Gautheret, Daniel; de Vries, Ronald P; Battaglia, Evy; Coutinho, Pedro M; Danchin, Etienne Gj; Henrissat, Bernard; Khoury, Riyad El; Sainsard-Chanet, Annie; Boivin, Antoine; Pinan-Lucarré, Bérangère; Sellem, Carole H; Debuchy, Robert; Wincker, Patrick; Weissenbach, Jean; Silar, Philippe

2008-01-01

The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development. We present a 10X draft sequence of P. anserina genome, linked to the sequences of a large expressed sequence tag collection. Similar to higher eukaryotes, the P. anserina transcription/splicing machinery generates numerous non-conventional transcripts. Comparison of the P. anserina genome and orthologous gene set with the one of its close relatives, Neurospora crassa, shows that synteny is poorly conserved, the main result of evolution being gene shuffling in the same chromosome. The P. anserina genome contains fewer repeated sequences and has evolved new genes by duplication since its separation from N. crassa, despite the presence of the repeat induced point mutation mechanism that mutates duplicated sequences. We also provide evidence that frequent gene loss took place in the lineages leading to P. anserina and N. crassa. P. anserina contains a large and highly specialized set of genes involved in utilization of natural carbon sources commonly found in its natural biotope. It includes genes potentially involved in lignin degradation and efficient cellulose breakdown. The features of the P. anserina genome indicate a highly dynamic evolution since the divergence of P. anserina and N. crassa, leading to the ability of the former to use specific complex carbon sources that match its needs in its natural biotope.

Simultaneous non-contiguous deletions using large synthetic DNA and site-specific recombinases

PubMed Central

Krishnakumar, Radha; Grose, Carissa; Haft, Daniel H.; Zaveri, Jayshree; Alperovich, Nina; Gibson, Daniel G.; Merryman, Chuck; Glass, John I.

2014-01-01

Toward achieving rapid and large scale genome modification directly in a target organism, we have developed a new genome engineering strategy that uses a combination of bioinformatics aided design, large synthetic DNA and site-specific recombinases. Using Cre recombinase we swapped a target 126-kb segment of the Escherichia coli genome with a 72-kb synthetic DNA cassette, thereby effectively eliminating over 54 kb of genomic DNA from three non-contiguous regions in a single recombination event. We observed complete replacement of the native sequence with the modified synthetic sequence through the action of the Cre recombinase and no competition from homologous recombination. Because of the versatility and high-efficiency of the Cre-lox system, this method can be used in any organism where this system is functional as well as adapted to use with other highly precise genome engineering systems. Compared to present-day iterative approaches in genome engineering, we anticipate this method will greatly speed up the creation of reduced, modularized and optimized genomes through the integration of deletion analyses data, transcriptomics, synthetic biology and site-specific recombination. PMID:24914053

Discovery and mapping of single feature polymorphisms in wheat using Affymetrix arrays

PubMed Central

Bernardo, Amy N; Bradbury, Peter J; Ma, Hongxiang; Hu, Shengwa; Bowden, Robert L; Buckler, Edward S; Bai, Guihua

2009-01-01

Background Wheat (Triticum aestivum L.) is a staple food crop worldwide. The wheat genome has not yet been sequenced due to its huge genome size (~17,000 Mb) and high levels of repetitive sequences; the whole genome sequence may not be expected in the near future. Available linkage maps have low marker density due to limitation in available markers; therefore new technologies that detect genome-wide polymorphisms are still needed to discover a large number of new markers for construction of high-resolution maps. A high-resolution map is a critical tool for gene isolation, molecular breeding and genomic research. Single feature polymorphism (SFP) is a new microarray-based type of marker that is detected by hybridization of DNA or cRNA to oligonucleotide probes. This study was conducted to explore the feasibility of using the Affymetrix GeneChip to discover and map SFPs in the large hexaploid wheat genome. Results Six wheat varieties of diverse origins (Ning 7840, Clark, Jagger, Encruzilhada, Chinese Spring, and Opata 85) were analyzed for significant probe by variety interactions and 396 probe sets with SFPs were identified. A subset of 164 unigenes was sequenced and 54% showed polymorphism within probes. Microarray analysis of 71 recombinant inbred lines from the cross Ning 7840/Clark identified 955 SFPs and 877 of them were mapped together with 269 simple sequence repeat markers. The SFPs were randomly distributed within a chromosome but were unevenly distributed among different genomes. The B genome had the most SFPs, and the D genome had the least. Map positions of a selected set of SFPs were validated by mapping single nucleotide polymorphism using SNaPshot and comparing with expressed sequence tags mapping data. Conclusion The Affymetrix array is a cost-effective platform for SFP discovery and SFP mapping in wheat. The new high-density map constructed in this study will be a useful tool for genetic and genomic research in wheat. PMID:19480702

Evolutionary and comparative analyses of the soybean genome

PubMed Central

Cannon, Steven B.; Shoemaker, Randy C.

2012-01-01

The soybean genome assembly has been available since the end of 2008. Significant features of the genome include large, gene-poor, repeat-dense pericentromeric regions, spanning roughly 57% of the genome sequence; a relatively large genome size of ~1.15 billion bases; remnants of a genome duplication that occurred ~13 million years ago (Mya); and fainter remnants of older polyploidies that occurred ~58 Mya and >130 Mya. The genome sequence has been used to identify the genetic basis for numerous traits, including disease resistance, nutritional characteristics, and developmental features. The genome sequence has provided a scaffold for placement of many genomic feature elements, both from within soybean and from related species. These may be accessed at several websites, including http://www.phytozome.net, http://soybase.org, http://comparative-legumes.org, and http://www.legumebase.brc.miyazaki-u.ac.jp. The taxonomic position of soybean in the Phaseoleae tribe of the legumes means that there are approximately two dozen other beans and relatives that have undergone independent domestication, and which may have traits that will be useful for transfer to soybean. Methods of translating information between species in the Phaseoleae range from design of markers for marker assisted selection, to transformation with Agrobacterium or with other experimental transformation methods. PMID:23136483

An Integrated Physical, Genetic and Cytogenetic Map of Brachypodium distachyon, a Model System for Grass Research

PubMed Central

Febrer, Melanie; Goicoechea, Jose Luis; Wright, Jonathan; McKenzie, Neil; Song, Xiang; Lin, Jinke; Collura, Kristi; Wissotski, Marina; Yu, Yeisoo; Ammiraju, Jetty S. S.; Wolny, Elzbieta; Idziak, Dominika; Betekhtin, Alexander; Kudrna, Dave; Hasterok, Robert; Wing, Rod A.; Bevan, Michael W.

2010-01-01

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent vaildation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation. PMID:20976139

Mechanisms of haplotype divergence at the RGA08 nucleotide-binding leucine-rich repeat gene locus in wild banana (Musa balbisiana).

PubMed

Baurens, Franc-Christophe; Bocs, Stéphanie; Rouard, Mathieu; Matsumoto, Takashi; Miller, Robert N G; Rodier-Goud, Marguerite; MBéguié-A-MBéguié, Didier; Yahiaoui, Nabila

2010-07-16

Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW). Sequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year divergence time for these M. balbisiana haplotypes. A large RGA08 gene cluster identified in wild banana corresponds to a highly variable genomic region between haplotypes surrounded by conserved flanking regions. High level of sequence identity (70 to 99%) of the genic and intergenic regions suggests a recent and rapid evolution of this cluster in M. balbisiana.

Construction of a plant-transformation-competent BIBAC library and genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.)

PubMed Central

2013-01-01

Background Cotton, one of the world’s leading crops, is important to the world’s textile and energy industries, and is a model species for studies of plant polyploidization, cellulose biosynthesis and cell wall biogenesis. Here, we report the construction of a plant-transformation-competent binary bacterial artificial chromosome (BIBAC) library and comparative genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L.) with one of its diploid putative progenitor species, G. raimondii Ulbr. Results We constructed the cotton BIBAC library in a vector competent for high-molecular-weight DNA transformation in different plant species through either Agrobacterium or particle bombardment. The library contains 76,800 clones with an average insert size of 135 kb, providing an approximate 99% probability of obtaining at least one positive clone from the library using a single-copy probe. The quality and utility of the library were verified by identifying BIBACs containing genes important for fiber development, fiber cellulose biosynthesis, seed fatty acid metabolism, cotton-nematode interaction, and bacterial blight resistance. In order to gain an insight into the Upland cotton genome and its relationship with G. raimondii, we sequenced nearly 10,000 BIBAC ends (BESs) randomly selected from the library, generating approximately one BES for every 250 kb along the Upland cotton genome. The retroelement Gypsy/DIRS1 family predominates in the Upland cotton genome, accounting for over 77% of all transposable elements. From the BESs, we identified 1,269 simple sequence repeats (SSRs), of which 1,006 were new, thus providing additional markers for cotton genome research. Surprisingly, comparative sequence analysis showed that Upland cotton is much more diverged from G. raimondii at the genomic sequence level than expected. There seems to be no significant difference between the relationships of the Upland cotton D- and A-subgenomes with the G. raimondii genome, even though G. raimondii contains a D genome (D5). Conclusions The library represents the first BIBAC library in cotton and related species, thus providing tools useful for integrative physical mapping, large-scale genome sequencing and large-scale functional analysis of the Upland cotton genome. Comparative sequence analysis provides insights into the Upland cotton genome, and a possible mechanism underlying the divergence and evolution of polyploid Upland cotton from its diploid putative progenitor species, G. raimondii. PMID:23537070

Comparative Chloroplast Genomics of Gossypium Species: Insights Into Repeat Sequence Variations and Phylogeny

PubMed Central

Wu, Ying; Liu, Fang; Yang, Dai-Gang; Li, Wei; Zhou, Xiao-Jian; Pei, Xiao-Yu; Liu, Yan-Gai; He, Kun-Lun; Zhang, Wen-Sheng; Ren, Zhong-Ying; Zhou, Ke-Hai; Ma, Xiong-Feng; Li, Zhong-Hu

2018-01-01

Cotton is one of the most economically important fiber crop plants worldwide. The genus Gossypium contains a single allotetraploid group (AD) and eight diploid genome groups (A–G and K). However, the evolution of repeat sequences in the chloroplast genomes and the phylogenetic relationships of Gossypium species are unclear. Thus, we determined the variations in the repeat sequences and the evolutionary relationships of 40 cotton chloroplast genomes, which represented the most diverse in the genus, including five newly sequenced diploid species, i.e., G. nandewarense (C1-n), G. armourianum (D2-1), G. lobatum (D7), G. trilobum (D8), and G. schwendimanii (D11), and an important semi-wild race of upland cotton, G. hirsutum race latifolium (AD1). The genome structure, gene order, and GC content of cotton species were similar to those of other higher plant plastid genomes. In total, 2860 long sequence repeats (>10 bp in length) were identified, where the F-genome species had the largest number of repeats (G. longicalyx F1: 108) and E-genome species had the lowest (G. stocksii E1: 53). Large-scale repeat sequences possibly enrich the genetic information and maintain genome stability in cotton species. We also identified 10 divergence hotspot regions, i.e., rpl33-rps18, psbZ-trnG (GCC), rps4-trnT (UGU), trnL (UAG)-rpl32, trnE (UUC)-trnT (GGU), atpE, ndhI, rps2, ycf1, and ndhF, which could be useful molecular genetic markers for future population genetics and phylogenetic studies. Site-specific selection analysis showed that some of the coding sites of 10 chloroplast genes (atpB, atpE, rps2, rps3, petB, petD, ccsA, cemA, ycf1, and rbcL) were under protein sequence evolution. Phylogenetic analysis based on the whole plastomes suggested that the Gossypium species grouped into six previously identified genetic clades. Interestingly, all 13 D-genome species clustered into a strong monophyletic clade. Unexpectedly, the cotton species with C, G, and K-genomes were admixed and nested in a large clade, which could have been due to their recent radiation, incomplete lineage sorting, and introgression hybridization among different cotton lineages. In conclusion, the results of this study provide new insights into the evolution of repeat sequences in chloroplast genomes and interspecific relationships in the genus Gossypium. PMID:29619041

Complete Genomic Sequence and Comparative Analysis of the Genome Segments of Sweet Potato Chlorotic Stunt Virus in China

PubMed Central

Qin, Yanhong; Wang, Li; Zhang, Zhenchen; Qiao, Qi; Zhang, Desheng; Tian, Yuting; Wang, Shuang; Wang, Yongjiang; Yan, Zhaoling

2014-01-01

Background Sweet potato chlorotic stunt virus (family Closteroviridae, genus Crinivirus) features a large bipartite, single-stranded, positive-sense RNA genome. To date, only three complete genomic sequences of SPCSV can be accessed through GenBank. SPCSV was first detected from China in 2011, only partial genomic sequences have been determined in the country. No report on the complete genomic sequence and genome structure of Chinese SPCSV isolates or the genetic relation between isolates from China and other countries is available. Methodology/Principal Findings The complete genomic sequences of five isolates from different areas in China were characterized. This study is the first to report the complete genome sequences of SPCSV from whitefly vectors. Genome structure analysis showed that isolates of WA and EA strains from China have the same coding protein as isolates Can181-9 and m2-47, respectively. Twenty cp genes and four RNA1 partial segments were sequenced and analyzed, and the nucleotide identities of complete genomic, cp, and RNA1 partial sequences were determined. Results indicated high conservation among strains and significant differences between WA and EA strains. Genetic analysis demonstrated that, except for isolates from Guangdong Province, SPCSVs from other areas belong to the WA strain. Genome organization analysis showed that the isolates in this study lack the p22 gene. Conclusions/Significance We presented the complete genome sequences of SPCSV in China. Comparison of nucleotide identities and genome structures between these isolates and previously reported isolates showed slight differences. The nucleotide identities of different SPCSV isolates showed high conservation among strains and significant differences between strains. All nine isolates in this study lacked p22 gene. WA strains were more extensively distributed than EA strains in China. These data provide important insights into the molecular variation and genomic structure of SPCSV in China as well as genetic relationships among isolates from China and other countries. PMID:25170926

de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer.

PubMed

Istace, Benjamin; Friedrich, Anne; d'Agata, Léo; Faye, Sébastien; Payen, Emilie; Beluche, Odette; Caradec, Claudia; Davidas, Sabrina; Cruaud, Corinne; Liti, Gianni; Lemainque, Arnaud; Engelen, Stefan; Wincker, Patrick; Schacherer, Joseph; Aury, Jean-Marc

2017-02-01

Oxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments from small genomes in a matter of seconds. The Oxford Nanopore technology is truly disruptive; it has the potential to revolutionize genomic applications due to its portability, low cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65 % of the chromosomes. This high contiguity allowed us to accurately detect large structural variations across the 21 studied genomes. Because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy. Our analyses show that the Oxford Nanopore technology is already usable for de novo sequencing and assembly; however, non-random errors in homopolymers require polishing the consensus using an alternate sequencing technology. © The Author 2017. Published by Oxford University Press.

de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer

PubMed Central

Istace, Benjamin; Friedrich, Anne; d'Agata, Léo; Faye, Sébastien; Payen, Emilie; Beluche, Odette; Caradec, Claudia; Davidas, Sabrina; Cruaud, Corinne; Liti, Gianni; Lemainque, Arnaud; Engelen, Stefan; Wincker, Patrick; Schacherer, Joseph

2017-01-01

Abstract Background: Oxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments from small genomes in a matter of seconds. The Oxford Nanopore technology is truly disruptive; it has the potential to revolutionize genomic applications due to its portability, low cost, and ease of use compared with existing long reads sequencing technologies. The MinION sequencer enables the rapid sequencing of small eukaryotic genomes, such as the yeast genome. Combined with existing assembler algorithms, near complete genome assemblies can be generated and comprehensive population genomic analyses can be performed. Results: Here, we resequenced the genome of the Saccharomyces cerevisiae S288C strain to evaluate the performance of nanopore-only assemblers. Then we de novo sequenced and assembled the genomes of 21 isolates representative of the S. cerevisiae genetic diversity using the MinION platform. The contiguity of our assemblies was 14 times higher than the Illumina-only assemblies and we obtained one or two long contigs for 65 % of the chromosomes. This high contiguity allowed us to accurately detect large structural variations across the 21 studied genomes. Conclusion: Because of the high completeness of the nanopore assemblies, we were able to produce a complete cartography of transposable elements insertions and inspect structural variants that are generally missed using a short-read sequencing strategy. Our analyses show that the Oxford Nanopore technology is already usable for de novo sequencing and assembly; however, non-random errors in homopolymers require polishing the consensus using an alternate sequencing technology. PMID:28369459

SOBA: sequence ontology bioinformatics analysis.

PubMed

Moore, Barry; Fan, Guozhen; Eilbeck, Karen

2010-07-01

The advent of cheaper, faster sequencing technologies has pushed the task of sequence annotation from the exclusive domain of large-scale multi-national sequencing projects to that of research laboratories and small consortia. The bioinformatics burden placed on these laboratories, some with very little programming experience can be daunting. Fortunately, there exist software libraries and pipelines designed with these groups in mind, to ease the transition from an assembled genome to an annotated and accessible genome resource. We have developed the Sequence Ontology Bioinformatics Analysis (SOBA) tool to provide a simple statistical and graphical summary of an annotated genome. We envisage its use during annotation jamborees, genome comparison and for use by developers for rapid feedback during annotation software development and testing. SOBA also provides annotation consistency feedback to ensure correct use of terminology within annotations, and guides users to add new terms to the Sequence Ontology when required. SOBA is available at http://www.sequenceontology.org/cgi-bin/soba.cgi.

The complete chloroplast genome of Capsicum annuum var. glabriusculum using Illumina sequencing.

PubMed

Raveendar, Sebastin; Na, Young-Wang; Lee, Jung-Ro; Shim, Donghwan; Ma, Kyung-Ho; Lee, Sok-Young; Chung, Jong-Wook

2015-07-20

Chloroplast (cp) genome sequences provide a valuable source for DNA barcoding. Molecular phylogenetic studies have concentrated on DNA sequencing of conserved gene loci. However, this approach is time consuming and more difficult to implement when gene organization differs among species. Here we report the complete re-sequencing of the cp genome of Capsicum pepper (Capsicum annuum var. glabriusculum) using the Illumina platform. The total length of the cp genome is 156,817 bp with a 37.7% overall GC content. A pair of inverted repeats (IRs) of 50,284 bp were separated by a small single copy (SSC; 18,948 bp) and a large single copy (LSC; 87,446 bp). The number of cp genes in C. annuum var. glabriusculum is the same as that in other Capsicum species. Variations in the lengths of LSC; SSC and IR regions were the main contributors to the size variation in the cp genome of this species. A total of 125 simple sequence repeat (SSR) and 48 insertions or deletions variants were found by sequence alignment of Capsicum cp genome. These findings provide a foundation for further investigation of cp genome evolution in Capsicum and other higher plants.

Analyzing Somatic Genome Rearrangements in Human Cancers by Using Whole-Exome Sequencing | Office of Cancer Genomics

Cancer.gov

Although exome sequencing data are generated primarily to detect single-nucleotide variants and indels, they can also be used to identify a subset of genomic rearrangements whose breakpoints are located in or near exons. Using >4,600 tumor and normal pairs across 15 cancer types, we identified over 9,000 high confidence somatic rearrangements, including a large number of gene fusions.

Complete Genome Sequence of the Symbiotic Strain Bradyrhizobium icense LMTR 13T, Isolated from Lima Bean (Phaseolus lunatus) in Peru

PubMed Central

Rogel, Marco A.; Zúñiga-Dávila, Doris; Martínez-Romero, Esperanza

2018-01-01

ABSTRACT The complete genome sequence of Bradyrhizobium icense LMTR 13T, a root nodule bacterium isolated from the legume Phaseolus lunatus, is reported here. The genome consists of a circular 8,322,773-bp chromosome which codes for a large and novel symbiotic island as well as genes putatively involved in soil and root colonization. PMID:29519840

Data mining for discovery of endophytic and epiphytic fungal diversity in short-read genomic data from deciduous trees

Treesearch

Nicholas R. ​LaBonte; James Jacobs; Aziz Ebrahimi; Shaneka Lawson; Keith Woeste

2018-01-01

High-throughput sequencing of DNA barcodes, such as the internal transcribed spacer (ITS) of the 16s rRNA sequence, has expanded the ability of researchers to investigate the endophytic fungal communities of living plants. With a large and growing database of complete fungal genomes, it may be possible to utilize portions of fungal symbiont genomes outside conventional...

Techniques for Large-Scale Bacterial Genome Manipulation and Characterization of the Mutants with Respect to In Silico Metabolic Reconstructions.

PubMed

diCenzo, George C; Finan, Turlough M

2018-01-01

The rate at which all genes within a bacterial genome can be identified far exceeds the ability to characterize these genes. To assist in associating genes with cellular functions, a large-scale bacterial genome deletion approach can be employed to rapidly screen tens to thousands of genes for desired phenotypes. Here, we provide a detailed protocol for the generation of deletions of large segments of bacterial genomes that relies on the activity of a site-specific recombinase. In this procedure, two recombinase recognition target sequences are introduced into known positions of a bacterial genome through single cross-over plasmid integration. Subsequent expression of the site-specific recombinase mediates recombination between the two target sequences, resulting in the excision of the intervening region and its loss from the genome. We further illustrate how this deletion system can be readily adapted to function as a large-scale in vivo cloning procedure, in which the region excised from the genome is captured as a replicative plasmid. We next provide a procedure for the metabolic analysis of bacterial large-scale genome deletion mutants using the Biolog Phenotype MicroArray™ system. Finally, a pipeline is described, and a sample Matlab script is provided, for the integration of the obtained data with a draft metabolic reconstruction for the refinement of the reactions and gene-protein-reaction relationships in a metabolic reconstruction.

Genomic Definition of Hypervirulent and Multidrug-Resistant Klebsiella pneumoniae Clonal Groups

PubMed Central

Bialek-Davenet, Suzanne; Criscuolo, Alexis; Ailloud, Florent; Passet, Virginie; Jones, Louis; Delannoy-Vieillard, Anne-Sophie; Garin, Benoit; Le Hello, Simon; Arlet, Guillaume; Nicolas-Chanoine, Marie-Hélène; Decré, Dominique

2014-01-01

Multidrug-resistant and highly virulent Klebsiella pneumoniae isolates are emerging, but the clonal groups (CGs) corresponding to these high-risk strains have remained imprecisely defined. We aimed to identify K. pneumoniae CGs on the basis of genome-wide sequence variation and to provide a simple bioinformatics tool to extract virulence and resistance gene data from genomic data. We sequenced 48 K. pneumoniae isolates, mostly of serotypes K1 and K2, and compared the genomes with 119 publicly available genomes. A total of 694 highly conserved genes were included in a core-genome multilocus sequence typing scheme, and cluster analysis of the data enabled precise definition of globally distributed hypervirulent and multidrug-resistant CGs. In addition, we created a freely accessible database, BIGSdb-Kp, to enable rapid extraction of medically and epidemiologically relevant information from genomic sequences of K. pneumoniae. Although drug-resistant and virulent K. pneumoniae populations were largely nonoverlapping, isolates with combined virulence and resistance features were detected. PMID:25341126

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

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

A draft physical map of a D-genome cotton species (Gossypium raimondii)

PubMed Central

2010-01-01

Background Genetically anchored physical maps of large eukaryotic genomes have proven useful both for their intrinsic merit and as an adjunct to genome sequencing. Cultivated tetraploid cottons, Gossypium hirsutum and G. barbadense, share a common ancestor formed by a merger of the A and D genomes about 1-2 million years ago. Toward the long-term goal of characterizing the spectrum of diversity among cotton genomes, the worldwide cotton community has prioritized the D genome progenitor Gossypium raimondii for complete sequencing. Results A whole genome physical map of G. raimondii, the putative D genome ancestral species of tetraploid cottons was assembled, integrating genetically-anchored overgo hybridization probes, agarose based fingerprints and 'high information content fingerprinting' (HICF). A total of 13,662 BAC-end sequences and 2,828 DNA probes were used in genetically anchoring 1585 contigs to a cotton consensus genetic map, and 370 and 438 contigs, respectively to Arabidopsis thaliana (AT) and Vitis vinifera (VV) whole genome sequences. Conclusion Several lines of evidence suggest that the G. raimondii genome is comprised of two qualitatively different components. Much of the gene rich component is aligned to the Arabidopsis and Vitis vinifera genomes and shows promise for utilizing translational genomic approaches in understanding this important genome and its resident genes. The integrated genetic-physical map is of value both in assembling and validating a planned reference sequence. PMID:20569427

Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species

PubMed Central

2013-01-01

Background The process of generating raw genome sequence data continues to become cheaper, faster, and more accurate. However, assembly of such data into high-quality, finished genome sequences remains challenging. Many genome assembly tools are available, but they differ greatly in terms of their performance (speed, scalability, hardware requirements, acceptance of newer read technologies) and in their final output (composition of assembled sequence). More importantly, it remains largely unclear how to best assess the quality of assembled genome sequences. The Assemblathon competitions are intended to assess current state-of-the-art methods in genome assembly. Results In Assemblathon 2, we provided a variety of sequence data to be assembled for three vertebrate species (a bird, a fish, and snake). This resulted in a total of 43 submitted assemblies from 21 participating teams. We evaluated these assemblies using a combination of optical map data, Fosmid sequences, and several statistical methods. From over 100 different metrics, we chose ten key measures by which to assess the overall quality of the assemblies. Conclusions Many current genome assemblers produced useful assemblies, containing a significant representation of their genes and overall genome structure. However, the high degree of variability between the entries suggests that there is still much room for improvement in the field of genome assembly and that approaches which work well in assembling the genome of one species may not necessarily work well for another. PMID:23870653

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PubMed

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

PubMed Central

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology. PMID:25423096

Draft Genome Sequence, and a Sequence-Defined Genetic Linkage Map of the Legume Crop Species Lupinus angustifolius L

PubMed Central

Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W.; Howieson, John G.; Li, Chengdao

2013-01-01

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species. PMID:23734219

Draft genome sequence, and a sequence-defined genetic linkage map of the legume crop species Lupinus angustifolius L.

PubMed

Yang, Huaan; Tao, Ye; Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W; Howieson, John G; Li, Chengdao

2013-01-01

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species.

Initial sequence and comparative analysis of the cat genome

PubMed Central

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

2007-01-01

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

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

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.

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

Ultraaccurate genome sequencing and haplotyping of single human cells.

PubMed

Chu, Wai Keung; Edge, Peter; Lee, Ho Suk; Bansal, Vikas; Bafna, Vineet; Huang, Xiaohua; Zhang, Kun

2017-11-21

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10 -8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs. Copyright © 2017 the Author(s). Published by PNAS.

Targeting vector construction through recombineering.

PubMed

Malureanu, Liviu A

2011-01-01

Gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. Conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific PCR amplification of repetitive DNA sequences, and cloning of large DNA fragments. Recombineering is a technique that exploits the highly efficient homologous recombination function encoded by λ phage in Escherichia coli. Bacteriophage-based recombination can recombine homologous sequences as short as 30-50 bases, allowing manipulations such as insertion, deletion, or mutation of virtually any genomic region. The large availability of mouse genomic bacterial artificial chromosome (BAC) libraries covering most of the genome facilitates the retrieval of genomic DNA sequences from the bacterial chromosomes through recombineering. This chapter describes a successfully applied protocol and aims to be a detailed guide through the steps of generation of targeting vectors through recombineering.

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 .

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.

The complete mitochondrial genome sequence of Malus hupehensis var. pinyiensis.

PubMed

Duan, Naibin; Sun, Honghe; Wang, Nan; Fei, Zhangjun; Chen, Xuesen

2016-07-01

The complete mitochondrial genome sequence of Malus hupehensis var. pinyiensis, a widely used apple rootstock, was determined using the Illumina high-throughput sequencing approach. The genome is 422,555 bp in length and has a GC content of 45.21%. It is separated by a pair of inverted repeats of 32,504 bp, to form a large single copy region of 213,055 bp and a small single copy region of 144,492 bp. The genome contains 38 protein-coding genes, four pseudogenes, 25 tRNA genes, and three rRNA genes. The genome is 25,608 bp longer than that of M. domestica, and several structural variations between these two mitogenomes were detected.

Mind the gap; seven reasons to close fragmented genome assemblies.

PubMed

Thomma, Bart P H J; Seidl, Michael F; Shi-Kunne, Xiaoqian; Cook, David E; Bolton, Melvin D; van Kan, Jan A L; Faino, Luigi

2016-05-01

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions. Copyright © 2015 Elsevier Inc. All rights reserved.

A vision for ubiquitous sequencing

PubMed Central

Erlich, Yaniv

2015-01-01

Genomics has recently celebrated reaching the $1000 genome milestone, making affordable DNA sequencing a reality. With this goal successfully completed, the next goal of the sequencing revolution can be sequencing sensors—miniaturized sequencing devices that are manufactured for real-time applications and deployed in large quantities at low costs. The first part of this manuscript envisions applications that will benefit from moving the sequencers to the samples in a range of domains. In the second part, the manuscript outlines the critical barriers that need to be addressed in order to reach the goal of ubiquitous sequencing sensors. PMID:26430149

Efficient identification of Y chromosome sequences in the human and Drosophila genomes.

PubMed

Carvalho, Antonio Bernardo; Clark, Andrew G

2013-11-01

Notwithstanding their biological importance, Y chromosomes remain poorly known in most species. A major obstacle to their study is the identification of Y chromosome sequences; due to its high content of repetitive DNA, in most genome projects, the Y chromosome sequence is fragmented into a large number of small, unmapped scaffolds. Identification of Y-linked genes among these fragments has yielded important insights about the origin and evolution of Y chromosomes, but the process is labor intensive, restricting studies to a small number of species. Apart from these fragmentary assemblies, in a few mammalian species, the euchromatic sequence of the Y is essentially complete, owing to painstaking BAC mapping and sequencing. Here we use female short-read sequencing and k-mer comparison to identify Y-linked sequences in two very different genomes, Drosophila virilis and human. Using this method, essentially all D. virilis scaffolds were unambiguously classified as Y-linked or not Y-linked. We found 800 new scaffolds (totaling 8.5 Mbp), and four new genes in the Y chromosome of D. virilis, including JYalpha, a gene involved in hybrid male sterility. Our results also strongly support the preponderance of gene gains over gene losses in the evolution of the Drosophila Y. In the intensively studied human genome, used here as a positive control, we recovered all previously known genes or gene families, plus a small amount (283 kb) of new, unfinished sequence. Hence, this method works in large and complex genomes and can be applied to any species with sex chromosomes.

Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

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

Catfish Genome Consortium; Wang, Shaolin; Peatman, Eric

2010-03-23

Background-Through the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energy's Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification. Results-A total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35percent of the unique sequences had significant similarities tomore » known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis. Conclusions-This project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.« less

Use of Whole-Genus Genome Sequence Data To Develop a Multilocus Sequence Typing Tool That Accurately Identifies Yersinia Isolates to the Species and Subspecies Levels

PubMed Central

Hall, Miquette; Chattaway, Marie A.; Reuter, Sandra; Savin, Cyril; Strauch, Eckhard; Carniel, Elisabeth; Connor, Thomas; Van Damme, Inge; Rajakaruna, Lakshani; Rajendram, Dunstan; Jenkins, Claire; Thomson, Nicholas R.

2014-01-01

The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica. PMID:25339391

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

A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq.

PubMed

Logan, Grace; Freimanis, Graham L; King, David J; Valdazo-González, Begoña; Bachanek-Bankowska, Katarzyna; Sanderson, Nicholas D; Knowles, Nick J; King, Donald P; Cottam, Eleanor M

2014-09-30

Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5' genomic termini and area immediately flanking the poly(C) region. We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

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

EUPAN enables pan-genome studies of a large number of eukaryotic genomes.

PubMed

Hu, Zhiqiang; Sun, Chen; Lu, Kuang-Chen; Chu, Xixia; Zhao, Yue; Lu, Jinyuan; Shi, Jianxin; Wei, Chaochun

2017-08-01

Pan-genome analyses are routinely carried out for bacteria to interpret the within-species gene presence/absence variations (PAVs). However, pan-genome analyses are rare for eukaryotes due to the large sizes and higher complexities of their genomes. Here we proposed EUPAN, a eukaryotic pan-genome analysis toolkit, enabling automatic large-scale eukaryotic pan-genome analyses and detection of gene PAVs at a relatively low sequencing depth. In the previous studies, we demonstrated the effectiveness and high accuracy of EUPAN in the pan-genome analysis of 453 rice genomes, in which we also revealed widespread gene PAVs among individual rice genomes. Moreover, EUPAN can be directly applied to the current re-sequencing projects primarily focusing on single nucleotide polymorphisms. EUPAN is implemented in Perl, R and C ++. It is supported under Linux and preferred for a computer cluster with LSF and SLURM job scheduling system. EUPAN together with its standard operating procedure (SOP) is freely available for non-commercial use (CC BY-NC 4.0) at http://cgm.sjtu.edu.cn/eupan/index.html . ccwei@sjtu.edu.cn or jianxin.shi@sjtu.edu.cn. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Whole genome sequencing identifies influenza A H3N2 transmission and offers superior resolution to classical typing methods.

PubMed

Meinel, Dominik M; Heinzinger, Susanne; Eberle, Ute; Ackermann, Nikolaus; Schönberger, Katharina; Sing, Andreas

2018-02-01

Influenza with its annual epidemic waves is a major cause of morbidity and mortality worldwide. However, only little whole genome data are available regarding the molecular epidemiology promoting our understanding of viral spread in human populations. We implemented a RT-PCR strategy starting from patient material to generate influenza A whole genome sequences for molecular epidemiological surveillance. Samples were obtained within the Bavarian Influenza Sentinel. The complete influenza virus genome was amplified by a one-tube multiplex RT-PCR and sequenced on an Illumina MiSeq. We report whole genomic sequences for 50 influenza A H3N2 viruses, which was the predominating virus in the season 2014/15, directly from patient specimens. The dataset included random samples from Bavaria (Germany) throughout the influenza season and samples from three suspected transmission clusters. We identified the outbreak samples based on sequence identity. Whole genome sequencing (WGS) was superior in resolution compared to analysis of single segments or partial segment analysis. Additionally, we detected manifestation of substantial amounts of viral quasispecies in several patients, carrying mutations varying from the dominant virus in each patient. Our rapid whole genome sequencing approach for influenza A virus shows that WGS can effectively be used to detect and understand outbreaks in large communities. Additionally, the genomic data provide in-depth details about the circulating virus within one season.

First Insights into the Large Genome of Epimedium sagittatum (Sieb. et Zucc) Maxim, a Chinese Traditional Medicinal Plant

PubMed Central

Liu, Di; Zeng, Shao-Hua; Chen, Jian-Jun; Zhang, Yan-Jun; Xiao, Gong; Zhu, Lin-Yao; Wang, Ying

2013-01-01

Epimedium sagittatum (Sieb. et Zucc) Maxim is a member of the Berberidaceae family of basal eudicot plants, widely distributed and used as a traditional medicinal plant in China for therapeutic effects on many diseases with a long history. Recent data shows that E. sagittatum has a relatively large genome, with a haploid genome size of ~4496 Mbp, divided into a small number of only 12 diploid chromosomes (2n = 2x = 12). However, little is known about Epimedium genome structure and composition. Here we present the analysis of 691 kb of high-quality genomic sequence derived from 672 randomly selected plasmid clones of E. sagittatum genomic DNA, representing ~0.0154% of the genome. The sampled sequences comprised at least 78.41% repetitive DNA elements and 2.51% confirmed annotated gene sequences, with a total GC% content of 39%. Retrotransposons represented the major class of transposable element (TE) repeats identified (65.37% of all TE repeats), particularly LTR (Long Terminal Repeat) retrotransposons (52.27% of all TE repeats). Chromosome analysis and Fluorescence in situ Hybridization of Gypsy-Ty3 retrotransposons were performed to survey the E. sagittatum genome at the cytological level. Our data provide the first insights into the composition and structure of the E. sagittatum genome, and will facilitate the functional genomic analysis of this valuable medicinal plant. PMID:23807511

Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

PubMed

Christen, Matthias; Del Medico, Luca; Christen, Heinz; Christen, Beat

2017-01-01

Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

Recapitulating phylogenies using k-mers: from trees to networks.

PubMed

Bernard, Guillaume; Ragan, Mark A; Chan, Cheong Xin

2016-01-01

Ernst Haeckel based his landmark Tree of Life on the supposed ontogenic recapitulation of phylogeny, i.e. that successive embryonic stages during the development of an organism re-trace the morphological forms of its ancestors over the course of evolution. Much of this idea has since been discredited. Today, phylogenies are often based on families of molecular sequences. The standard approach starts with a multiple sequence alignment, in which the sequences are arranged relative to each other in a way that maximises a measure of similarity position-by-position along their entire length. A tree (or sometimes a network) is then inferred. Rigorous multiple sequence alignment is computationally demanding, and evolutionary processes that shape the genomes of many microbes (bacteria, archaea and some morphologically simple eukaryotes) can add further complications. In particular, recombination, genome rearrangement and lateral genetic transfer undermine the assumptions that underlie multiple sequence alignment, and imply that a tree-like structure may be too simplistic. Here, using genome sequences of 143 bacterial and archaeal genomes, we construct a network of phylogenetic relatedness based on the number of shared k -mers (subsequences at fixed length k ). Our findings suggest that the network captures not only key aspects of microbial genome evolution as inferred from a tree, but also features that are not treelike. The method is highly scalable, allowing for investigation of genome evolution across a large number of genomes. Instead of using specific regions or sequences from genome sequences, or indeed Haeckel's idea of ontogeny, we argue that genome phylogenies can be inferred using k -mers from whole-genome sequences. Representing these networks dynamically allows biological questions of interest to be formulated and addressed quickly and in a visually intuitive manner.

Purification of Marek's disease virus DNA for 454 pyrosequencing using micrococcal nuclease digestion and polyethylene glycol precipitation

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV-1) is a cell-associated alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. The genomes of 6 strains have been sequenced using both Sanger didoxy sequencing and 454 Life Science pyrosequencing. These genomes largely represent cell culture adapted strains...

Genome Sequence of Fusarium oxysporum f. sp. melonis, a fungus causing wilt disease on melon

USDA-ARS?s Scientific Manuscript database

This manuscript reports the genome sequence of F. oxysporum f. sp. melonis, a fungal pathogen that causes Fusarium wilt disease on melon (Cucumis melo). The project is part of a large comparative study designed to explore the genetic composition and evolutionary origin of this group of horizontally ...

Genome sequence of Fusarium oxysporum f. sp. melonis, a fungus causing wilt disease on melon

USDA-ARS?s Scientific Manuscript database

This manuscript reports the genome sequence of F. oxysporum f. sp. melonis, a fungal pathogen that causes Fusarium wilt disease on melon (Cucumis melo). The project is part of a large comparative study designed to explore the genetic composition and evolutionary origin of this group of horizontally ...

Impacts of Genome-Wide Analyses on Our Understanding of Human Herpesvirus Diversity and Evolution.

PubMed

Renner, Daniel W; Szpara, Moriah L

2018-01-01

Until fairly recently, genome-wide evolutionary dynamics and within-host diversity were more commonly examined in the context of small viruses than in the context of large double-stranded DNA viruses such as herpesviruses. The high mutation rates and more compact genomes of RNA viruses have inspired the investigation of population dynamics for these species, and recent data now suggest that herpesviruses might also be considered candidates for population modeling. High-throughput sequencing (HTS) and bioinformatics have expanded our understanding of herpesviruses through genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures. Here we discuss recent data on the mechanisms that generate herpesvirus genomic diversity and underlie the evolution of these virus families. We focus on human herpesviruses, with key insights drawn from veterinary herpesviruses and other large DNA virus families. We consider the impacts of cell culture on herpesvirus genomes and how to accurately describe the viral populations under study. The need for a strong foundation of high-quality genomes is also discussed, since it underlies all secondary genomic analyses such as RNA sequencing (RNA-Seq), chromatin immunoprecipitation, and ribosome profiling. Areas where we foresee future progress, such as the linking of viral genetic differences to phenotypic or clinical outcomes, are highlighted as well. Copyright © 2017 Renner and Szpara.

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

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

Genome-Wide Search Identifies 1.9 Mb from the Polar Bear Y Chromosome for Evolutionary Analyses.

PubMed

Bidon, Tobias; Schreck, Nancy; Hailer, Frank; Nilsson, Maria A; Janke, Axel

2015-05-27

The male-inherited Y chromosome is the major haploid fraction of the mammalian genome, rendering Y-linked sequences an indispensable resource for evolutionary research. However, despite recent large-scale genome sequencing approaches, only a handful of Y chromosome sequences have been characterized to date, mainly in model organisms. Using polar bear (Ursus maritimus) genomes, we compare two different in silico approaches to identify Y-linked sequences: 1) Similarity to known Y-linked genes and 2) difference in the average read depth of autosomal versus sex chromosomal scaffolds. Specifically, we mapped available genomic sequencing short reads from a male and a female polar bear against the reference genome and identify 112 Y-chromosomal scaffolds with a combined length of 1.9 Mb. We verified the in silico findings for the longer polar bear scaffolds by male-specific in vitro amplification, demonstrating the reliability of the average read depth approach. The obtained Y chromosome sequences contain protein-coding sequences, single nucleotide polymorphisms, microsatellites, and transposable elements that are useful for evolutionary studies. A high-resolution phylogeny of the polar bear patriline shows two highly divergent Y chromosome lineages, obtained from analysis of the identified Y scaffolds in 12 previously published male polar bear genomes. Moreover, we find evidence of gene conversion among ZFX and ZFY sequences in the giant panda lineage and in the ancestor of ursine and tremarctine bears. Thus, the identification of Y-linked scaffold sequences from unordered genome sequences yields valuable data to infer phylogenomic and population-genomic patterns in bears. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Augmenting Chinese hamster genome assembly by identifying regions of high confidence.

PubMed

Vishwanathan, Nandita; Bandyopadhyay, Arpan A; Fu, Hsu-Yuan; Sharma, Mohit; Johnson, Kathryn C; Mudge, Joann; Ramaraj, Thiruvarangan; Onsongo, Getiria; Silverstein, Kevin A T; Jacob, Nitya M; Le, Huong; Karypis, George; Hu, Wei-Shou

2016-09-01

Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot-gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re-scaffolding and gap-filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as "high confidence regions" which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high-quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

pico-PLAZA, a genome database of microbial photosynthetic eukaryotes.

PubMed

Vandepoele, Klaas; Van Bel, Michiel; Richard, Guilhem; Van Landeghem, Sofie; Verhelst, Bram; Moreau, Hervé; Van de Peer, Yves; Grimsley, Nigel; Piganeau, Gwenael

2013-08-01

With the advent of next generation genome sequencing, the number of sequenced algal genomes and transcriptomes is rapidly growing. Although a few genome portals exist to browse individual genome sequences, exploring complete genome information from multiple species for the analysis of user-defined sequences or gene lists remains a major challenge. pico-PLAZA is a web-based resource (http://bioinformatics.psb.ugent.be/pico-plaza/) for algal genomics that combines different data types with intuitive tools to explore genomic diversity, perform integrative evolutionary sequence analysis and study gene functions. Apart from homologous gene families, multiple sequence alignments, phylogenetic trees, Gene Ontology, InterPro and text-mining functional annotations, different interactive viewers are available to study genome organization using gene collinearity and synteny information. Different search functions, documentation pages, export functions and an extensive glossary are available to guide non-expert scientists. To illustrate the versatility of the platform, different case studies are presented demonstrating how pico-PLAZA can be used to functionally characterize large-scale EST/RNA-Seq data sets and to perform environmental genomics. Functional enrichments analysis of 16 Phaeodactylum tricornutum transcriptome libraries offers a molecular view on diatom adaptation to different environments of ecological relevance. Furthermore, we show how complementary genomic data sources can easily be combined to identify marker genes to study the diversity and distribution of algal species, for example in metagenomes, or to quantify intraspecific diversity from environmental strains. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

Human Y chromosome copy number variation in the next generation sequencing era and beyond.

PubMed

Massaia, Andrea; Xue, Yali

2017-05-01

The human Y chromosome provides a fertile ground for structural rearrangements owing to its haploidy and high content of repeated sequences. The methodologies used for copy number variation (CNV) studies have developed over the years. Low-throughput techniques based on direct observation of rearrangements were developed early on, and are still used, often to complement array-based or sequencing approaches which have limited power in regions with high repeat content and specifically in the presence of long, identical repeats, such as those found in human sex chromosomes. Some specific rearrangements have been investigated for decades; because of their effects on fertility, or their outstanding evolutionary features, the interest in these has not diminished. However, following the flourishing of large-scale genomics, several studies have investigated CNVs across the whole chromosome. These studies sometimes employ data generated within large genomic projects such as the DDD study or the 1000 Genomes Project, and often survey large samples of healthy individuals without any prior selection. Novel technologies based on sequencing long molecules and combinations of technologies, promise to stimulate the study of Y-CNVs in the immediate future.

The First Endogenous Herpesvirus, Identified in the Tarsier Genome, and Novel Sequences from Primate Rhadinoviruses and Lymphocryptoviruses

PubMed Central

Aswad, Amr; Katzourakis, Aris

2014-01-01

Herpesviridae is a diverse family of large and complex pathogens whose genomes are extremely difficult to sequence. This is particularly true for clinical samples, and if the virus, host, or both genomes are being sequenced for the first time. Although herpesviruses are known to occasionally integrate in host genomes, and can also be inherited in a Mendelian fashion, they are notably absent from the genomic fossil record comprised of endogenous viral elements (EVEs). Here, we combine paleovirological and metagenomic approaches to both explore the constituent viral diversity of mammalian genomes and search for endogenous herpesviruses. We describe the first endogenous herpesvirus from the genome of the Philippine tarsier, belonging to the Roseolovirus genus, and characterize its highly defective genome that is integrated and flanked by unambiguous host DNA. From a draft assembly of the aye-aye genome, we use bioinformatic tools to reveal over 100,000 bp of a novel rhadinovirus that is the first lemur gammaherpesvirus, closely related to Kaposi's sarcoma-associated virus. We also identify 58 genes of Pan paniscus lymphocryptovirus 1, the bonobo equivalent of human Epstein-Barr virus. For each of the viruses, we postulate gene function via comparative analysis to known viral relatives. Most notably, the evidence from gene content and phylogenetics suggests that the aye-aye sequences represent the most basal known rhadinovirus, and indicates that tumorigenic herpesviruses have been infecting primates since their emergence in the late Cretaceous. Overall, these data show that a genomic fossil record of herpesviruses exists despite their extremely large genomes, and expands the known diversity of Herpesviridae, which will aid the characterization of pathogenesis. Our analytical approach illustrates the benefit of intersecting evolutionary approaches with metagenomics, genetics and paleovirology. PMID:24945689

Complete Genome Sequence of the Symbiotic Strain Bradyrhizobium icense LMTR 13T, Isolated from Lima Bean (Phaseolus lunatus) in Peru.

PubMed

Ormeño-Orrillo, Ernesto; Rogel, Marco A; Zúñiga-Dávila, Doris; Martínez-Romero, Esperanza

2018-03-08

The complete genome sequence of Bradyrhizobium icense LMTR 13 T , a root nodule bacterium isolated from the legume Phaseolus lunatus , is reported here. The genome consists of a circular 8,322,773-bp chromosome which codes for a large and novel symbiotic island as well as genes putatively involved in soil and root colonization. Copyright © 2018 Ormeño-Orrillo et al.

Toward an Integrated BAC Library Resource for Genome Sequencing and Analysis

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

Simon, M. I.; Kim, U.-J.

We developed a great deal of expertise in building large BAC libraries from a variety of DNA sources including humans, mice, corn, microorganisms, worms, and Arabidopsis. We greatly improved the technology for screening these libraries rapidly and for selecting appropriate BACs and mapping BACs to develop large overlapping contigs. We became involved in supplying BACs and BAC contigs to a variety of sequencing and mapping projects and we began to collaborate with Drs. Adams and Venter at TIGR and with Dr. Leroy Hood and his group at University of Washington to provide BACs for end sequencing and for mapping andmore » sequencing of large fragments of chromosome 16. Together with Dr. Ian Dunham and his co-workers at the Sanger Center we completed the mapping and they completed the sequencing of the first human chromosome, chromosome 22. This was published in Nature in 1999 and our BAC contigs made a major contribution to this sequencing effort. Drs. Shizuya and Ding invented an automated highly accurate BAC mapping technique. We also developed long-term collaborations with Dr. Uli Weier at UCSF in the design of BAC probes for characterization of human tumors and specific chromosome deletions and breakpoints. Finally the contribution of our work to the human genome project has been recognized in the publication both by the international consortium and the NIH of a draft sequence of the human genome in Nature last year. Dr. Shizuya was acknowledged in the authorship of that landmark paper. Dr. Simon was also an author on the Venter/Adams Celera project sequencing the human genome that was published in Science last year.« less

Wheat EST resources for functional genomics of abiotic stress

PubMed Central

Houde, Mario; Belcaid, Mahdi; Ouellet, François; Danyluk, Jean; Monroy, Antonio F; Dryanova, Ani; Gulick, Patrick; Bergeron, Anne; Laroche, André; Links, Matthew G; MacCarthy, Luke; Crosby, William L; Sarhan, Fathey

2006-01-01

Background Wheat is an excellent species to study freezing tolerance and other abiotic stresses. However, the sequence of the wheat genome has not been completely characterized due to its complexity and large size. To circumvent this obstacle and identify genes involved in cold acclimation and associated stresses, a large scale EST sequencing approach was undertaken by the Functional Genomics of Abiotic Stress (FGAS) project. Results We generated 73,521 quality-filtered ESTs from eleven cDNA libraries constructed from wheat plants exposed to various abiotic stresses and at different developmental stages. In addition, 196,041 ESTs for which tracefiles were available from the National Science Foundation wheat EST sequencing program and DuPont were also quality-filtered and used in the analysis. Clustering of the combined ESTs with d2_cluster and TGICL yielded a few large clusters containing several thousand ESTs that were refractory to routine clustering techniques. To resolve this problem, the sequence proximity and "bridges" were identified by an e-value distance graph to manually break clusters into smaller groups. Assembly of the resolved ESTs generated a 75,488 unique sequence set (31,580 contigs and 43,908 singletons/singlets). Digital expression analyses indicated that the FGAS dataset is enriched in stress-regulated genes compared to the other public datasets. Over 43% of the unique sequence set was annotated and classified into functional categories according to Gene Ontology. Conclusion We have annotated 29,556 different sequences, an almost 5-fold increase in annotated sequences compared to the available wheat public databases. Digital expression analysis combined with gene annotation helped in the identification of several pathways associated with abiotic stress. The genomic resources and knowledge developed by this project will contribute to a better understanding of the different mechanisms that govern stress tolerance in wheat and other cereals. PMID:16772040

The Large Mitochondrial Genome of Symbiodinium minutum Reveals Conserved Noncoding Sequences between Dinoflagellates and Apicomplexans.

PubMed

Shoguchi, Eiichi; Shinzato, Chuya; Hisata, Kanako; Satoh, Nori; Mungpakdee, Sutada

2015-07-20

Even though mitochondrial genomes, which characterize eukaryotic cells, were first discovered more than 50 years ago, mitochondrial genomics remains an important topic in molecular biology and genome sciences. The Phylum Alveolata comprises three major groups (ciliates, apicomplexans, and dinoflagellates), the mitochondrial genomes of which have diverged widely. Even though the gene content of dinoflagellate mitochondrial genomes is reportedly comparable to that of apicomplexans, the highly fragmented and rearranged genome structures of dinoflagellates have frustrated whole genomic analysis. Consequently, noncoding sequences and gene arrangements of dinoflagellate mitochondrial genomes have not been well characterized. Here we report that the continuous assembled genome (∼326 kb) of the dinoflagellate, Symbiodinium minutum, is AT-rich (∼64.3%) and that it contains three protein-coding genes. Based upon in silico analysis, the remaining 99% of the genome comprises transcriptomic noncoding sequences. RNA edited sites and unique, possible start and stop codons clarify conserved regions among dinoflagellates. Our massive transcriptome analysis shows that almost all regions of the genome are transcribed, including 27 possible fragmented ribosomal RNA genes and 12 uncharacterized small RNAs that are similar to mitochondrial RNA genes of the malarial parasite, Plasmodium falciparum. Gene map comparisons show that gene order is only slightly conserved between S. minutum and P. falciparum. However, small RNAs and intergenic sequences share sequence similarities with P. falciparum, suggesting that the function of noncoding sequences has been preserved despite development of very different genome structures. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Rapid sequencing of the bamboo mitochondrial genome using Illumina technology and parallel episodic evolution of organelle genomes in grasses.

PubMed

Ma, Peng-Fei; Guo, Zhen-Hua; Li, De-Zhu

2012-01-01

Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change. We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses. Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast genomes in grasses is consistent with lineage effects.

Rapid Sequencing of the Bamboo Mitochondrial Genome Using Illumina Technology and Parallel Episodic Evolution of Organelle Genomes in Grasses

PubMed Central

Ma, Peng-Fei; Guo, Zhen-Hua; Li, De-Zhu

2012-01-01

Background Compared to their counterparts in animals, the mitochondrial (mt) genomes of angiosperms exhibit a number of unique features. However, unravelling their evolution is hindered by the few completed genomes, of which are essentially Sanger sequenced. While next-generation sequencing technologies have revolutionized chloroplast genome sequencing, they are just beginning to be applied to angiosperm mt genomes. Chloroplast genomes of grasses (Poaceae) have undergone episodic evolution and the evolutionary rate was suggested to be correlated between chloroplast and mt genomes in Poaceae. It is interesting to investigate whether correlated rate change also occurred in grass mt genomes as expected under lineage effects. A time-calibrated phylogenetic tree is needed to examine rate change. Methodology/Principal Findings We determined a largely completed mt genome from a bamboo, Ferrocalamus rimosivaginus (Poaceae), through Illumina sequencing of total DNA. With combination of de novo and reference-guided assembly, 39.5-fold coverage Illumina reads were finally assembled into scaffolds totalling 432,839 bp. The assembled genome contains nearly the same genes as the completed mt genomes in Poaceae. For examining evolutionary rate in grass mt genomes, we reconstructed a phylogenetic tree including 22 taxa based on 31 mt genes. The topology of the well-resolved tree was almost identical to that inferred from chloroplast genome with only minor difference. The inconsistency possibly derived from long branch attraction in mtDNA tree. By calculating absolute substitution rates, we found significant rate change (∼4-fold) in mt genome before and after the diversification of Poaceae both in synonymous and nonsynonymous terms. Furthermore, the rate change was correlated with that of chloroplast genomes in grasses. Conclusions/Significance Our result demonstrates that it is a rapid and efficient approach to obtain angiosperm mt genome sequences using Illumina sequencing technology. The parallel episodic evolution of mt and chloroplast genomes in grasses is consistent with lineage effects. PMID:22272330

Complete genome sequence of a Watermelon silver mottle virus isolate from China.

PubMed

Rao, Xueqin; Wu, Zhuyan; Li, Yuan

2013-06-01

The complete genome of a Watermelon silver mottle virus (WSMoV) (genus Tospovirus, family Bunyaviridae) isolate (WSMoV-GZ) from Guangdong province, China was sequenced. The genomes of WSMoV-GZ contained 3,603, 4,909, and 8,914 nt of small (S), medium (M), and large (L) RNA segments, respectively, and had a genomic organization characteristic of members of the genus Tospovirus. The amino acid sequence of the nucleocapsid (N) protein, S RNA-encoded nonstructural (NSs) protein, M RNA-encoded nonstructural (NSm) protein, Gn/Gc glycoprotein precursor, and RNA-dependent RNA polymerase (RdRp) protein showed 94.3-97.5 % identity with those of other WSMoV isolates. Phylogenetic analysis showed that the N protein of WSMoV-GZ was clustered together with those of the WSMoV isolates. The full sequence of WSMoV-GZ provides a reference genome for comparison with other tospoviruses.

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

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing.

PubMed

Feehan, Joanna M; Scheibel, Katherine E; Bourras, Salim; Underwood, William; Keller, Beat; Somerville, Shauna C

2017-03-31

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 µg DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., > 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Protein family clustering for structural genomics.

PubMed

Yan, Yongpan; Moult, John

2005-10-28

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

Library preparation and data analysis packages for rapid genome sequencing.

PubMed

Pomraning, Kyle R; Smith, Kristina M; Bredeweg, Erin L; Connolly, Lanelle R; Phatale, Pallavi A; Freitag, Michael

2012-01-01

High-throughput sequencing (HTS) has quickly become a valuable tool for comparative genetics and genomics and is now regularly carried out in laboratories that are not connected to large sequencing centers. Here we describe an updated version of our protocol for constructing single- and paired-end Illumina sequencing libraries, beginning with purified genomic DNA. The present protocol can also be used for "multiplexing," i.e. the analysis of several samples in a single flowcell lane by generating "barcoded" or "indexed" Illumina sequencing libraries in a way that is independent from Illumina-supported methods. To analyze sequencing results, we suggest several independent approaches but end users should be aware that this is a quickly evolving field and that currently many alignment (or "mapping") and counting algorithms are being developed and tested.

RATT: Rapid Annotation Transfer Tool

PubMed Central

Otto, Thomas D.; Dillon, Gary P.; Degrave, Wim S.; Berriman, Matthew

2011-01-01

Second-generation sequencing technologies have made large-scale sequencing projects commonplace. However, making use of these datasets often requires gene function to be ascribed genome wide. Although tool development has kept pace with the changes in sequence production, for tasks such as mapping, de novo assembly or visualization, genome annotation remains a challenge. We have developed a method to rapidly provide accurate annotation for new genomes using previously annotated genomes as a reference. The method, implemented in a tool called RATT (Rapid Annotation Transfer Tool), transfers annotations from a high-quality reference to a new genome on the basis of conserved synteny. We demonstrate that a Mycobacterium tuberculosis genome or a single 2.5 Mb chromosome from a malaria parasite can be annotated in less than five minutes with only modest computational resources. RATT is available at http://ratt.sourceforge.net. PMID:21306991

Downsizing genomic medicine: approaching the ethical complexity of whole-genome sequencing by starting small.

PubMed

Sharp, Richard R

2011-03-01

As we look to a time when whole-genome sequencing is integrated into patient care, it is possible to anticipate a number of ethical challenges that will need to be addressed. The most intractable of these concern informed consent and the responsible management of very large amounts of genetic information. Given the range of possible findings, it remains unclear to what extent it will be possible to obtain meaningful patient consent to genomic testing. Equally unclear is how clinicians will disseminate the enormous volume of genetic information produced by whole-genome sequencing. Toward developing practical strategies for managing these ethical challenges, we propose a research agenda that approaches multiplexed forms of clinical genetic testing as natural laboratories in which to develop best practices for managing the ethical complexities of genomic medicine.

Comparative Genomics of Interreplichore Translocations in Bacteria: A Measure of Chromosome Topology?

PubMed

Khedkar, Supriya; Seshasayee, Aswin Sai Narain

2016-06-01

Genomes evolve not only in base sequence but also in terms of their architecture, defined by gene organization and chromosome topology. Whereas genome sequence data inform us about the changes in base sequences for a large variety of organisms, the study of chromosome topology is restricted to a few model organisms studied using microscopy and chromosome conformation capture techniques. Here, we exploit whole genome sequence data to study the link between gene organization and chromosome topology in bacteria. Using comparative genomics across ∼250 pairs of closely related bacteria we show that: (a) many organisms show a high degree of interreplichore translocations throughout the chromosome and not limited to the inversion-prone terminus (ter) or the origin of replication (oriC); (b) translocation maps may reflect chromosome topologies; and (c) symmetric interreplichore translocations do not disrupt the distance of a gene from oriC or affect gene expression states or strand biases in gene densities. In summary, we suggest that translocation maps might be a first line in defining a gross chromosome topology given a pair of closely related genome sequences. Copyright © 2016 Khedkar and Seshasayee.

Comparative Genomics of Interreplichore Translocations in Bacteria: A Measure of Chromosome Topology?

PubMed Central

Khedkar, Supriya; Seshasayee, Aswin Sai Narain

2016-01-01

Genomes evolve not only in base sequence but also in terms of their architecture, defined by gene organization and chromosome topology. Whereas genome sequence data inform us about the changes in base sequences for a large variety of organisms, the study of chromosome topology is restricted to a few model organisms studied using microscopy and chromosome conformation capture techniques. Here, we exploit whole genome sequence data to study the link between gene organization and chromosome topology in bacteria. Using comparative genomics across ∼250 pairs of closely related bacteria we show that: (a) many organisms show a high degree of interreplichore translocations throughout the chromosome and not limited to the inversion-prone terminus (ter) or the origin of replication (oriC); (b) translocation maps may reflect chromosome topologies; and (c) symmetric interreplichore translocations do not disrupt the distance of a gene from oriC or affect gene expression states or strand biases in gene densities. In summary, we suggest that translocation maps might be a first line in defining a gross chromosome topology given a pair of closely related genome sequences. PMID:27172194

Complete genome sequence of keunjorong mosaic virus, a potyvirus from Cynanchum wilfordii.

PubMed

Nam, Moon; Lee, Joo-Hee; Choi, Hong Soo; Lim, Hyoun-Sub; Moon, Jae Sun; Lee, Su-Heon

2013-08-01

We have determined the complete genome sequence of keunjorong mosaic virus (KjMV). The KjMV genome is composed of 9,611 nucleotides, excluding the 3'-terminal poly(A) tail. It contains two open reading frames (ORFs), with the large one encoding a polyprotein of 3,070 amino acids and the small overlapping ORF encoding a PIPO protein of 81 amino acids. The KjMV genome shared the highest nucleotide sequence identity (57.5  %) with pepper mottle virus and freesia mosaic virus, two members of the genus Potyvirus. Based on the phylogenetic relatedness to known potyviruses, KjMV appears to be a member of a new species in the genus Potyvirus.

Genome-wide DNA polymorphisms in two cultivars of mei (Prunus mume sieb. et zucc.).

PubMed

Sun, Lidan; Zhang, Qixiang; Xu, Zongda; Yang, Weiru; Guo, Yu; Lu, Jiuxing; Pan, Huitang; Cheng, Tangren; Cai, Ming

2013-10-06

Mei (Prunus mume Sieb. et Zucc.) is a famous ornamental plant and fruit crop grown in East Asian countries. Limited genetic resources, especially molecular markers, have hindered the progress of mei breeding projects. Here, we performed low-depth whole-genome sequencing of Prunus mume 'Fenban' and Prunus mume 'Kouzi Yudie' to identify high-quality polymorphic markers between the two cultivars on a large scale. A total of 1464.1 Mb and 1422.1 Mb of 'Fenban' and 'Kouzi Yudie' sequencing data were uniquely mapped to the mei reference genome with about 6-fold coverage, respectively. We detected a large number of putative polymorphic markers from the 196.9 Mb of sequencing data shared by the two cultivars, which together contained 200,627 SNPs, 4,900 InDels, and 7,063 SSRs. Among these markers, 38,773 SNPs, 174 InDels, and 418 SSRs were distributed in the 22.4 Mb CDS region, and 63.0% of these marker-containing CDS sequences were assigned to GO terms. Subsequently, 670 selected SNPs were validated using an Agilent's SureSelect solution phase hybridization assay. A subset of 599 SNPs was used to assess the genetic similarity of a panel of mei germplasm samples and a plum (P. salicina) cultivar, producing a set of informative diversity data. We also analyzed the frequency and distribution of detected InDels and SSRs in mei genome and validated their usefulness as DNA markers. These markers were successfully amplified in the cultivars and in their segregating progeny. A large set of high-quality polymorphic SNPs, InDels, and SSRs were identified in parallel between 'Fenban' and 'Kouzi Yudie' using low-depth whole-genome sequencing. The study presents extensive data on these polymorphic markers, which can be useful for constructing high-resolution genetic maps, performing genome-wide association studies, and designing genomic selection strategies in mei.

The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species.

PubMed

Gilchrist, Anthony Stuart; Shearman, Deborah C A; Frommer, Marianne; Raphael, Kathryn A; Deshpande, Nandan P; Wilkins, Marc R; Sherwin, William B; Sved, John A

2014-12-20

The tephritid fruit flies include a number of economically important pests of horticulture, with a large accumulated body of research on their biology and control. Amongst the Tephritidae, the genus Bactrocera, containing over 400 species, presents various species groups of potential utility for genetic studies of speciation, behaviour or pest control. In Australia, there exists a triad of closely-related, sympatric Bactrocera species which do not mate in the wild but which, despite distinct morphologies and behaviours, can be force-mated in the laboratory to produce fertile hybrid offspring. To exploit the opportunities offered by genomics, such as the efficient identification of genetic loci central to pest behaviour and to the earliest stages of speciation, investigators require genomic resources for future investigations. We produced a draft de novo genome assembly of Australia's major tephritid pest species, Bactrocera tryoni. The male genome (650-700 Mbp) includes approximately 150 Mb of interspersed repetitive DNA sequences and 60 Mb of satellite DNA. Assessment using conserved core eukaryotic sequences indicated 98% completeness. Over 16,000 MAKER-derived gene models showed a large degree of overlap with other Dipteran reference genomes. The sequence of the ribosomal RNA transcribed unit was also determined. Unscaffolded assemblies of B. neohumeralis and B. jarvisi were then produced; comparison with B. tryoni showed that the species are more closely related than any Drosophila species pair. The similarity of the genomes was exploited to identify 4924 potentially diagnostic indels between the species, all of which occur in non-coding regions. This first draft B. tryoni genome resembles other dipteran genomes in terms of size and putative coding sequences. For all three species included in this study, we have identified a comprehensive set of non-redundant repetitive sequences, including the ribosomal RNA unit, and have quantified the major satellite DNA families. These genetic resources will facilitate the further investigations of genetic mechanisms responsible for the behavioural and morphological differences between these three species and other tephritids. We have also shown how whole genome sequence data can be used to generate simple diagnostic tests between very closely-related species where only one of the species is scaffolded.

The genome sequence of the model ascomycete fungus Podospora anserina

PubMed Central

Espagne, Eric; Lespinet, Olivier; Malagnac, Fabienne; Da Silva, Corinne; Jaillon, Olivier; Porcel, Betina M; Couloux, Arnaud; Aury, Jean-Marc; Ségurens, Béatrice; Poulain, Julie; Anthouard, Véronique; Grossetete, Sandrine; Khalili, Hamid; Coppin, Evelyne; Déquard-Chablat, Michelle; Picard, Marguerite; Contamine, Véronique; Arnaise, Sylvie; Bourdais, Anne; Berteaux-Lecellier, Véronique; Gautheret, Daniel; de Vries, Ronald P; Battaglia, Evy; Coutinho, Pedro M; Danchin, Etienne GJ; Henrissat, Bernard; Khoury, Riyad EL; Sainsard-Chanet, Annie; Boivin, Antoine; Pinan-Lucarré, Bérangère; Sellem, Carole H; Debuchy, Robert; Wincker, Patrick; Weissenbach, Jean; Silar, Philippe

2008-01-01

Background The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development. Results We present a 10X draft sequence of P. anserina genome, linked to the sequences of a large expressed sequence tag collection. Similar to higher eukaryotes, the P. anserina transcription/splicing machinery generates numerous non-conventional transcripts. Comparison of the P. anserina genome and orthologous gene set with the one of its close relatives, Neurospora crassa, shows that synteny is poorly conserved, the main result of evolution being gene shuffling in the same chromosome. The P. anserina genome contains fewer repeated sequences and has evolved new genes by duplication since its separation from N. crassa, despite the presence of the repeat induced point mutation mechanism that mutates duplicated sequences. We also provide evidence that frequent gene loss took place in the lineages leading to P. anserina and N. crassa. P. anserina contains a large and highly specialized set of genes involved in utilization of natural carbon sources commonly found in its natural biotope. It includes genes potentially involved in lignin degradation and efficient cellulose breakdown. Conclusion The features of the P. anserina genome indicate a highly dynamic evolution since the divergence of P. anserina and N. crassa, leading to the ability of the former to use specific complex carbon sources that match its needs in its natural biotope. PMID:18460219

Remnants of an Ancient Deltaretrovirus in the Genomes of Horseshoe Bats (Rhinolophidae).

PubMed

Hron, Tomáš; Farkašová, Helena; Gifford, Robert J; Benda, Petr; Hulva, Pavel; Görföl, Tamás; Pačes, Jan; Elleder, Daniel

2018-04-10

Endogenous retrovirus (ERV) sequences provide a rich source of information about the long-term interactions between retroviruses and their hosts. However, most ERVs are derived from a subset of retrovirus groups, while ERVs derived from certain other groups remain extremely rare. In particular, only a single ERV sequence has been identified that shows evidence of being related to an ancient Deltaretrovirus , despite the large number of vertebrate genome sequences now available. In this report, we identify a second example of an ERV sequence putatively derived from a past deltaretroviral infection, in the genomes of several species of horseshoe bats (Rhinolophidae). This sequence represents a fragment of viral genome derived from a single integration. The time of the integration was estimated to be 11-19 million years ago. This finding, together with the previously identified endogenous Deltaretrovirus in long-fingered bats (Miniopteridae), suggest a close association of bats with ancient deltaretroviruses.

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

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

In vitro propagation of the microsporidian pathogen Brachiola algerae and studies of its chromosome and ribosomal DNA organization in the context of the complete genome sequencing project.

PubMed

Belkorchia, Abdel; Biderre, Corinne; Militon, Cécile; Polonais, Valérie; Wincker, Patrick; Jubin, Claire; Delbac, Frédéric; Peyretaillade, Eric; Peyret, Pierre

2008-03-01

Brachiola algerae has a broad host spectrum from human to mosquitoes. The successful infection of two mosquito cell lines (Mos55: embryonic cells and Sua 4.0: hemocyte-like cells) and a human cell line (HFF) highlights the efficient adaptive capacity of this microsporidian pathogen. The molecular karyotype of this microsporidian species was determined in the context of the B. algerae genome sequencing project, showing that its haploid genome consists of 30 chromosomal-sized DNAs ranging from 160 to 2240 kbp giving an estimated genome size of 23 Mbp. A contig of 12,269 bp including the DNA sequence of the B. algerae ribosomal transcription unit has been built from initial genomic sequences and the secondary structure of the large subunit rRNA constructed. The data obtained indicate that B. algerae should be an excellent parasitic model to understand genome evolution in relation to infectious capacity.

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

Characterization of full-length sequenced cDNA inserts (FLIcs) from Atlantic salmon (Salmo salar)

PubMed Central

Andreassen, Rune; Lunner, Sigbjørn; Høyheim, Bjørn

2009-01-01

Background Sequencing of the Atlantic salmon genome is now being planned by an international research consortium. Full-length sequenced inserts from cDNAs (FLIcs) are an important tool for correct annotation and clustering of the genomic sequence in any species. The large amount of highly similar duplicate sequences caused by the relatively recent genome duplication in the salmonid ancestor represents a particular challenge for the genome project. FLIcs will therefore be an extremely useful resource for the Atlantic salmon sequencing project. In addition to be helpful in order to distinguish between duplicate genome regions and in determining correct gene structures, FLIcs are an important resource for functional genomic studies and for investigation of regulatory elements controlling gene expression. In contrast to the large number of ESTs available, including the ESTs from 23 developmental and tissue specific cDNA libraries contributed by the Salmon Genome Project (SGP), the number of sequences where the full-length of the cDNA insert has been determined has been small. Results High quality full-length insert sequences from 560 pre-smolt white muscle tissue specific cDNAs were generated, accession numbers [GenBank: BT043497 - BT044056]. Five hundred and ten (91%) of the transcripts were annotated using Gene Ontology (GO) terms and 440 of the FLIcs are likely to contain a complete coding sequence (cCDS). The sequence information was used to identify putative paralogs, characterize salmon Kozak motifs, polyadenylation signal variation and to identify motifs likely to be involved in the regulation of particular genes. Finally, conserved 7-mers in the 3'UTRs were identified, of which some were identical to miRNA target sequences. Conclusion This paper describes the first Atlantic salmon FLIcs from a tissue and developmental stage specific cDNA library. We have demonstrated that many FLIcs contained a complete coding sequence (cCDS). This suggests that the remaining cDNA libraries generated by SGP represent a valuable cCDS FLIc source. The conservation of 7-mers in 3'UTRs indicates that these motifs are functionally important. Identity between some of these 7-mers and miRNA target sequences suggests that they are miRNA targets in Salmo salar transcripts as well. PMID:19878547

PepLine: a software pipeline for high-throughput direct mapping of tandem mass spectrometry data on genomic sequences.

PubMed

Ferro, Myriam; Tardif, Marianne; Reguer, Erwan; Cahuzac, Romain; Bruley, Christophe; Vermat, Thierry; Nugues, Estelle; Vigouroux, Marielle; Vandenbrouck, Yves; Garin, Jérôme; Viari, Alain

2008-05-01

PepLine is a fully automated software which maps MS/MS fragmentation spectra of trypsic peptides to genomic DNA sequences. The approach is based on Peptide Sequence Tags (PSTs) obtained from partial interpretation of QTOF MS/MS spectra (first module). PSTs are then mapped on the six-frame translations of genomic sequences (second module) giving hits. Hits are then clustered to detect potential coding regions (third module). Our work aimed at optimizing the algorithms of each component to allow the whole pipeline to proceed in a fully automated manner using raw nucleic acid sequences (i.e., genomes that have not been "reduced" to a database of ORFs or putative exons sequences). The whole pipeline was tested on controlled MS/MS spectra sets from standard proteins and from Arabidopsis thaliana envelope chloroplast samples. Our results demonstrate that PepLine competed with protein database searching softwares and was fast enough to potentially tackle large data sets and/or high size genomes. We also illustrate the potential of this approach for the detection of the intron/exon structure of genes.

Distilled single-cell genome sequencing and de novo assembly for sparse microbial communities.

PubMed

Taghavi, Zeinab; Movahedi, Narjes S; Draghici, Sorin; Chitsaz, Hamidreza

2013-10-01

Identification of every single genome present in a microbial sample is an important and challenging task with crucial applications. It is challenging because there are typically millions of cells in a microbial sample, the vast majority of which elude cultivation. The most accurate method to date is exhaustive single-cell sequencing using multiple displacement amplification, which is simply intractable for a large number of cells. However, there is hope for breaking this barrier, as the number of different cell types with distinct genome sequences is usually much smaller than the number of cells. Here, we present a novel divide and conquer method to sequence and de novo assemble all distinct genomes present in a microbial sample with a sequencing cost and computational complexity proportional to the number of genome types, rather than the number of cells. The method is implemented in a tool called Squeezambler. We evaluated Squeezambler on simulated data. The proposed divide and conquer method successfully reduces the cost of sequencing in comparison with the naïve exhaustive approach. Squeezambler and datasets are available at http://compbio.cs.wayne.edu/software/squeezambler/.

A Mitogenomic Phylogeny of Living Primates

PubMed Central

Finstermeier, Knut; Zinner, Dietmar; Brameier, Markus; Meyer, Matthias; Kreuz, Eva; Hofreiter, Michael; Roos, Christian

2013-01-01

Primates, the mammalian order including our own species, comprise 480 species in 78 genera. Thus, they represent the third largest of the 18 orders of eutherian mammals. Although recent phylogenetic studies on primates are increasingly built on molecular datasets, most of these studies have focused on taxonomic subgroups within the order. Complete mitochondrial (mt) genomes have proven to be extremely useful in deciphering within-order relationships even up to deep nodes. Using 454 sequencing, we sequenced 32 new complete mt genomes adding 20 previously not represented genera to the phylogenetic reconstruction of the primate tree. With 13 new sequences, the number of complete mt genomes within the parvorder Platyrrhini was widely extended, resulting in a largely resolved branching pattern among New World monkey families. We added 10 new Strepsirrhini mt genomes to the 15 previously available ones, thus almost doubling the number of mt genomes within this clade. Our data allow precise date estimates of all nodes and offer new insights into primate evolution. One major result is a relatively young date for the most recent common ancestor of all living primates which was estimated to 66-69 million years ago, suggesting that the divergence of extant primates started close to the K/T-boundary. Although some relationships remain unclear, the large number of mt genomes used allowed us to reconstruct a robust primate phylogeny which is largely in agreement with previous publications. Finally, we show that mt genomes are a useful tool for resolving primate phylogenetic relationships on various taxonomic levels. PMID:23874967

Whole-Genome Sequences of Two Borrelia afzelii and Two Borrelia garinii Lyme Disease Agent Isolates

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

Casjens, S.R.; Dunn, J.; Mongodin, E. F.

2011-12-01

Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B. burgdorferi, and B. bavariensis sp. nov. Whole-genome sequencing is an excellent tool for investigating and understanding the influence of bacterial diversity on the pathogenesis and etiology of Lyme disease. We report here the whole-genome sequences of four isolates from two of the Borrelia species that cause human Lyme disease, B. afzelii isolates ACA-1 and PKo and B. garinii isolates PBr and Far04.

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 complete chloroplast genome sequences of Lychnis wilfordii and Silene capitata and comparative analyses with other Caryophyllaceae genomes.

PubMed

Kang, Jong-Soo; Lee, Byoung Yoon; Kwak, Myounghai

2017-01-01

The complete chloroplast genomes of Lychnis wilfordii and Silene capitata were determined and compared with ten previously reported Caryophyllaceae chloroplast genomes. The chloroplast genome sequences of L. wilfordii and S. capitata contain 152,320 bp and 150,224 bp, respectively. The gene contents and orders among 12 Caryophyllaceae species are consistent, but several microstructural changes have occurred. Expansion of the inverted repeat (IR) regions at the large single copy (LSC)/IRb and small single copy (SSC)/IR boundaries led to partial or entire gene duplications. Additionally, rearrangements of the LSC region were caused by gene inversions and/or transpositions. The 18 kb inversions, which occurred three times in different lineages of tribe Sileneae, were thought to be facilitated by the intermolecular duplicated sequences. Sequence analyses of the L. wilfordii and S. capitata genomes revealed 39 and 43 repeats, respectively, including forward, palindromic, and reverse repeats. In addition, a total of 67 and 56 simple sequence repeats were discovered in the L. wilfordii and S. capitata chloroplast genomes, respectively. Finally, we constructed phylogenetic trees of the 12 Caryophyllaceae species and two Amaranthaceae species based on 73 protein-coding genes using both maximum parsimony and likelihood methods.

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells.

PubMed

Yajima, Misako; Ikuta, Kazufumi; Kanda, Teru

2018-04-03

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically.

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells

PubMed Central

Ikuta, Kazufumi; Kanda, Teru

2018-01-01

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically. PMID:29614006

Assembly of the Lactuca sativa, L. cv. Tizian draft genome sequence reveals differences within major resistance complex 1 as compared to the cv. Salinas reference genome.

PubMed

Verwaaijen, Bart; Wibberg, Daniel; Nelkner, Johanna; Gordin, Miriam; Rupp, Oliver; Winkler, Anika; Bremges, Andreas; Blom, Jochen; Grosch, Rita; Pühler, Alfred; Schlüter, Andreas

2018-02-10

Lettuce (Lactuca sativa, L.) is an important annual plant of the family Asteraceae (Compositae). The commercial lettuce cultivar Tizian has been used in various scientific studies investigating the interaction of the plant with phytopathogens or biological control agents. Here, we present the de novo draft genome sequencing and gene prediction for this specific cultivar derived from transcriptome sequence data. The assembled scaffolds amount to a size of 2.22 Gb. Based on RNAseq data, 31,112 transcript isoforms were identified. Functional predictions for these transcripts were determined within the GenDBE annotation platform. Comparison with the cv. Salinas reference genome revealed a high degree of sequence similarity on genome and transcriptome levels, with an average amino acid identity of 99%. Furthermore, it was observed that two large regions are either missing or are highly divergent within the cv. Tizian genome compared to cv. Salinas. One of these regions covers the major resistance complex 1 region of cv. Salinas. The cv. Tizian draft genome sequence provides a valuable resource for future functional and transcriptome analyses focused on this lettuce cultivar. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Lightweight genome viewer: portable software for browsing genomics data in its chromosomal context

PubMed Central

Faith, Jeremiah J; Olson, Andrew J; Gardner, Timothy S; Sachidanandam, Ravi

2007-01-01

Background Lightweight genome viewer (lwgv) is a web-based tool for visualization of sequence annotations in their chromosomal context. It performs most of the functions of larger genome browsers, while relying on standard flat-file formats and bypassing the database needs of most visualization tools. Visualization as an aide to discovery requires display of novel data in conjunction with static annotations in their chromosomal context. With database-based systems, displaying dynamic results requires temporary tables that need to be tracked for removal. Results lwgv simplifies the visualization of user-generated results on a local computer. The dynamic results of these analyses are written to transient files, which can import static content from a more permanent file. lwgv is currently used in many different applications, from whole genome browsers to single-gene RNAi design visualization, demonstrating its applicability in a large variety of contexts and scales. Conclusion lwgv provides a lightweight alternative to large genome browsers for visualizing biological annotations and dynamic analyses in their chromosomal context. It is particularly suited for applications ranging from short sequences to medium-sized genomes when the creation and maintenance of a large software and database infrastructure is not necessary or desired. PMID:17877794

Lightweight genome viewer: portable software for browsing genomics data in its chromosomal context.

PubMed

Faith, Jeremiah J; Olson, Andrew J; Gardner, Timothy S; Sachidanandam, Ravi

2007-09-18

Lightweight genome viewer (lwgv) is a web-based tool for visualization of sequence annotations in their chromosomal context. It performs most of the functions of larger genome browsers, while relying on standard flat-file formats and bypassing the database needs of most visualization tools. Visualization as an aide to discovery requires display of novel data in conjunction with static annotations in their chromosomal context. With database-based systems, displaying dynamic results requires temporary tables that need to be tracked for removal. lwgv simplifies the visualization of user-generated results on a local computer. The dynamic results of these analyses are written to transient files, which can import static content from a more permanent file. lwgv is currently used in many different applications, from whole genome browsers to single-gene RNAi design visualization, demonstrating its applicability in a large variety of contexts and scales. lwgv provides a lightweight alternative to large genome browsers for visualizing biological annotations and dynamic analyses in their chromosomal context. It is particularly suited for applications ranging from short sequences to medium-sized genomes when the creation and maintenance of a large software and database infrastructure is not necessary or desired.

The use of PacBio and Hi-C data in denovo assembly of the goat genome

USDA-ARS?s Scientific Manuscript database

Generating de novo reference genome assemblies for non-model organisms is a laborious task that often requires a large amount of data from several sequencing platforms and cytogenetic surveys. By using PacBio sequence data and new library creation techniques, we present a de novo, high quality refer...

dBBQs: dataBase of Bacterial Quality scores.

PubMed

Wanchai, Visanu; Patumcharoenpol, Preecha; Nookaew, Intawat; Ussery, David

2017-12-28

It is well-known that genome sequencing technologies are becoming significantly cheaper and faster. As a result of this, the exponential growth in sequencing data in public databases allows us to explore ever growing large collections of genome sequences. However, it is less known that the majority of available sequenced genome sequences in public databases are not complete, drafts of varying qualities. We have calculated quality scores for around 100,000 bacterial genomes from all major genome repositories and put them in a fast and easy-to-use database. Prokaryotic genomic data from all sources were collected and combined to make a non-redundant set of bacterial genomes. The genome quality score for each was calculated by four different measurements: assembly quality, number of rRNA and tRNA genes, and the occurrence of conserved functional domains. The dataBase of Bacterial Quality scores (dBBQs) was designed to store and retrieve quality scores. It offers fast searching and download features which the result can be used for further analysis. In addition, the search results are shown in interactive JavaScript chart framework using DC.js. The analysis of quality scores across major public genome databases find that around 68% of the genomes are of acceptable quality for many uses. dBBQs (available at http://arc-gem.uams.edu/dbbqs ) provides genome quality scores for all available prokaryotic genome sequences with a user-friendly Web-interface. These scores can be used as cut-offs to get a high-quality set of genomes for testing bioinformatics tools or improving the analysis. Moreover, all data of the four measurements that were combined to make the quality score for each genome, which can potentially be used for further analysis. dBBQs will be updated regularly and is freely use for non-commercial purpose.

Draft genome sequence of Lactobacillus mali KCTC 3596.

PubMed

Kim, Dong-Wook; Choi, Sang-Haeng; Kang, Aram; Nam, Seong-Hyeuk; Kim, Dae-Soo; Kim, Ryong Nam; Kim, Aeri; Park, Hong-Seog

2011-09-01

We announce the draft genome sequence of the type strain Lactobacillus mali KCTC 3596 (2,652,969 bp, with a G+C content of 36.0%), which is one of the most prevalent lactic acid bacteria present during the manufacturing process of apple juice. The genome consists of 122 large contigs (>100 bp). All of the contigs were assembled by Newbler Assembler 2.3 (454 Life Science). Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Inaugural Genomics Automation Congress and the coming deluge of sequencing data.

PubMed

Creighton, Chad J

2010-10-01

Presentations at Select Biosciences's first 'Genomics Automation Congress' (Boston, MA, USA) in 2010 focused on next-generation sequencing and the platforms and methodology around them. The meeting provided an overview of sequencing technologies, both new and emerging. Speakers shared their recent work on applying sequencing to profile cells for various levels of biomolecular complexity, including DNA sequences, DNA copy, DNA methylation, mRNA and microRNA. With sequencing time and costs continuing to drop dramatically, a virtual explosion of very large sequencing datasets is at hand, which will probably present challenges and opportunities for high-level data analysis and interpretation, as well as for information technology infrastructure.

BAC end sequencing of Pacific white shrimp Litopenaeus vannamei: a glimpse into the genome of Penaeid shrimp

NASA Astrophysics Data System (ADS)

Zhao, Cui; Zhang, Xiaojun; Liu, Chengzhang; Huan, Pin; Li, Fuhua; Xiang, Jianhai; Huang, Chao

2012-05-01

Little is known about the genome of Pacific white shrimp ( Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 pairedends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for this species.

Bringing the fathead minnow into the genomic era | Science ...

EPA Pesticide Factsheets

The fathead minnow is a well-established ecotoxicological model organism that has been widely used for regulatory ecotoxicity testing and research for over a half century. While a large amount of molecular information has been gathered on the fathead minnow over the years, the lack of genomic sequence data has limited the utility of the fathead minnow for certain applications. To address this limitation, high-throughput Illumina sequencing technology was employed to sequence the fathead minnow genome. Approximately 100X coverage was achieved by sequencing several libraries of paired-end reads with differing genome insert sizes. Two draft genome assemblies were generated using the SOAPdenovo and String Graph Assembler (SGA) methods, respectively. When these were compared, the SOAPdenovo assembly had a higher scaffold N50 value of 60.4 kbp versus 15.4 kbp, and it also performed better in a Core Eukaryotic Genes Mapping Analysis (CEGMA), mapping 91% versus 67% of genes. As such, this assembly was selected for further development and annotation. The foundation for genome annotation was generated using AUGUSTUS, an ab initio method for gene prediction. A total of 43,345 potential coding sequences were predicted on the genome assembly. These predicted sequences were translated to peptides and queried in a BLAST search against all vertebrates, with 28,290 of these sequences corresponding to zebrafish peptides and 5,242 producing no significant alignments. Additional ty

Defining the diverse spectrum of inversions, complex structural variation, and chromothripsis in the morbid human genome.

PubMed

Collins, Ryan L; Brand, Harrison; Redin, Claire E; Hanscom, Carrie; Antolik, Caroline; Stone, Matthew R; Glessner, Joseph T; Mason, Tamara; Pregno, Giulia; Dorrani, Naghmeh; Mandrile, Giorgia; Giachino, Daniela; Perrin, Danielle; Walsh, Cole; Cipicchio, Michelle; Costello, Maura; Stortchevoi, Alexei; An, Joon-Yong; Currall, Benjamin B; Seabra, Catarina M; Ragavendran, Ashok; Margolin, Lauren; Martinez-Agosto, Julian A; Lucente, Diane; Levy, Brynn; Sanders, Stephan J; Wapner, Ronald J; Quintero-Rivera, Fabiola; Kloosterman, Wigard; Talkowski, Michael E

2017-03-06

Structural variation (SV) influences genome organization and contributes to human disease. However, the complete mutational spectrum of SV has not been routinely captured in disease association studies. We sequenced 689 participants with autism spectrum disorder (ASD) and other developmental abnormalities to construct a genome-wide map of large SV. Using long-insert jumping libraries at 105X mean physical coverage and linked-read whole-genome sequencing from 10X Genomics, we document seven major SV classes at ~5 kb SV resolution. Our results encompass 11,735 distinct large SV sites, 38.1% of which are novel and 16.8% of which are balanced or complex. We characterize 16 recurrent subclasses of complex SV (cxSV), revealing that: (1) cxSV are larger and rarer than canonical SV; (2) each genome harbors 14 large cxSV on average; (3) 84.4% of large cxSVs involve inversion; and (4) most large cxSV (93.8%) have not been delineated in previous studies. Rare SVs are more likely to disrupt coding and regulatory non-coding loci, particularly when truncating constrained and disease-associated genes. We also identify multiple cases of catastrophic chromosomal rearrangements known as chromoanagenesis, including somatic chromoanasynthesis, and extreme balanced germline chromothripsis events involving up to 65 breakpoints and 60.6 Mb across four chromosomes, further defining rare categories of extreme cxSV. These data provide a foundational map of large SV in the morbid human genome and demonstrate a previously underappreciated abundance and diversity of cxSV that should be considered in genomic studies of human disease.

Resolving the tips of the tree of life: How much mitochondrialdata doe we need?

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

Bonett, Ronald M.; Macey, J. Robert; Boore, Jeffrey L.

2005-04-29

Mitochondrial (mt) DNA sequences are used extensively to reconstruct evolutionary relationships among recently diverged animals,and have constituted the most widely used markers for species- and generic-level relationships for the last decade or more. However, most studies to date have employed relatively small portions of the mt-genome. In contrast, complete mt-genomes primarily have been used to investigate deep divergences, including several studies of the amount of mt sequence necessary to recover ancient relationships. We sequenced and analyzed 24 complete mt-genomes from a group of salamander species exhibiting divergences typical of those in many species-level studies. We present the first comprehensive investigationmore » of the amount of mt sequence data necessary to consistently recover the mt-genome tree at this level, using parsimony and Bayesian methods. Both methods of phylogenetic analysis revealed extremely similar results. A surprising number of well supported, yet conflicting, relationships were found in trees based on fragments less than {approx}2000 nucleotides (nt), typical of the vast majority of the thousands of mt-based studies published to date. Large amounts of data (11,500+ nt) were necessary to consistently recover the whole mt-genome tree. Some relationships consistently were recovered with fragments of all sizes, but many nodes required the majority of the mt-genome to stabilize, particularly those associated with short internal branches. Although moderate amounts of data (2000-3000 nt) were adequate to recover mt-based relationships for which most nodes were congruent with the whole mt-genome tree, many thousands of nucleotides were necessary to resolve rapid bursts of evolution. Recent advances in genomics are making collection of large amounts of sequence data highly feasible, and our results provide the basis for comparative studies of other closely related groups to optimize mt sequence sampling and phylogenetic resolution at the ''tips'' of the Tree of Life.« less

A Deep-Coverage Tomato BAC Library and Prospects Toward Development of an STC Framework for Genome Sequencing

PubMed Central

Budiman, Muhammad A.; Mao, Long; Wood, Todd C.; Wing, Rod A.

2000-01-01

Recently a new strategy using BAC end sequences as sequence-tagged connectors (STCs) was proposed for whole-genome sequencing projects. In this study, we present the construction and detailed characterization of a 15.0 haploid genome equivalent BAC library for the cultivated tomato, Lycopersicon esculentum cv. Heinz 1706. The library contains 129,024 clones with an average insert size of 117.5 kb and a chloroplast content of 1.11%. BAC end sequences from 1490 ends were generated and analyzed as a preliminary evaluation for using this library to develop an STC framework to sequence the tomato genome. A total of 1205 BAC end sequences (80.9%) were obtained, with an average length of 360 high-quality bases, and were searched against the GenBank database. Using a cutoff expectation value of <10−6, and combining the results from BLASTN, BLASTX, and TBLASTX searches, 24.3% of the BAC end sequences were similar to known sequences, of which almost half (48.7%) share sequence similarities to retrotransposons and 7% to known genes. Some of the transposable element sequences were the first reported in tomato, such as sequences similar to maize transposon Activator (Ac) ORF and tobacco pararetrovirus-like sequences. Interestingly, there were no BAC end sequences similar to the highly repeated TGRI and TGRII elements. However, the majority (70.3%) of STCs did not share significant sequence similarities to any sequences in GenBank at either the DNA or predicted protein levels, indicating that a large portion of the tomato genome is still unknown. Our data demonstrate that this BAC library is suitable for developing an STC database to sequence the tomato genome. The advantages of developing an STC framework for whole-genome sequencing of tomato are discussed. [The BAC end sequences described in this paper have been deposited in the GenBank data library under accession nos. AQ367111–AQ368361.] PMID:10645957

Whole-Genome Sequencing and Assembly with High-Throughput, Short-Read Technologies

PubMed Central

Sundquist, Andreas; Ronaghi, Mostafa; Tang, Haixu; Pevzner, Pavel; Batzoglou, Serafim

2007-01-01

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology. PMID:17534434

An integrated semiconductor device enabling non-optical genome sequencing.

PubMed

Rothberg, Jonathan M; Hinz, Wolfgang; Rearick, Todd M; Schultz, Jonathan; Mileski, William; Davey, Mel; Leamon, John H; Johnson, Kim; Milgrew, Mark J; Edwards, Matthew; Hoon, Jeremy; Simons, Jan F; Marran, David; Myers, Jason W; Davidson, John F; Branting, Annika; Nobile, John R; Puc, Bernard P; Light, David; Clark, Travis A; Huber, Martin; Branciforte, Jeffrey T; Stoner, Isaac B; Cawley, Simon E; Lyons, Michael; Fu, Yutao; Homer, Nils; Sedova, Marina; Miao, Xin; Reed, Brian; Sabina, Jeffrey; Feierstein, Erika; Schorn, Michelle; Alanjary, Mohammad; Dimalanta, Eileen; Dressman, Devin; Kasinskas, Rachel; Sokolsky, Tanya; Fidanza, Jacqueline A; Namsaraev, Eugeni; McKernan, Kevin J; Williams, Alan; Roth, G Thomas; Bustillo, James

2011-07-20

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

JGI Plant Genomics Gene Annotation Pipeline

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

Shu, Shengqiang; Rokhsar, Dan; Goodstein, David

2014-07-14

Plant genomes vary in size and are highly complex with a high amount of repeats, genome duplication and tandem duplication. Gene encodes a wealth of information useful in studying organism and it is critical to have high quality and stable gene annotation. Thanks to advancement of sequencing technology, many plant species genomes have been sequenced and transcriptomes are also sequenced. To use these vastly large amounts of sequence data to make gene annotation or re-annotation in a timely fashion, an automatic pipeline is needed. JGI plant genomics gene annotation pipeline, called integrated gene call (IGC), is our effort toward thismore » aim with aid of a RNA-seq transcriptome assembly pipeline. It utilizes several gene predictors based on homolog peptides and transcript ORFs. See Methods for detail. Here we present genome annotation of JGI flagship green plants produced by this pipeline plus Arabidopsis and rice except for chlamy which is done by a third party. The genome annotations of these species and others are used in our gene family build pipeline and accessible via JGI Phytozome portal whose URL and front page snapshot are shown below.« less

The Global Invertebrate Genomics Alliance (GIGA): Developing Community Resources to Study Diverse Invertebrate Genomes

PubMed Central

2014-01-01

Over 95% of all metazoan (animal) species comprise the “invertebrates,” but very few genomes from these organisms have been sequenced. We have, therefore, formed a “Global Invertebrate Genomics Alliance” (GIGA). Our intent is to build a collaborative network of diverse scientists to tackle major challenges (e.g., species selection, sample collection and storage, sequence assembly, annotation, analytical tools) associated with genome/transcriptome sequencing across a large taxonomic spectrum. We aim to promote standards that will facilitate comparative approaches to invertebrate genomics and collaborations across the international scientific community. Candidate study taxa include species from Porifera, Ctenophora, Cnidaria, Placozoa, Mollusca, Arthropoda, Echinodermata, Annelida, Bryozoa, and Platyhelminthes, among others. GIGA will target 7000 noninsect/nonnematode species, with an emphasis on marine taxa because of the unrivaled phyletic diversity in the oceans. Priorities for selecting invertebrates for sequencing will include, but are not restricted to, their phylogenetic placement; relevance to organismal, ecological, and conservation research; and their importance to fisheries and human health. We highlight benefits of sequencing both whole genomes (DNA) and transcriptomes and also suggest policies for genomic-level data access and sharing based on transparency and inclusiveness. The GIGA Web site (http://giga.nova.edu) has been launched to facilitate this collaborative venture. PMID:24336862

The Global Invertebrate Genomics Alliance (GIGA): developing community resources to study diverse invertebrate genomes.

PubMed

Bracken-Grissom, Heather; Collins, Allen G; Collins, Timothy; Crandall, Keith; Distel, Daniel; Dunn, Casey; Giribet, Gonzalo; Haddock, Steven; Knowlton, Nancy; Martindale, Mark; Medina, Mónica; Messing, Charles; O'Brien, Stephen J; Paulay, Gustav; Putnam, Nicolas; Ravasi, Timothy; Rouse, Greg W; Ryan, Joseph F; Schulze, Anja; Wörheide, Gert; Adamska, Maja; Bailly, Xavier; Breinholt, Jesse; Browne, William E; Diaz, M Christina; Evans, Nathaniel; Flot, Jean-François; Fogarty, Nicole; Johnston, Matthew; Kamel, Bishoy; Kawahara, Akito Y; Laberge, Tammy; Lavrov, Dennis; Michonneau, François; Moroz, Leonid L; Oakley, Todd; Osborne, Karen; Pomponi, Shirley A; Rhodes, Adelaide; Santos, Scott R; Satoh, Nori; Thacker, Robert W; Van de Peer, Yves; Voolstra, Christian R; Welch, David Mark; Winston, Judith; Zhou, Xin

2014-01-01

Over 95% of all metazoan (animal) species comprise the "invertebrates," but very few genomes from these organisms have been sequenced. We have, therefore, formed a "Global Invertebrate Genomics Alliance" (GIGA). Our intent is to build a collaborative network of diverse scientists to tackle major challenges (e.g., species selection, sample collection and storage, sequence assembly, annotation, analytical tools) associated with genome/transcriptome sequencing across a large taxonomic spectrum. We aim to promote standards that will facilitate comparative approaches to invertebrate genomics and collaborations across the international scientific community. Candidate study taxa include species from Porifera, Ctenophora, Cnidaria, Placozoa, Mollusca, Arthropoda, Echinodermata, Annelida, Bryozoa, and Platyhelminthes, among others. GIGA will target 7000 noninsect/nonnematode species, with an emphasis on marine taxa because of the unrivaled phyletic diversity in the oceans. Priorities for selecting invertebrates for sequencing will include, but are not restricted to, their phylogenetic placement; relevance to organismal, ecological, and conservation research; and their importance to fisheries and human health. We highlight benefits of sequencing both whole genomes (DNA) and transcriptomes and also suggest policies for genomic-level data access and sharing based on transparency and inclusiveness. The GIGA Web site (http://giga.nova.edu) has been launched to facilitate this collaborative venture.

Host-Associated Genomic Features of the Novel Uncultured Intracellular Pathogen Ca. Ichthyocystis Revealed by Direct Sequencing of Epitheliocysts

PubMed Central

Qi, Weihong; Vaughan, Lloyd; Katharios, Pantelis; Schlapbach, Ralph; Seth-Smith, Helena M.B.

2016-01-01

Advances in single-cell and mini-metagenome sequencing have enabled important investigations into uncultured bacteria. In this study, we applied the mini-metagenome sequencing method to assemble genome drafts of the uncultured causative agents of epitheliocystis, an emerging infectious disease in the Mediterranean aquaculture species gilthead seabream. We sequenced multiple cyst samples and constructed 11 genome drafts from a novel beta-proteobacterial lineage, Candidatus Ichthyocystis. The draft genomes demonstrate features typical of pathogenic bacteria with an obligate intracellular lifestyle: a reduced genome of up to 2.6 Mb, reduced G + C content, and reduced metabolic capacity. Reconstruction of metabolic pathways reveals that Ca. Ichthyocystis genomes lack all amino acid synthesis pathways, compelling them to scavenge from the fish host. All genomes encode type II, III, and IV secretion systems, a large repertoire of predicted effectors, and a type IV pilus. These are all considered to be virulence factors, required for adherence, invasion, and host manipulation. However, no evidence of lipopolysaccharide synthesis could be found. Beyond the core functions shared within the genus, alignments showed distinction into different species, characterized by alternative large gene families. These comprise up to a third of each genome, appear to have arisen through duplication and diversification, encode many effector proteins, and are seemingly critical for virulence. Thus, Ca. Ichthyocystis represents a novel obligatory intracellular pathogenic beta-proteobacterial lineage. The methods used: mini-metagenome analysis and manual annotation, have generated important insights into the lifestyle and evolution of the novel, uncultured pathogens, elucidating many putative virulence factors including an unprecedented array of novel gene families. PMID:27190004

Implications of the plastid genome sequence of typha (typhaceae, poales) for understanding genome evolution in poaceae.

PubMed

Guisinger, Mary M; Chumley, Timothy W; Kuehl, Jennifer V; Boore, Jeffrey L; Jansen, Robert K

2010-02-01

Plastid genomes of the grasses (Poaceae) are unusual in their organization and rates of sequence evolution. There has been a recent surge in the availability of grass plastid genome sequences, but a comprehensive comparative analysis of genome evolution has not been performed that includes any related families in the Poales. We report on the plastid genome of Typha latifolia, the first non-grass Poales sequenced to date, and we present comparisons of genome organization and sequence evolution within Poales. Our results confirm that grass plastid genomes exhibit acceleration in both genomic rearrangements and nucleotide substitutions. Poaceae have multiple structural rearrangements, including three inversions, three genes losses (accD, ycf1, ycf2), intron losses in two genes (clpP, rpoC1), and expansion of the inverted repeat (IR) into both large and small single-copy regions. These rearrangements are restricted to the Poaceae, and IR expansion into the small single-copy region correlates with the phylogeny of the family. Comparisons of 73 protein-coding genes for 47 angiosperms including nine Poaceae genera confirm that the branch leading to Poaceae has significantly accelerated rates of change relative to other monocots and angiosperms. Furthermore, rates of sequence evolution within grasses are lower, indicating a deceleration during diversification of the family. Overall there is a strong correlation between accelerated rates of genomic rearrangements and nucleotide substitutions in Poaceae, a phenomenon that has been noted recently throughout angiosperms. The cause of the correlation is unknown, but faulty DNA repair has been suggested in other systems including bacterial and animal mitochondrial genomes.

Molecular evolution of the plastid genome during diversification of the cotton genus.

PubMed

Chen, Zhiwen; Grover, Corrinne E; Li, Pengbo; Wang, Yumei; Nie, Hushuai; Zhao, Yanpeng; Wang, Meiyan; Liu, Fang; Zhou, Zhongli; Wang, Xingxing; Cai, Xiaoyan; Wang, Kunbo; Wendel, Jonathan F; Hua, Jinping

2017-07-01

Cotton (Gossypium spp.) is commonly grouped into eight diploid genomic groups, designated A-G and K, and one tetraploid genomic group, namely AD. To gain insight into the phylogeny of Gossypium and molecular evolution of the chloroplast genome duringdiversification, chloroplast genomes (cpDNA) from 6 D-genome and 2 G-genome species of Gossypium (G. armourianum D 2-1 , G. harknessii D 2-2 , G. davidsonii D 3-d , G. klotzschianum D 3-k , G. aridum D 4 , G. trilobum D 8 , and G. australe G 2 , G. nelsonii G 3 ) were newly reported here. In combination with the 26 previously released cpDNA sequences, we performed comparative phylogenetic analyses of 34 Gossypium chloroplast genomes that collectively represent most of the diversity in the genus. Gossypium chloroplasts span a small range in size that is mostly attributable to indels that occur in the large single copy (LSC) region of the genome. Phylogenetic analysis using a concatenation of all genes provides robust support for six major Gossypium clades, largely supporting earlier inferences but also revealing new information on intrageneric relationships. Using Theobroma cacao as an outgroup, diversification of the genus was dated, yielding results that are in accord with previous estimates of divergence times, but also offering new perspectives on the basal, early radiation of all major clades within the genus as well as gaps in the record indicative of extinctions. Like most higher-plant chloroplast genomes, all cotton species exhibit a conserved quadripartite structure, i.e., two large inverted repeats (IR) containing most of the ribosomal RNA genes, and two unique regions, LSC (large single sequence) and SSC (small single sequence). Within Gossypium, the IR-single copy region junctions are both variable and homoplasious among species. Two genes, accD and psaJ, exhibited greater rates of synonymous and non-synonymous substitutions than did other genes. Most genes exhibited Ka/Ks ratios suggestive of neutral evolution, with 8 exceptions distributed among one to several species. This research provides an overview of the molecular evolution of a single, large non-recombining molecular during the diversification of this important genus. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanisms of haplotype divergence at the RGA08 nucleotide-binding leucine-rich repeat gene locus in wild banana (Musa balbisiana)

PubMed Central

2010-01-01

Background Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW). Results Sequence comparison revealed two regions of contrasting features. The first is a highly colinear gene-rich region where the two haplotypes diverge only by single nucleotide polymorphisms and two repetitive element insertions. The second corresponds to a large cluster of RGA08 genes, with 13 and 18 predicted RGA genes and pseudogenes spread over 131 and 152 kb respectively on each haplotype. The RGA08 cluster is enriched in repetitive element insertions, in duplicated non-coding intergenic sequences including low complexity regions and shows structural variations between haplotypes. Although some allelic relationships are retained, a large diversity of RGA08 genes occurs in this single M. balbisiana genotype, with several RGA08 paralogs specific to each haplotype. The RGA08 gene family has evolved by mechanisms of unequal recombination, intragenic sequence exchange and diversifying selection. An unequal recombination event taking place between duplicated non-coding intergenic sequences resulted in a different RGA08 gene content between haplotypes pointing out the role of such duplicated regions in the evolution of RGA clusters. Based on the synonymous substitution rate in coding sequences, we estimated a 1 million year divergence time for these M. balbisiana haplotypes. Conclusions A large RGA08 gene cluster identified in wild banana corresponds to a highly variable genomic region between haplotypes surrounded by conserved flanking regions. High level of sequence identity (70 to 99%) of the genic and intergenic regions suggests a recent and rapid evolution of this cluster in M. balbisiana. PMID:20637079

Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications

PubMed Central

Del Medico, Luca; Christen, Heinz; Christen, Beat

2017-01-01

Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner. PMID:28531174

Molecular characterization of the complete genome of falconid herpesvirus strain S-18

USDA-ARS?s Scientific Manuscript database

Falconid herpesvirus type 1 (FHV-1) is the causative agent of falcon inclusion body disease, an acute, highly contagious disease of raptors. The complete nucleotide sequence of the genome of FHV-1 has been determined. The genome is arranged as a D-type genome with large inverted repeats flanking a ...

GWATCH: a web platform for automated gene association discovery analysis.

PubMed

Svitin, Anton; Malov, Sergey; Cherkasov, Nikolay; Geerts, Paul; Rotkevich, Mikhail; Dobrynin, Pavel; Shevchenko, Andrey; Guan, Li; Troyer, Jennifer; Hendrickson, Sher; Dilks, Holli Hutcheson; Oleksyk, Taras K; Donfield, Sharyne; Gomperts, Edward; Jabs, Douglas A; Sezgin, Efe; Van Natta, Mark; Harrigan, P Richard; Brumme, Zabrina L; O'Brien, Stephen J

2014-01-01

As genome-wide sequence analyses for complex human disease determinants are expanding, it is increasingly necessary to develop strategies to promote discovery and validation of potential disease-gene associations. Here we present a dynamic web-based platform - GWATCH - that automates and facilitates four steps in genetic epidemiological discovery: 1) Rapid gene association search and discovery analysis of large genome-wide datasets; 2) Expanded visual display of gene associations for genome-wide variants (SNPs, indels, CNVs), including Manhattan plots, 2D and 3D snapshots of any gene region, and a dynamic genome browser illustrating gene association chromosomal regions; 3) Real-time validation/replication of candidate or putative genes suggested from other sources, limiting Bonferroni genome-wide association study (GWAS) penalties; 4) Open data release and sharing by eliminating privacy constraints (The National Human Genome Research Institute (NHGRI) Institutional Review Board (IRB), informed consent, The Health Insurance Portability and Accountability Act (HIPAA) of 1996 etc.) on unabridged results, which allows for open access comparative and meta-analysis. GWATCH is suitable for both GWAS and whole genome sequence association datasets. We illustrate the utility of GWATCH with three large genome-wide association studies for HIV-AIDS resistance genes screened in large multicenter cohorts; however, association datasets from any study can be uploaded and analyzed by GWATCH.

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 principle and application of the single-molecule real-time sequencing technology].

PubMed

Yanhu, Liu; Lu, Wang; Li, Yu

2015-03-01

Last decade witnessed the explosive development of the third-generation sequencing strategy, including single-molecule real-time sequencing (SMRT), true single-molecule sequencing (tSMSTM) and the single-molecule nanopore DNA sequencing. In this review, we summarize the principle, performance and application of the SMRT sequencing technology. Compared with the traditional Sanger method and the next-generation sequencing (NGS) technologies, the SMRT approach has several advantages, including long read length, high speed, PCR-free and the capability of direct detection of epigenetic modifications. However, the disadvantage of its low accuracy, most of which resulted from insertions and deletions, is also notable. So, the raw sequence data need to be corrected before assembly. Up to now, the SMRT is a good fit for applications in the de novo genomic sequencing and the high-quality assemblies of small genomes. In the future, it is expected to play an important role in epigenetics, transcriptomic sequencing, and assemblies of large genomes.

Flow Sorting and Sequencing Meadow Fescue Chromosome 4F1[C][W

PubMed Central

Kopecký, David; Martis, Mihaela; Číhalíková, Jarmila; Hřibová, Eva; Vrána, Jan; Bartoš, Jan; Kopecká, Jitka; Cattonaro, Federica; Stočes, Štěpán; Novák, Petr; Neumann, Pavel; Macas, Jiří; Šimková, Hana; Studer, Bruno; Asp, Torben; Baird, James H.; Navrátil, Petr; Karafiátová, Miroslava; Kubaláková, Marie; Šafář, Jan; Mayer, Klaus; Doležel, Jaroslav

2013-01-01

The analysis of large genomes is hampered by a high proportion of repetitive DNA, which makes the assembly of short sequence reads difficult. This is also the case in meadow fescue (Festuca pratensis), which is known for good abiotic stress resistance and has been used in intergeneric hybridization with ryegrasses (Lolium spp.) to produce Festulolium cultivars. In this work, we describe a new approach to analyze the large genome of meadow fescue, which involves the reduction of sample complexity without compromising information content. This is achieved by dissecting the genome to smaller parts: individual chromosomes and groups of chromosomes. As the first step, we flow sorted chromosome 4F and sequenced it by Illumina with approximately 50× coverage. This provided, to our knowledge, the first insight into the composition of the fescue genome, enabled the construction of the virtual gene order of the chromosome, and facilitated detailed comparative analysis with the sequenced genomes of rice (Oryza sativa), Brachypodium distachyon, sorghum (Sorghum bicolor), and barley (Hordeum vulgare). Using GenomeZipper, we were able to confirm the collinearity of chromosome 4F with barley chromosome 4H and the long arm of chromosome 5H. Several new tandem repeats were identified and physically mapped using fluorescence in situ hybridization. They were found as robust cytogenetic markers for karyotyping of meadow fescue and ryegrass species and their hybrids. The ability to purify chromosome 4F opens the way for more efficient analysis of genomic loci on this chromosome underlying important traits, including freezing tolerance. Our results confirm that next-generation sequencing of flow-sorted chromosomes enables an overview of chromosome structure and evolution at a resolution never achieved before. PMID:24096412

Application of population sequencing (POPSEQ) for ordering and inputting genotyping-by-sequencing markers in hexaploid wheat

USDA-ARS?s Scientific Manuscript database

The advancement of next-generation sequencing technologies in conjunction with new bioinformatics tools enabled fine-tuning of sequence-based high resolution mapping strategies for complex genomes. Although genotyping-by-sequencing (GBS) provides a large number of markers, its application for assoc...

DNA sequence-level analyses reveal potential phenotypic modifiers in a large family with psychiatric disorders.

PubMed

Ryan, Niamh M; Lihm, Jayon; Kramer, Melissa; McCarthy, Shane; Morris, Stewart W; Arnau-Soler, Aleix; Davies, Gail; Duff, Barbara; Ghiban, Elena; Hayward, Caroline; Deary, Ian J; Blackwood, Douglas H R; Lawrie, Stephen M; McIntosh, Andrew M; Evans, Kathryn L; Porteous, David J; McCombie, W Richard; Thomson, Pippa A

2018-06-07

Psychiatric disorders are a group of genetically related diseases with highly polygenic architectures. Genome-wide association analyses have made substantial progress towards understanding the genetic architecture of these disorders. More recently, exome- and whole-genome sequencing of cases and families have identified rare, high penetrant variants that provide direct functional insight. There remains, however, a gap in the heritability explained by these complementary approaches. To understand how multiple genetic variants combine to modify both severity and penetrance of a highly penetrant variant, we sequenced 48 whole genomes from a family with a high loading of psychiatric disorder linked to a balanced chromosomal translocation. The (1;11)(q42;q14.3) translocation directly disrupts three genes: DISC1, DISC2, DISC1FP and has been linked to multiple brain imaging and neurocognitive outcomes in the family. Using DNA sequence-level linkage analysis, functional annotation and population-based association, we identified common and rare variants in GRM5 (minor allele frequency (MAF) > 0.05), PDE4D (MAF > 0.2) and CNTN5 (MAF < 0.01) that may help explain the individual differences in phenotypic expression in the family. We suggest that whole-genome sequencing in large families will improve the understanding of the combined effects of the rare and common sequence variation underlying psychiatric phenotypes.

Genomic analysis of NAC transcription factors in banana (Musa acuminata) and definition of NAC orthologous groups for monocots and dicots.

PubMed

Cenci, Albero; Guignon, Valentin; Roux, Nicolas; Rouard, Mathieu

2014-05-01

Identifying the molecular mechanisms underlying tolerance to abiotic stresses is important in crop breeding. A comprehensive understanding of the gene families associated with drought tolerance is therefore highly relevant. NAC transcription factors form a large plant-specific gene family involved in the regulation of tissue development and responses to biotic and abiotic stresses. The main goal of this study was to set up a framework of orthologous groups determined by an expert sequence comparison of NAC genes from both monocots and dicots. In order to clarify the orthologous relationships among NAC genes of different species, we performed an in-depth comparative study of four divergent taxa, in dicots and monocots, whose genomes have already been completely sequenced: Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa. Due to independent evolution, NAC copy number is highly variable in these plant genomes. Based on an expert NAC sequence comparison, we propose forty orthologous groups of NAC sequences that were probably derived from an ancestor gene present in the most recent common ancestor of dicots and monocots. These orthologous groups provide a curated resource for large-scale protein sequence annotation of NAC transcription factors. The established orthology relationships also provide a useful reference for NAC function studies in newly sequenced genomes such as M. acuminata and other plant species.

Defining the transcriptome assembly and its use for genome dynamics and transcriptome profiling studies in pigeonpea (Cajanus cajan L.).

PubMed

Dubey, Anuja; Farmer, Andrew; Schlueter, Jessica; Cannon, Steven B; Abernathy, Brian; Tuteja, Reetu; Woodward, Jimmy; Shah, Trushar; Mulasmanovic, Benjamin; Kudapa, Himabindu; Raju, Nikku L; Gothalwal, Ragini; Pande, Suresh; Xiao, Yongli; Town, Chris D; Singh, Nagendra K; May, Gregory D; Jackson, Scott; Varshney, Rajeev K

2011-06-01

This study reports generation of large-scale genomic resources for pigeonpea, a so-called 'orphan crop species' of the semi-arid tropic regions. FLX/454 sequencing carried out on a normalized cDNA pool prepared from 31 tissues produced 494 353 short transcript reads (STRs). Cluster analysis of these STRs, together with 10 817 Sanger ESTs, resulted in a pigeonpea trancriptome assembly (CcTA) comprising of 127 754 tentative unique sequences (TUSs). Functional analysis of these TUSs highlights several active pathways and processes in the sampled tissues. Comparison of the CcTA with the soybean genome showed similarity to 10 857 and 16 367 soybean gene models (depending on alignment methods). Additionally, Illumina 1G sequencing was performed on Fusarium wilt (FW)- and sterility mosaic disease (SMD)-challenged root tissues of 10 resistant and susceptible genotypes. More than 160 million sequence tags were used to identify FW- and SMD-responsive genes. Sequence analysis of CcTA and the Illumina tags identified a large new set of markers for use in genetics and breeding, including 8137 simple sequence repeats, 12 141 single-nucleotide polymorphisms and 5845 intron-spanning regions. Genomic resources developed in this study should be useful for basic and applied research, not only for pigeonpea improvement but also for other related, agronomically important legumes.

Omics approaches in food safety: fulfilling the promise?

PubMed Central

Bergholz, Teresa M.; Moreno Switt, Andrea I.; Wiedmann, Martin

2014-01-01

Genomics, transcriptomics, and proteomics are rapidly transforming our approaches to detection, prevention and treatment of foodborne pathogens. Microbial genome sequencing in particular has evolved from a research tool into an approach that can be used to characterize foodborne pathogen isolates as part of routine surveillance systems. Genome sequencing efforts will not only improve outbreak detection and source tracking, but will also create large amounts of foodborne pathogen genome sequence data, which will be available for data mining efforts that could facilitate better source attribution and provide new insights into foodborne pathogen biology and transmission. While practical uses and application of metagenomics, transcriptomics, and proteomics data and associated tools are less prominent, these tools are also starting to yield practical food safety solutions. PMID:24572764

Living with genome instability: the adaptation of phytoplasmas todiverse environments of their insect and plant hosts

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

Bai, Xiaodong; Zhang, Jianhua; Ewing, Adam

Phytoplasmas (Candidatus Phytoplasma, Class Mollicutes) cause disease in hundreds of economically important plants, and are obligately transmitted by sap-feeding insects of the order Hemiptera, mainly leafhoppers and psyllids. The 706,569-bp chromosome and four plasmids of aster yellows phytoplasma strain witches broom (AY-WB) were sequenced and compared to the onion yellows phytoplasma strain M (OY-M) genome. The phytoplasmas have small repeat-rich genomes. The repeated DNAs are organized into large clusters, potential mobile units (PMUs), which contain tra5 insertion sequences (ISs), and specialized sigma factors and membrane proteins. So far, PMUs are unique to phytoplasmas. Compared to mycoplasmas, phytoplasmas lack several recombinationmore » and DNA modification functions, and therefore phytoplasmas probably use different mechanisms of recombination, likely involving PMUs, for the creation of variability, allowing phytoplasmas to adjust to the diverse environments of plants and insects. The irregular GC skews and presence of ISs and large repeated sequences in the AY-WB and OY-M genomes are indicative of high genomic plasticity. Nevertheless, segments of {approx}250 kb, located between genes lplA and glnQ are syntenic between the two phytoplasmas, contain the majority of the metabolic genes and no ISs. AY-WB is further along in the reductive evolution process than OY-M. The AY-WB genome is {approx}154 kb smaller than the OY-M genome, primarily as a result of fewer multicopy sequences, including PMUs. Further, AY-WB lacks genes that are truncated and are part of incomplete pathways in OY-M. This is the first comparative phytoplasma genome analysis and report of the existence of PMUs in phytoplasma genomes.« less

Sequencing technologies - the next generation.

PubMed

Metzker, Michael L

2010-01-01

Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.

Uniform standards for genome databases in forest and fruit trees

USDA-ARS?s Scientific Manuscript database

TreeGenes and tfGDR serve the international forestry and fruit tree genomics research communities, respectively. These databases hold similar sequence data and provide resources for the submission and recovery of this information in order to enable comparative genomics research. Large-scale genotype...

A resource of large-scale molecular markers for monitoring Agropyron cristatum chromatin introgression in wheat background based on transcriptome sequences.

PubMed

Zhang, Jinpeng; Liu, Weihua; Lu, Yuqing; Liu, Qunxing; Yang, Xinming; Li, Xiuquan; Li, Lihui

2017-09-20

Agropyron cristatum is a wild grass of the tribe Triticeae and serves as a gene donor for wheat improvement. However, very few markers can be used to monitor A. cristatum chromatin introgressions in wheat. Here, we reported a resource of large-scale molecular markers for tracking alien introgressions in wheat based on transcriptome sequences. By aligning A. cristatum unigenes with the Chinese Spring reference genome sequences, we designed 9602 A. cristatum expressed sequence tag-sequence-tagged site (EST-STS) markers for PCR amplification and experimental screening. As a result, 6063 polymorphic EST-STS markers were specific for the A. cristatum P genome in the single-receipt wheat background. A total of 4956 randomly selected polymorphic EST-STS markers were further tested in eight wheat variety backgrounds, and 3070 markers displaying stable and polymorphic amplification were validated. These markers covered more than 98% of the A. cristatum genome, and the marker distribution density was approximately 1.28 cM. An application case of all EST-STS markers was validated on the A. cristatum 6 P chromosome. These markers were successfully applied in the tracking of alien A. cristatum chromatin. Altogether, this study provided a universal method of large-scale molecular marker development to monitor wild relative chromatin in wheat.

Discovery of common sequences absent in the human reference genome using pooled samples from next generation sequencing.

PubMed

Liu, Yu; Koyutürk, Mehmet; Maxwell, Sean; Xiang, Min; Veigl, Martina; Cooper, Richard S; Tayo, Bamidele O; Li, Li; LaFramboise, Thomas; Wang, Zhenghe; Zhu, Xiaofeng; Chance, Mark R

2014-08-16

Sequences up to several megabases in length have been found to be present in individual genomes but absent in the human reference genome. These sequences may be common in populations, and their absence in the reference genome may indicate rare variants in the genomes of individuals who served as donors for the human genome project. As the reference genome is used in probe design for microarray technology and mapping short reads in next generation sequencing (NGS), this missing sequence could be a source of bias in functional genomic studies and variant analysis. One End Anchor (OEA) and/or orphan reads from paired-end sequencing have been used to identify novel sequences that are absent in reference genome. However, there is no study to investigate the distribution, evolution and functionality of those sequences in human populations. To systematically identify and study the missing common sequences (micSeqs), we extended the previous method by pooling OEA reads from large number of individuals and applying strict filtering methods to remove false sequences. The pipeline was applied to data from phase 1 of the 1000 Genomes Project. We identified 309 micSeqs that are present in at least 1% of the human population, but absent in the reference genome. We confirmed 76% of these 309 micSeqs by comparison to other primate genomes, individual human genomes, and gene expression data. Furthermore, we randomly selected fifteen micSeqs and confirmed their presence using PCR validation in 38 additional individuals. Functional analysis using published RNA-seq and ChIP-seq data showed that eleven micSeqs are highly expressed in human brain and three micSeqs contain transcription factor (TF) binding regions, suggesting they are functional elements. In addition, the identified micSeqs are absent in non-primates and show dynamic acquisition during primate evolution culminating with most micSeqs being present in Africans, suggesting some micSeqs may be important sources of human diversity. 76% of micSeqs were confirmed by a comparative genomics approach. Fourteen micSeqs are expressed in human brain or contain TF binding regions. Some micSeqs are primate-specific, conserved and may play a role in the evolution of primates.

Genome evolution in Reptilia: in silico chicken mapping of 12,000 BAC-end sequences from two reptiles and a basal bird

PubMed Central

2009-01-01

Background With the publication of the draft chicken genome and the recent production of several BAC clone libraries from non-avian reptiles and birds, it is now possible to undertake more detailed comparative genomic studies in Reptilia. Of interest in particular are the genomic events that transformed the large, repeat-rich genomes of mammals and non-avian reptiles into the minimalist chicken genome. We have used paired BAC end sequences (BESs) from the American alligator (Alligator mississippiensis), painted turtle (Chrysemys picta) and emu (Dromaius novaehollandiae) to investigate patterns of sequence divergence, gene and retroelement content, and microsynteny between these species and chicken. Results From a total of 11,967 curated BESs, we successfully mapped 725, 773 and 2597 sequences in alligator, turtle, and emu, respectively, to sites in the draft chicken genome using a stringent BLAST protocol. Most commonly, sequences mapped to a single site in the chicken genome. Of 1675, 1828 and 2936 paired BESs obtained for alligator, turtle, and emu, respectively, a total of 34 (alligator, 2%), 24 (turtle, 1.3%) and 479 (emu, 16.3%) pairs were found to map with high confidence and in the correct orientation and with BAC-sized intermarker distances to single chicken chromosomes, including 25 such paired hits in emu mapping to the chicken Z chromosome. By determining the insert sizes of a subset of BAC clones from these three species, we also found a significant correlation between the intermarker distance in alligator and turtle and in chicken, with slopes as expected on the basis of the ratio of the genome sizes. Conclusion Our results suggest that a large number of small-scale chromosomal rearrangements and deletions in the lineage leading to chicken have drastically reduced the number of detected syntenies observed between the chicken and alligator, turtle, and emu genomes and imply that small deletions occurring widely throughout the genomes of reptilian and avian ancestors led to the ~50% reduction in genome size observed in birds compared to reptiles. We have also mapped and identified likely gene regions in hundreds of new BAC clones from these species. PMID:19607659

Genome evolution in Reptilia: in silico chicken mapping of 12,000 BAC-end sequences from two reptiles and a basal bird.

PubMed

Chapus, Charles; Edwards, Scott V

2009-07-14

With the publication of the draft chicken genome and the recent production of several BAC clone libraries from non-avian reptiles and birds, it is now possible to undertake more detailed comparative genomic studies in Reptilia. Of interest in particular are the genomic events that transformed the large, repeat-rich genomes of mammals and non-avian reptiles into the minimalist chicken genome. We have used paired BAC end sequences (BESs) from the American alligator (Alligator mississippiensis), painted turtle (Chrysemys picta) and emu (Dromaius novaehollandiae) to investigate patterns of sequence divergence, gene and retroelement content, and microsynteny between these species and chicken. From a total of 11,967 curated BESs, we successfully mapped 725, 773 and 2597 sequences in alligator, turtle, and emu, respectively, to sites in the draft chicken genome using a stringent BLAST protocol. Most commonly, sequences mapped to a single site in the chicken genome. Of 1675, 1828 and 2936 paired BESs obtained for alligator, turtle, and emu, respectively, a total of 34 (alligator, 2%), 24 (turtle, 1.3%) and 479 (emu, 16.3%) pairs were found to map with high confidence and in the correct orientation and with BAC-sized intermarker distances to single chicken chromosomes, including 25 such paired hits in emu mapping to the chicken Z chromosome. By determining the insert sizes of a subset of BAC clones from these three species, we also found a significant correlation between the intermarker distance in alligator and turtle and in chicken, with slopes as expected on the basis of the ratio of the genome sizes. Our results suggest that a large number of small-scale chromosomal rearrangements and deletions in the lineage leading to chicken have drastically reduced the number of detected syntenies observed between the chicken and alligator, turtle, and emu genomes and imply that small deletions occurring widely throughout the genomes of reptilian and avian ancestors led to the ~50% reduction in genome size observed in birds compared to reptiles. We have also mapped and identified likely gene regions in hundreds of new BAC clones from these species.

Optical mapping reveals a large genetic inversion between two methicillin-resistant Staphylococcus aureus strains.

PubMed

Shukla, Sanjay K; Kislow, Jennifer; Briska, Adam; Henkhaus, John; Dykes, Colin

2009-09-01

Staphylococcus aureus is a highly versatile and evolving bacterium of great clinical importance. S. aureus can evolve by acquiring single nucleotide polymorphisms and mobile genetic elements and by recombination events. Identification and location of novel genomic elements in a bacterial genome are not straightforward, unless the whole genome is sequenced. Optical mapping is a new tool that creates a high-resolution, in situ ordered restriction map of a bacterial genome. These maps can be used to determine genomic organization and perform comparative genomics to identify genomic rearrangements, such as insertions, deletions, duplications, and inversions, compared to an in silico (virtual) restriction map of a known genome sequence. Using this technology, we report here the identification, approximate location, and characterization of a genetic inversion of approximately 500 kb of a DNA element between the NRS387 (USA800) and FPR3757 (USA300) strains. The presence of the inversion and location of its junction sites were confirmed by site-specific PCR and sequencing. At both the left and right junction sites in NRS387, an IS1181 element and a 73-bp sequence were identified as inverted repeats, which could explain the possible mechanism of the inversion event.

Genome Sequences of Marine Shrimp Exopalaemon carinicauda Holthuis Provide Insights into Genome Size Evolution of Caridea.

PubMed

Yuan, Jianbo; Gao, Yi; Zhang, Xiaojun; Wei, Jiankai; Liu, Chengzhang; Li, Fuhua; Xiang, Jianhai

2017-07-05

Crustacea, particularly Decapoda, contains many economically important species, such as shrimps and crabs. Crustaceans exhibit enormous (nearly 500-fold) variability in genome size. However, limited genome resources are available for investigating these species. Exopalaemon carinicauda Holthuis, an economical caridean shrimp, is a potential ideal experimental animal for research on crustaceans. In this study, we performed low-coverage sequencing and de novo assembly of the E. carinicauda genome. The assembly covers more than 95% of coding regions. E. carinicauda possesses a large complex genome (5.73 Gb), with size twice higher than those of many decapod shrimps. As such, comparative genomic analyses were implied to investigate factors affecting genome size evolution of decapods. However, clues associated with genome duplication were not identified, and few horizontally transferred sequences were detected. Ultimately, the burst of transposable elements, especially retrotransposons, was determined as the major factor influencing genome expansion. A total of 2 Gb repeats were identified, and RTE-BovB, Jockey, Gypsy, and DIRS were the four major retrotransposons that significantly expanded. Both recent (Jockey and Gypsy) and ancestral (DIRS) originated retrotransposons responsible for the genome evolution. The E. carinicauda genome also exhibited potential for the genomic and experimental research of shrimps.

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 Single Transcriptome of a Green Toad (Bufo viridis) Yields Candidate Genes for Sex Determination and -Differentiation and Non-Anonymous Population Genetic Markers

PubMed Central

Gerchen, Jörn F.; Reichert, Samuel J.; Röhr, Johannes T.; Dieterich, Christoph; Kloas, Werner

2016-01-01

Large genome size, including immense repetitive and non-coding fractions, still present challenges for capacity, bioinformatics and thus affordability of whole genome sequencing in most amphibians. Here, we test the performance of a single transcriptome to understand whether it can provide a cost-efficient resource for species with large unknown genomes. Using RNA from six different tissues from a single Palearctic green toad (Bufo viridis) specimen and Hiseq2000, we obtained 22,5 Mio reads and publish >100,000 unigene sequences. To evaluate efficacy and quality, we first use this data to identify green toad specific candidate genes, known from other vertebrates for their role in sex determination and differentiation. Of a list of 37 genes, the transcriptome yielded 32 (87%), many of which providing the first such data for this non-model anuran species. However, for many of these genes, only fragments could be retrieved. In order to allow also applications to population genetics, we further used the transcriptome for the targeted development of 21 non-anonymous microsatellites and tested them in genetic families and backcrosses. Eleven markers were specifically developed to be located on the B. viridis sex chromosomes; for eight markers we can indeed demonstrate sex-specific transmission in genetic families. Depending on phylogenetic distance, several markers, which are sex-linked in green toads, show high cross-amplification success across the anuran phylogeny, involving nine systematic anuran families. Our data support the view that single transcriptome sequencing (based on multiple tissues) provides a reliable genomic resource and cost-efficient method for non-model amphibian species with large genome size and, despite limitations, should be considered as long as genome sequencing remains unaffordable for most species. PMID:27232626

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.

Genome Sequences of Three Cluster AU Arthrobacter Phages, Caterpillar, Nightmare, and Teacup

PubMed Central

Adair, Tamarah L.; Stowe, Emily; Pizzorno, Marie C.; Krukonis, Gregory; Harrison, Melinda; Garlena, Rebecca A.; Russell, Daniel A.; Jacobs-Sera, Deborah

2017-01-01

ABSTRACT Caterpillar, Nightmare, and Teacup are cluster AU siphoviral phages isolated from enriched soil on Arthrobacter sp. strain ATCC 21022. These genomes are 58 kbp long with an average G+C content of 50%. Sequence analysis predicts 86 to 92 protein-coding genes, including a large number of small proteins with predicted transmembrane domains. PMID:29122860

Genome Sequence of Lactobacillus sakei LK-145 Isolated from a Japanese Sake Cellar as a High Producer of d-Amino Acids

PubMed Central

Kato, Shiro

2017-01-01

ABSTRACT This announcement reports the complete genome sequence of strain LK-145 of Lactobacillus sakei isolated from a Japanese sake cellar as a potent strain for the production of large amounts of d-amino acids. Three putative genes encoding an amino acid racemase were identified. PMID:28818888

Complete Genome Sequence of Elizabethkingia miricola Strain EM798-26 Isolated from the Blood of a Cancer Patient

PubMed Central

Lai, Chung-Hsu; Yang, Chih-Hui; Huang, Yi-Han; Lin, Hsi-Hsun

2018-01-01

ABSTRACT Elizabethkingia miricola EM798-26 was isolated from the blood of a patient with diffuse large B-cell lymphoma in Taiwan. We report here the complete genome sequence of EM798-26, which contains a G+C content of 35.7% and 3,877 candidate protein-coding genes. PMID:29301886

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

PubMed

Clark, Clifford G; Berry, Chrystal; Walker, Matthew; Petkau, Aaron; Barker, Dillon O R; Guan, Cai; Reimer, Aleisha; Taboada, Eduardo N

2016-12-03

Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates. Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain. The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

pyGeno: A Python package for precision medicine and proteogenomics.

PubMed

Daouda, Tariq; Perreault, Claude; Lemieux, Sébastien

2016-01-01

pyGeno is a Python package mainly intended for precision medicine applications that revolve around genomics and proteomics. It integrates reference sequences and annotations from Ensembl, genomic polymorphisms from the dbSNP database and data from next-gen sequencing into an easy to use, memory-efficient and fast framework, therefore allowing the user to easily explore subject-specific genomes and proteomes. Compared to a standalone program, pyGeno gives the user access to the complete expressivity of Python, a general programming language. Its range of application therefore encompasses both short scripts and large scale genome-wide studies.

pyGeno: A Python package for precision medicine and proteogenomics

PubMed Central

Daouda, Tariq; Perreault, Claude; Lemieux, Sébastien

2016-01-01

pyGeno is a Python package mainly intended for precision medicine applications that revolve around genomics and proteomics. It integrates reference sequences and annotations from Ensembl, genomic polymorphisms from the dbSNP database and data from next-gen sequencing into an easy to use, memory-efficient and fast framework, therefore allowing the user to easily explore subject-specific genomes and proteomes. Compared to a standalone program, pyGeno gives the user access to the complete expressivity of Python, a general programming language. Its range of application therefore encompasses both short scripts and large scale genome-wide studies. PMID:27785359

Clan Genomics and the Complex Architecture of Human Disease

PubMed Central

Belmont, John W.; Boerwinkle, Eric

2013-01-01

Human diseases are caused by alleles that encompass the full range of variant types, from single-nucleotide changes to copy-number variants, and these variations span a broad frequency spectrum, from the very rare to the common. The picture emerging from analysis of whole-genome sequences, the 1000 Genomes Project pilot studies, and targeted genomic sequencing derived from very large sample sizes reveals an abundance of rare and private variants. One implication of this realization is that recent mutation may have a greater influence on disease susceptibility or protection than is conferred by variations that arose in distant ancestors. PMID:21962505

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

Experiences Building Globus Genomics: A Next-Generation Sequencing Analysis Service using Galaxy, Globus, and Amazon Web Services

PubMed Central

Madduri, Ravi K.; Sulakhe, Dinanath; Lacinski, Lukasz; Liu, Bo; Rodriguez, Alex; Chard, Kyle; Dave, Utpal J.; Foster, Ian T.

2014-01-01

We describe Globus Genomics, a system that we have developed for rapid analysis of large quantities of next-generation sequencing (NGS) genomic data. This system achieves a high degree of end-to-end automation that encompasses every stage of data analysis including initial data retrieval from remote sequencing centers or storage (via the Globus file transfer system); specification, configuration, and reuse of multi-step processing pipelines (via the Galaxy workflow system); creation of custom Amazon Machine Images and on-demand resource acquisition via a specialized elastic provisioner (on Amazon EC2); and efficient scheduling of these pipelines over many processors (via the HTCondor scheduler). The system allows biomedical researchers to perform rapid analysis of large NGS datasets in a fully automated manner, without software installation or a need for any local computing infrastructure. We report performance and cost results for some representative workloads. PMID:25342933

Experiences Building Globus Genomics: A Next-Generation Sequencing Analysis Service using Galaxy, Globus, and Amazon Web Services.

PubMed

Madduri, Ravi K; Sulakhe, Dinanath; Lacinski, Lukasz; Liu, Bo; Rodriguez, Alex; Chard, Kyle; Dave, Utpal J; Foster, Ian T

2014-09-10

We describe Globus Genomics, a system that we have developed for rapid analysis of large quantities of next-generation sequencing (NGS) genomic data. This system achieves a high degree of end-to-end automation that encompasses every stage of data analysis including initial data retrieval from remote sequencing centers or storage (via the Globus file transfer system); specification, configuration, and reuse of multi-step processing pipelines (via the Galaxy workflow system); creation of custom Amazon Machine Images and on-demand resource acquisition via a specialized elastic provisioner (on Amazon EC2); and efficient scheduling of these pipelines over many processors (via the HTCondor scheduler). The system allows biomedical researchers to perform rapid analysis of large NGS datasets in a fully automated manner, without software installation or a need for any local computing infrastructure. We report performance and cost results for some representative workloads.

Real-Time Pathogen Detection in the Era of Whole-Genome Sequencing and Big Data: Comparison of k-mer and Site-Based Methods for Inferring the Genetic Distances among Tens of Thousands of Salmonella Samples.

PubMed

Pettengill, James B; Pightling, Arthur W; Baugher, Joseph D; Rand, Hugh; Strain, Errol

2016-01-01

The adoption of whole-genome sequencing within the public health realm for molecular characterization of bacterial pathogens has been followed by an increased emphasis on real-time detection of emerging outbreaks (e.g., food-borne Salmonellosis). In turn, large databases of whole-genome sequence data are being populated. These databases currently contain tens of thousands of samples and are expected to grow to hundreds of thousands within a few years. For these databases to be of optimal use one must be able to quickly interrogate them to accurately determine the genetic distances among a set of samples. Being able to do so is challenging due to both biological (evolutionary diverse samples) and computational (petabytes of sequence data) issues. We evaluated seven measures of genetic distance, which were estimated from either k-mer profiles (Jaccard, Euclidean, Manhattan, Mash Jaccard, and Mash distances) or nucleotide sites (NUCmer and an extended multi-locus sequence typing (MLST) scheme). When analyzing empirical data (whole-genome sequence data from 18,997 Salmonella isolates) there are features (e.g., genomic, assembly, and contamination) that cause distances inferred from k-mer profiles, which treat absent data as informative, to fail to accurately capture the distance between samples when compared to distances inferred from differences in nucleotide sites. Thus, site-based distances, like NUCmer and extended MLST, are superior in performance, but accessing the computing resources necessary to perform them may be challenging when analyzing large databases.

Chromosome-level assembly of Arabidopsis thaliana Ler reveals the extent of translocation and inversion polymorphisms.

PubMed

Zapata, Luis; Ding, Jia; Willing, Eva-Maria; Hartwig, Benjamin; Bezdan, Daniela; Jiao, Wen-Biao; Patel, Vipul; Velikkakam James, Geo; Koornneef, Maarten; Ossowski, Stephan; Schneeberger, Korbinian

2016-07-12

Resequencing or reference-based assemblies reveal large parts of the small-scale sequence variation. However, they typically fail to separate such local variation into colinear and rearranged variation, because they usually do not recover the complement of large-scale rearrangements, including transpositions and inversions. Besides the availability of hundreds of genomes of diverse Arabidopsis thaliana accessions, there is so far only one full-length assembled genome: the reference sequence. We have assembled 117 Mb of the A. thaliana Landsberg erecta (Ler) genome into five chromosome-equivalent sequences using a combination of short Illumina reads, long PacBio reads, and linkage information. Whole-genome comparison against the reference sequence revealed 564 transpositions and 47 inversions comprising ∼3.6 Mb, in addition to 4.1 Mb of nonreference sequence, mostly originating from duplications. Although rearranged regions are not different in local divergence from colinear regions, they are drastically depleted for meiotic recombination in heterozygotes. Using a 1.2-Mb inversion as an example, we show that such rearrangement-mediated reduction of meiotic recombination can lead to genetically isolated haplotypes in the worldwide population of A. thaliana Moreover, we found 105 single-copy genes, which were only present in the reference sequence or the Ler assembly, and 334 single-copy orthologs, which showed an additional copy in only one of the genomes. To our knowledge, this work gives first insights into the degree and type of variation, which will be revealed once complete assemblies will replace resequencing or other reference-dependent methods.

Using Markov chains of nucleotide sequences as a possible precursor to predict functional roles of human genome: a case study on inactive chromatin regions.

PubMed

Lee, K-E; Lee, E-J; Park, H-S

2016-08-30

Recent advances in computational epigenetics have provided new opportunities to evaluate n-gram probabilistic language models. In this paper, we describe a systematic genome-wide approach for predicting functional roles in inactive chromatin regions by using a sequence-based Markovian chromatin map of the human genome. We demonstrate that Markov chains of sequences can be used as a precursor to predict functional roles in heterochromatin regions and provide an example comparing two publicly available chromatin annotations of large-scale epigenomics projects: ENCODE project consortium and Roadmap Epigenomics consortium.

Large-scale genomic analyses reveal the population structure and evolutionary trends of Streptococcus agalactiae strains in Brazilian fish farms.

PubMed

Barony, Gustavo M; Tavares, Guilherme C; Pereira, Felipe L; Carvalho, Alex F; Dorella, Fernanda A; Leal, Carlos A G; Figueiredo, Henrique C P

2017-10-19

Streptococcus agalactiae is a major pathogen and a hindrance on tilapia farming worldwide. The aims of this work were to analyze the genomic evolution of Brazilian strains of S. agalactiae and to establish spatial and temporal relations between strains isolated from different outbreaks of streptococcosis. A total of 39 strains were obtained from outbreaks and their whole genomes were sequenced and annotated for comparative analysis of multilocus sequence typing, genomic similarity and whole genome multilocus sequence typing (wgMLST). The Brazilian strains presented two sequence types, including a newly described ST, and a non-typeable lineage. The use of wgMLST could differentiate each strain in a single clone and was used to establish temporal and geographical correlations among strains. Bayesian phylogenomic analysis suggests that the studied Brazilian population was co-introduced in the country with their host, approximately 60 years ago. Brazilian strains of S. agalactiae were shown to be heterogeneous in their genome sequences and were distributed in different regions of the country according to their genotype, which allowed the use of wgMLST analysis to track each outbreak event individually.

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

Templated sequence insertion polymorphisms in the human genome

NASA Astrophysics Data System (ADS)

Onozawa, Masahiro; Aplan, Peter

2016-11-01

Templated Sequence Insertion Polymorphism (TSIP) is a recently described form of polymorphism recognized in the human genome, in which a sequence that is templated from a distant genomic region is inserted into the genome, seemingly at random. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; Class 1 TSIPs show features of insertions that are mediated via the LINE-1 ORF2 protein, including 1) target-site duplication (TSD), 2) polyadenylation 10-30 nucleotides downstream of a “cryptic” polyadenylation signal, and 3) preference for insertion at a 5’-TTTT/A-3’ sequence. In contrast, class 2 TSIPs show features consistent with repair of a DNA double-strand break via insertion of a DNA “patch” that is derived from a distant genomic region. Survey of a large number of normal human volunteers demonstrates that most individuals have 25-30 TSIPs, and that these TSIPs track with specific geographic regions. Similar to other forms of human polymorphism, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases.

Phylogenomic evidence for ancient hybridization in the genomes of living cats (Felidae)

PubMed Central

Li, Gang; Davis, Brian W.; Eizirik, Eduardo; Murphy, William J.

2016-01-01

Inter-species hybridization has been recently recognized as potentially common in wild animals, but the extent to which it shapes modern genomes is still poorly understood. Distinguishing historical hybridization events from other processes leading to phylogenetic discordance among different markers requires a well-resolved species tree that considers all modes of inheritance and overcomes systematic problems due to rapid lineage diversification by sampling large genomic character sets. Here, we assessed genome-wide phylogenetic variation across a diverse mammalian family, Felidae (cats). We combined genotypes from a genome-wide SNP array with additional autosomal, X- and Y-linked variants to sample ∼150 kb of nuclear sequence, in addition to complete mitochondrial genomes generated using light-coverage Illumina sequencing. We present the first robust felid time tree that accounts for unique maternal, paternal, and biparental evolutionary histories. Signatures of phylogenetic discordance were abundant in the genomes of modern cats, in many cases indicating hybridization as the most likely cause. Comparison of big cat whole-genome sequences revealed a substantial reduction of X-linked divergence times across several large recombination cold spots, which were highly enriched for signatures of selection-driven post-divergence hybridization between the ancestors of the snow leopard and lion lineages. These results highlight the mosaic origin of modern felid genomes and the influence of sex chromosomes and sex-biased dispersal in post-speciation gene flow. A complete resolution of the tree of life will require comprehensive genomic sampling of biparental and sex-limited genetic variation to identify and control for phylogenetic conflict caused by ancient admixture and sex-biased differences in genomic transmission. PMID:26518481

RPAN: rice pan-genome browser for ∼3000 rice genomes.

PubMed

Sun, Chen; Hu, Zhiqiang; Zheng, Tianqing; Lu, Kuangchen; Zhao, Yue; Wang, Wensheng; Shi, Jianxin; Wang, Chunchao; Lu, Jinyuan; Zhang, Dabing; Li, Zhikang; Wei, Chaochun

2017-01-25

A pan-genome is the union of the gene sets of all the individuals of a clade or a species and it provides a new dimension of genome complexity with the presence/absence variations (PAVs) of genes among these genomes. With the progress of sequencing technologies, pan-genome study is becoming affordable for eukaryotes with large-sized genomes. The Asian cultivated rice, Oryza sativa L., is one of the major food sources for the world and a model organism in plant biology. Recently, the 3000 Rice Genome Project (3K RGP) sequenced more than 3000 rice genomes with a mean sequencing depth of 14.3×, which provided a tremendous resource for rice research. In this paper, we present a genome browser, Rice Pan-genome Browser (RPAN), as a tool to search and visualize the rice pan-genome derived from 3K RGP. RPAN contains a database of the basic information of 3010 rice accessions, including genomic sequences, gene annotations, PAV information and gene expression data of the rice pan-genome. At least 12 000 novel genes absent in the reference genome were included. RPAN also provides multiple search and visualization functions. RPAN can be a rich resource for rice biology and rice breeding. It is available at http://cgm.sjtu.edu.cn/3kricedb/ or http://www.rmbreeding.cn/pan3k. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Draft sequencing and comparative genomics of Xylella fastidiosa strains reveal novel biological insights.

PubMed

Bhattacharyya, Anamitra; Stilwagen, Stephanie; Reznik, Gary; Feil, Helene; Feil, William S; Anderson, Iain; Bernal, Axel; D'Souza, Mark; Ivanova, Natalia; Kapatral, Vinayak; Larsen, Niels; Los, Tamara; Lykidis, Athanasios; Selkov, Eugene; Walunas, Theresa L; Purcell, Alexander; Edwards, Rob A; Hawkins, Trevor; Haselkorn, Robert; Overbeek, Ross; Kyrpides, Nikos C; Predki, Paul F

2002-10-01

Draft sequencing is a rapid and efficient method for determining the near-complete sequence of microbial genomes. Here we report a comparative analysis of one complete and two draft genome sequences of the phytopathogenic bacterium, Xylella fastidiosa, which causes serious disease in plants, including citrus, almond, and oleander. We present highlights of an in silico analysis based on a comparison of reconstructions of core biological subsystems. Cellular pathway reconstructions have been used to identify a small number of genes, which are likely to reside within the draft genomes but are not captured in the draft assembly. These represented only a small fraction of all genes and were predominantly large and small ribosomal subunit protein components. By using this approach, some of the inherent limitations of draft sequence can be significantly reduced. Despite the incomplete nature of the draft genomes, it is possible to identify several phage-related genes, which appear to be absent from the draft genomes and not the result of insufficient sequence sampling. This region may therefore identify potential host-specific functions. Based on this first functional reconstruction of a phytopathogenic microbe, we spotlight an unusual respiration machinery as a potential target for biological control. We also predicted and developed a new defined growth medium for Xylella.

The Neandertal genome and ancient DNA authenticity

PubMed Central

Green, Richard E; Briggs, Adrian W; Krause, Johannes; Prüfer, Kay; Burbano, Hernán A; Siebauer, Michael; Lachmann, Michael; Pääbo, Svante

2009-01-01

Recent advances in high-thoughput DNA sequencing have made genome-scale analyses of genomes of extinct organisms possible. With these new opportunities come new difficulties in assessing the authenticity of the DNA sequences retrieved. We discuss how these difficulties can be addressed, particularly with regard to analyses of the Neandertal genome. We argue that only direct assays of DNA sequence positions in which Neandertals differ from all contemporary humans can serve as a reliable means to estimate human contamination. Indirect measures, such as the extent of DNA fragmentation, nucleotide misincorporations, or comparison of derived allele frequencies in different fragment size classes, are unreliable. Fortunately, interim approaches based on mtDNA differences between Neandertals and current humans, detection of male contamination through Y chromosomal sequences, and repeated sequencing from the same fossil to detect autosomal contamination allow initial large-scale sequencing of Neandertal genomes. This will result in the discovery of fixed differences in the nuclear genome between Neandertals and current humans that can serve as future direct assays for contamination. For analyses of other fossil hominins, which may become possible in the future, we suggest a similar ‘boot-strap' approach in which interim approaches are applied until sufficient data for more definitive direct assays are acquired. PMID:19661919

Draft genome of the gayal, Bos frontalis

PubMed Central

Wang, Ming-Shan; Zeng, Yan; Wang, Xiao; Nie, Wen-Hui; Wang, Jin-Huan; Su, Wei-Ting; Xiong, Zi-Jun; Wang, Sheng; Qu, Kai-Xing; Yan, Shou-Qing; Yang, Min-Min; Wang, Wen; Dong, Yang; Zhang, Ya-Ping

2017-01-01

Abstract Gayal (Bos frontalis), also known as mithan or mithun, is a large endangered semi-domesticated bovine that has a limited geographical distribution in the hill-forests of China, Northeast India, Bangladesh, Myanmar, and Bhutan. Many questions about the gayal such as its origin, population history, and genetic basis of local adaptation remain largely unresolved. De novo sequencing and assembly of the whole gayal genome provides an opportunity to address these issues. We report a high-depth sequencing, de novo assembly, and annotation of a female Chinese gayal genome. Based on the Illumina genomic sequencing platform, we have generated 350.38 Gb of raw data from 16 different insert-size libraries. A total of 276.86 Gb of clean data is retained after quality control. The assembled genome is about 2.85 Gb with scaffold and contig N50 sizes of 2.74 Mb and 14.41 kb, respectively. Repetitive elements account for 48.13% of the genome. Gene annotation has yielded 26 667 protein-coding genes, of which 97.18% have been functionally annotated. BUSCO assessment shows that our assembly captures 93% (3183 of 4104) of the core eukaryotic genes and 83.1% of vertebrate universal single-copy orthologs. We provide the first comprehensive de novo genome of the gayal. This genetic resource is integral for investigating the origin of the gayal and performing comparative genomic studies to improve understanding of the speciation and divergence of bovine species. The assembled genome could be used as reference in future population genetic studies of gayal. PMID:29048483

'Big data', Hadoop and cloud computing in genomics.

PubMed

O'Driscoll, Aisling; Daugelaite, Jurate; Sleator, Roy D

2013-10-01

Since the completion of the Human Genome project at the turn of the Century, there has been an unprecedented proliferation of genomic sequence data. A consequence of this is that the medical discoveries of the future will largely depend on our ability to process and analyse large genomic data sets, which continue to expand as the cost of sequencing decreases. Herein, we provide an overview of cloud computing and big data technologies, and discuss how such expertise can be used to deal with biology's big data sets. In particular, big data technologies such as the Apache Hadoop project, which provides distributed and parallelised data processing and analysis of petabyte (PB) scale data sets will be discussed, together with an overview of the current usage of Hadoop within the bioinformatics community. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Genome analysis and polar tube firing dynamics of mosquito-infecting microsporidia

USDA-ARS?s Scientific Manuscript database

Microsporidia are highly divergent fungi that are obligate intracellular pathogens of a wide range of host organisms. Here we review recent findings from the genome sequences of mosquito-infecting microsporidian species Edhazardia aedis and Vavraia culicis, which show large differences in genome siz...

Metavir 2: new tools for viral metagenome comparison and assembled virome analysis

PubMed Central

2014-01-01

Background Metagenomics, based on culture-independent sequencing, is a well-fitted approach to provide insights into the composition, structure and dynamics of environmental viral communities. Following recent advances in sequencing technologies, new challenges arise for existing bioinformatic tools dedicated to viral metagenome (i.e. virome) analysis as (i) the number of viromes is rapidly growing and (ii) large genomic fragments can now be obtained by assembling the huge amount of sequence data generated for each metagenome. Results To face these challenges, a new version of Metavir was developed. First, all Metavir tools have been adapted to support comparative analysis of viromes in order to improve the analysis of multiple datasets. In addition to the sequence comparison previously provided, viromes can now be compared through their k-mer frequencies, their taxonomic compositions, recruitment plots and phylogenetic trees containing sequences from different datasets. Second, a new section has been specifically designed to handle assembled viromes made of thousands of large genomic fragments (i.e. contigs). This section includes an annotation pipeline for uploaded viral contigs (gene prediction, similarity search against reference viral genomes and protein domains) and an extensive comparison between contigs and reference genomes. Contigs and their annotations can be explored on the website through specifically developed dynamic genomic maps and interactive networks. Conclusions The new features of Metavir 2 allow users to explore and analyze viromes composed of raw reads or assembled fragments through a set of adapted tools and a user-friendly interface. PMID:24646187

Utility of Combining Whole Genome Sequencing with Traditional Investigational Methods To Solve Foodborne Outbreaks of Salmonella Infections Associated with Chicken: A New Tool for Tackling This Challenging Food Vehicle.

PubMed

Crowe, Samuel J; Green, Alice; Hernandez, Kimberly; Peralta, Vi; Bottichio, Lyndsay; Defibaugh-Chavez, Stephanie; Douris, Aphrodite; Gieraltowski, Laura; Hise, Kelley; La-Pham, Karen; Neil, Karen P; Simmons, Mustafa; Tillman, Glenn; Tolar, Beth; Wagner, Darlene; Wasilenko, Jamie; Holt, Kristin; Trees, Eija; Wise, Matthew E

2017-04-01

High consumption rates and a multitude of brands make multistate foodborne outbreaks of Salmonella infections associated with chicken challenging to investigate, but whole genome sequencing is a powerful tool that can be used to assist investigators. Whole genome sequencing of pathogens isolated from clinical, environmental, and food samples is increasingly being used in multistate foodborne outbreak investigations to determine with unprecedented resolution how closely related these isolates are to one another genetically. In 2014, federal and state health officials investigated an outbreak of 146 Salmonella Heidelberg infections in 24 states. A follow-up analysis was conducted after the conclusion of the investigation in which 27 clinical and 24 food isolates from the outbreak underwent whole genome sequencing. These isolates formed seven clades, the largest of which contained clinical isolates from a subcluster of case patients who attended a catered party. One isolate from a chicken processed by a large producer was closely related genetically (zero to three single-nucleotide polymorphism differences) to the clinical isolates from these subcluster case patients. Chicken from this large producer was also present in the kitchen of the caterer on the day before the event, thus providing additional evidence that the chicken from this producer was the outbreak source. This investigation highlights how whole genome sequencing can be used with epidemiologic and traceback evidence to identify chicken sources of foodborne outbreaks.

Utility of Combining Whole Genome Sequencing with Traditional Investigational Methods To Solve Foodborne Outbreaks of Salmonella Infections Associated with Chicken: A New Tool for Tackling This Challenging Food Vehicle

PubMed Central

Crowe, Samuel J.; Green, Alice; Hernandez, Kimberly; Peralta, Vi; Bottichio, Lyndsay; Defibaugh-Chavez, Stephanie; Douris, Aphrodite; Gieraltowski, Laura; Hise, Kelley; La-Pham, Karen; Neil, Karen P.; Simmons, Mustafa; Tillman, Glenn; Tolar, Beth; Wagner, Darlene; Wasilenko, Jamie; Holt, Kristin; Trees, Eija; Wise, Matthew E.

2017-01-01

High consumption rates and a multitude of brands make multistate foodborne outbreaks of Salmonella infections associated with chicken challenging to investigate, but whole genome sequencing is a powerful tool that can be used to assist investigators. Whole genome sequencing of pathogens isolated from clinical, environmental, and food samples is increasingly being used in multistate foodborne outbreak investigations to determine with unprecedented resolution how closely related these isolates are to one another genetically. In 2014, federal and state health officials investigated an outbreak of 146 Salmonella Heidelberg infections in 24 states. A follow-up analysis was conducted after the conclusion of the investigation in which 27 clinical and 24 food isolates from the outbreak underwent whole genome sequencing. These isolates formed seven clades, the largest of which contained clinical isolates from a subcluster of case patients who attended a catered party. One isolate from a chicken processed by a large producer was closely related genetically (zero to three single-nucleotide polymorphism differences) to the clinical isolates from these subcluster case patients. Chicken from this large producer was also present in the kitchen of the caterer on the day before the event, thus providing additional evidence that the chicken from this producer was the outbreak source. This investigation highlights how whole genome sequencing can be used with epidemiologic and traceback evidence to identify chicken sources of foodborne outbreaks. PMID:28294686

Genome Sequence of African Swine Fever Virus BA71, the Virulent Parental Strain of the Nonpathogenic and Tissue-Culture Adapted BA71V.

PubMed

Rodríguez, Javier M; Moreno, Leticia Tais; Alejo, Alí; Lacasta, Anna; Rodríguez, Fernando; Salas, María L

2015-01-01

The strain BA71V has played a key role in African swine fever virus (ASFV) research. It was the first genome sequenced, and remains the only genome completely determined. A large part of the studies on the function of ASFV genes, viral transcription, replication, DNA repair and morphogenesis, has been performed using this model. This avirulent strain was obtained by adaptation to grow in Vero cells of the highly virulent BA71 strain. We report here the analysis of the genome sequence of BA71 in comparison with that of BA71V. They possess the smallest genomes for a virulent or an attenuated ASFV, and are essentially identical except for a relatively small number of changes. We discuss the possible contribution of these changes to virulence. Analysis of the BA71 sequence allowed us to identify new similarities among ASFV proteins, and with database proteins including two ASFV proteins that could function as a two-component signaling network.

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

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...

Chromosomal targeting by CRISPR-Cas systems can contribute to genome plasticity in bacteria

PubMed Central

Dy, Ron L; Pitman, Andrew R; Fineran, Peter C

2013-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated (Cas) proteins form adaptive immune systems in bacteria to combat phage and other foreign genetic elements. Typically, short spacer sequences are acquired from the invader DNA and incorporated into CRISPR arrays in the bacterial genome. Small RNAs are generated that contain these spacer sequences and enable sequence-specific destruction of the foreign nucleic acids. Occasionally, spacers are acquired from the chromosome, which instead leads to targeting of the host genome. Chromosomal targeting is highly toxic to the bacterium, providing a strong selective pressure for a variety of evolutionary routes that enable host cell survival. Mutations that inactivate the CRISPR-Cas functionality, such as within the cas genes, CRISPR repeat, protospacer adjacent motifs (PAM), and target sequence, mediate escape from toxicity. This self-targeting might provide some explanation for the incomplete distribution of CRISPR-Cas systems in less than half of sequenced bacterial genomes. More importantly, self-genome targeting can cause large-scale genomic alterations, including remodeling or deletion of pathogenicity islands and other non-mobile chromosomal regions. While control of horizontal gene transfer is perceived as their main function, our recent work illuminates an alternative role of CRISPR-Cas systems in causing host genomic changes and influencing bacterial evolution. PMID:24251073

RAD tag sequencing as a source of SNP markers in Cynara cardunculus L

PubMed Central

2012-01-01

Background The globe artichoke (Cynara cardunculus L. var. scolymus) genome is relatively poorly explored, especially compared to those of the other major Asteraceae crops sunflower and lettuce. No SNP markers are in the public domain. We have combined the recently developed restriction-site associated DNA (RAD) approach with the Illumina DNA sequencing platform to effect the rapid and mass discovery of SNP markers for C. cardunculus. Results RAD tags were sequenced from the genomic DNA of three C. cardunculus mapping population parents, generating 9.7 million reads, corresponding to ~1 Gbp of sequence. An assembly based on paired ends produced ~6.0 Mbp of genomic sequence, separated into ~19,000 contigs (mean length 312 bp), of which ~21% were fragments of putative coding sequence. The shared sequences allowed for the discovery of ~34,000 SNPs and nearly 800 indels, equivalent to a SNP frequency of 5.6 per 1,000 nt, and an indel frequency of 0.2 per 1,000 nt. A sample of heterozygous SNP loci was mapped by CAPS assays and this exercise provided validation of our mining criteria. The repetitive fraction of the genome had a high representation of retrotransposon sequence, followed by simple repeats, AT-low complexity regions and mobile DNA elements. The genomic k-mers distribution and CpG rate of C. cardunculus, compared with data derived from three whole genome-sequenced dicots species, provided a further evidence of the random representation of the C. cardunculus genome generated by RAD sampling. Conclusion The RAD tag sequencing approach is a cost-effective and rapid method to develop SNP markers in a highly heterozygous species. Our approach permitted to generate a large and robust SNP datasets by the adoption of optimized filtering criteria. PMID:22214349

Young, intact and nested retrotransposons are abundant in the onion and asparagus genomes

PubMed Central

Vitte, C.; Estep, M. C.; Leebens-Mack, J.; Bennetzen, J. L.

2013-01-01

Background and Aims Although monocotyledonous plants comprise one of the two major groups of angiosperms and include >65 000 species, comprehensive genome analysis has been focused mainly on the Poaceae (grass) family. Due to this bias, most of the conclusions that have been drawn for monocot genome evolution are based on grasses. It is not known whether these conclusions apply to many other monocots. Methods To extend our understanding of genome evolution in the monocots, Asparagales genomic sequence data were acquired and the structural properties of asparagus and onion genomes were analysed. Specifically, several available onion and asparagus bacterial artificial chromosomes (BACs) with contig sizes >35 kb were annotated and analysed, with a particular focus on the characterization of long terminal repeat (LTR) retrotransposons. Key Results The results reveal that LTR retrotransposons are the major components of the onion and garden asparagus genomes. These elements are mostly intact (i.e. with two LTRs), have mainly inserted within the past 6 million years and are piled up into nested structures. Analysis of shotgun genomic sequence data and the observation of two copies for some transposable elements (TEs) in annotated BACs indicates that some families have become particularly abundant, as high as 4–5 % (asparagus) or 3–4 % (onion) of the genome for the most abundant families, as also seen in large grass genomes such as wheat and maize. Conclusions Although previous annotations of contiguous genomic sequences have suggested that LTR retrotransposons were highly fragmented in these two Asparagales genomes, the results presented here show that this was largely due to the methodology used. In contrast, this current work indicates an ensemble of genomic features similar to those observed in the Poaceae. PMID:23887091

Evidence for a lineage of virulent bacteriophages that target Campylobacter.

PubMed

Timms, Andrew R; Cambray-Young, Joanna; Scott, Andrew E; Petty, Nicola K; Connerton, Phillippa L; Clarke, Louise; Seeger, Kathy; Quail, Mike; Cummings, Nicola; Maskell, Duncan J; Thomson, Nicholas R; Connerton, Ian F

2010-03-30

Our understanding of the dynamics of genome stability versus gene flux within bacteriophage lineages is limited. Recently, there has been a renewed interest in the use of bacteriophages as 'therapeutic' agents; a prerequisite for their use in such therapies is a thorough understanding of their genetic complement, genome stability and their ecology to avoid the dissemination or mobilisation of phage or bacterial virulence and toxin genes. Campylobacter, a food-borne pathogen, is one of the organisms for which the use of bacteriophage is being considered to reduce human exposure to this organism. Sequencing and genome analysis was performed for two Campylobacter bacteriophages. The genomes were extremely similar at the nucleotide level (> or = 96%) with most differences accounted for by novel insertion sequences, DNA methylases and an approximately 10 kb contiguous region of metabolic genes that were dissimilar at the sequence level but similar in gene function between the two phages. Both bacteriophages contained a large number of radical S-adenosylmethionine (SAM) genes, presumably involved in boosting host metabolism during infection, as well as evidence that many genes had been acquired from a wide range of bacterial species. Further bacteriophages, from the UK Campylobacter typing set, were screened for the presence of bacteriophage structural genes, DNA methylases, mobile genetic elements and regulatory genes identified from the genome sequences. The results indicate that many of these bacteriophages are related, with 10 out of 15 showing some relationship to the sequenced genomes. Two large virulent Campylobacter bacteriophages were found to show very high levels of sequence conservation despite separation in time and place of isolation. The bacteriophages show adaptations to their host and possess genes that may enhance Campylobacter metabolism, potentially advantaging both the bacteriophage and its host. Genetic conservation has been shown to extend to other Campylobacter bacteriophages, forming a highly conserved lineage of bacteriophages that predate upon campylobacters and indicating that highly adapted bacteriophage genomes can be stable over prolonged periods of time.

Evolutionary Dynamics of Microsatellite Distribution in Plants: Insight from the Comparison of Sequenced Brassica, Arabidopsis and Other Angiosperm Species

PubMed Central

Shi, Jiaqin; Huang, Shunmou; Fu, Donghui; Yu, Jinyin; Wang, Xinfa; Hua, Wei; Liu, Shengyi; Liu, Guihua; Wang, Hanzhong

2013-01-01

Despite their ubiquity and functional importance, microsatellites have been largely ignored in comparative genomics, mostly due to the lack of genomic information. In the current study, microsatellite distribution was characterized and compared in the whole genomes and both the coding and non-coding DNA sequences of the sequenced Brassica, Arabidopsis and other angiosperm species to investigate their evolutionary dynamics in plants. The variation in the microsatellite frequencies of these angiosperm species was much smaller than those for their microsatellite numbers and genome sizes, suggesting that microsatellite frequency may be relatively stable in plants. The microsatellite frequencies of these angiosperm species were significantly negatively correlated with both their genome sizes and transposable elements contents. The pattern of microsatellite distribution may differ according to the different genomic regions (such as coding and non-coding sequences). The observed differences in many important microsatellite characteristics (especially the distribution with respect to motif length, type and repeat number) of these angiosperm species were generally accordant with their phylogenetic distance, which suggested that the evolutionary dynamics of microsatellite distribution may be generally consistent with plant divergence/evolution. Importantly, by comparing these microsatellite characteristics (especially the distribution with respect to motif type) the angiosperm species (aside from a few species) all clustered into two obviously different groups that were largely represented by monocots and dicots, suggesting a complex and generally dichotomous evolutionary pattern of microsatellite distribution in angiosperms. Polyploidy may lead to a slight increase in microsatellite frequency in the coding sequences and a significant decrease in microsatellite frequency in the whole genome/non-coding sequences, but have little effect on the microsatellite distribution with respect to motif length, type and repeat number. Interestingly, several microsatellite characteristics seemed to be constant in plant evolution, which can be well explained by the general biological rules. PMID:23555856

Importing statistical measures into Artemis enhances gene identification in the Leishmania genome project.

PubMed

Aggarwal, Gautam; Worthey, E A; McDonagh, Paul D; Myler, Peter J

2003-06-07

Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces. Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODONUSAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence. An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

GenomeVista

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

Poliakov, Alexander; Couronne, Olivier

2002-11-04

Aligning large vertebrate genomes that are structurally complex poses a variety of problems not encountered on smaller scales. Such genomes are rich in repetitive elements and contain multiple segmental duplications, which increases the difficulty of identifying true orthologous SNA segments in alignments. The sizes of the sequences make many alignment algorithms designed for comparing single proteins extremely inefficient when processing large genomic intervals. We integrated both local and global alignment tools and developed a suite of programs for automatically aligning large vertebrate genomes and identifying conserved non-coding regions in the alignments. Our method uses the BLAT local alignment program tomore » find anchors on the base genome to identify regions of possible homology for a query sequence. These regions are postprocessed to find the best candidates which are then globally aligned using the AVID global alignment program. In the last step conserved non-coding segments are identified using VISTA. Our methods are fast and the resulting alignments exhibit a high degree of sensitivity, covering more than 90% of known coding exons in the human genome. The GenomeVISTA software is a suite of Perl programs that is built on a MySQL database platform. The scheduler gets control data from the database, builds a queve of jobs, and dispatches them to a PC cluster for execution. The main program, running on each node of the cluster, processes individual sequences. A Perl library acts as an interface between the database and the above programs. The use of a separate library allows the programs to function independently of the database schema. The library also improves on the standard Perl MySQL database interfere package by providing auto-reconnect functionality and improved error handling.« less

Characterization of genetic variability of Venezuelan equine encephalitis viruses

DOE PAGES

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.; ...

2016-04-07

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

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

PubMed Central

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

2017-01-01

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

Analyses of deep mammalian sequence alignments and constraint predictions for 1% of the human genome

PubMed Central

Margulies, Elliott H.; Cooper, Gregory M.; Asimenos, George; Thomas, Daryl J.; Dewey, Colin N.; Siepel, Adam; Birney, Ewan; Keefe, Damian; Schwartz, Ariel S.; Hou, Minmei; Taylor, James; Nikolaev, Sergey; Montoya-Burgos, Juan I.; Löytynoja, Ari; Whelan, Simon; Pardi, Fabio; Massingham, Tim; Brown, James B.; Bickel, Peter; Holmes, Ian; Mullikin, James C.; Ureta-Vidal, Abel; Paten, Benedict; Stone, Eric A.; Rosenbloom, Kate R.; Kent, W. James; Bouffard, Gerard G.; Guan, Xiaobin; Hansen, Nancy F.; Idol, Jacquelyn R.; Maduro, Valerie V.B.; Maskeri, Baishali; McDowell, Jennifer C.; Park, Morgan; Thomas, Pamela J.; Young, Alice C.; Blakesley, Robert W.; Muzny, Donna M.; Sodergren, Erica; Wheeler, David A.; Worley, Kim C.; Jiang, Huaiyang; Weinstock, George M.; Gibbs, Richard A.; Graves, Tina; Fulton, Robert; Mardis, Elaine R.; Wilson, Richard K.; Clamp, Michele; Cuff, James; Gnerre, Sante; Jaffe, David B.; Chang, Jean L.; Lindblad-Toh, Kerstin; Lander, Eric S.; Hinrichs, Angie; Trumbower, Heather; Clawson, Hiram; Zweig, Ann; Kuhn, Robert M.; Barber, Galt; Harte, Rachel; Karolchik, Donna; Field, Matthew A.; Moore, Richard A.; Matthewson, Carrie A.; Schein, Jacqueline E.; Marra, Marco A.; Antonarakis, Stylianos E.; Batzoglou, Serafim; Goldman, Nick; Hardison, Ross; Haussler, David; Miller, Webb; Pachter, Lior; Green, Eric D.; Sidow, Arend

2007-01-01

A key component of the ongoing ENCODE project involves rigorous comparative sequence analyses for the initially targeted 1% of the human genome. Here, we present orthologous sequence generation, alignment, and evolutionary constraint analyses of 23 mammalian species for all ENCODE targets. Alignments were generated using four different methods; comparisons of these methods reveal large-scale consistency but substantial differences in terms of small genomic rearrangements, sensitivity (sequence coverage), and specificity (alignment accuracy). We describe the quantitative and qualitative trade-offs concomitant with alignment method choice and the levels of technical error that need to be accounted for in applications that require multisequence alignments. Using the generated alignments, we identified constrained regions using three different methods. While the different constraint-detecting methods are in general agreement, there are important discrepancies relating to both the underlying alignments and the specific algorithms. However, by integrating the results across the alignments and constraint-detecting methods, we produced constraint annotations that were found to be robust based on multiple independent measures. Analyses of these annotations illustrate that most classes of experimentally annotated functional elements are enriched for constrained sequences; however, large portions of each class (with the exception of protein-coding sequences) do not overlap constrained regions. The latter elements might not be under primary sequence constraint, might not be constrained across all mammals, or might have expendable molecular functions. Conversely, 40% of the constrained sequences do not overlap any of the functional elements that have been experimentally identified. Together, these findings demonstrate and quantify how many genomic functional elements await basic molecular characterization. PMID:17567995

Inexpensive and Highly Reproducible Cloud-Based Variant Calling of 2,535 Human Genomes

PubMed Central

Shringarpure, Suyash S.; Carroll, Andrew; De La Vega, Francisco M.; Bustamante, Carlos D.

2015-01-01

Population scale sequencing of whole human genomes is becoming economically feasible; however, data management and analysis remains a formidable challenge for many research groups. Large sequencing studies, like the 1000 Genomes Project, have improved our understanding of human demography and the effect of rare genetic variation in disease. Variant calling on datasets of hundreds or thousands of genomes is time-consuming, expensive, and not easily reproducible given the myriad components of a variant calling pipeline. Here, we describe a cloud-based pipeline for joint variant calling in large samples using the Real Time Genomics population caller. We deployed the population caller on the Amazon cloud with the DNAnexus platform in order to achieve low-cost variant calling. Using our pipeline, we were able to identify 68.3 million variants in 2,535 samples from Phase 3 of the 1000 Genomes Project. By performing the variant calling in a parallel manner, the data was processed within 5 days at a compute cost of $7.33 per sample (a total cost of $18,590 for completed jobs and $21,805 for all jobs). Analysis of cost dependence and running time on the data size suggests that, given near linear scalability, cloud computing can be a cheap and efficient platform for analyzing even larger sequencing studies in the future. PMID:26110529

Sensitivity to sequencing depth in single-cell cancer genomics.

PubMed

Alves, João M; Posada, David

2018-04-16

Querying cancer genomes at single-cell resolution is expected to provide a powerful framework to understand in detail the dynamics of cancer evolution. However, given the high costs currently associated with single-cell sequencing, together with the inevitable technical noise arising from single-cell genome amplification, cost-effective strategies that maximize the quality of single-cell data are critically needed. Taking advantage of previously published single-cell whole-genome and whole-exome cancer datasets, we studied the impact of sequencing depth and sampling effort towards single-cell variant detection. Five single-cell whole-genome and whole-exome cancer datasets were independently downscaled to 25, 10, 5, and 1× sequencing depth. For each depth level, ten technical replicates were generated, resulting in a total of 6280 single-cell BAM files. The sensitivity of variant detection, including structural and driver mutations, genotyping, clonal inference, and phylogenetic reconstruction to sequencing depth was evaluated using recent tools specifically designed for single-cell data. Altogether, our results suggest that for relatively large sample sizes (25 or more cells) sequencing single tumor cells at depths > 5× does not drastically improve somatic variant discovery, characterization of clonal genotypes, or estimation of single-cell phylogenies. We suggest that sequencing multiple individual tumor cells at a modest depth represents an effective alternative to explore the mutational landscape and clonal evolutionary patterns of cancer genomes.

CNVcaller: highly efficient and widely applicable software for detecting copy number variations in large populations.

PubMed

Wang, Xihong; Zheng, Zhuqing; Cai, Yudong; Chen, Ting; Li, Chao; Fu, Weiwei; Jiang, Yu

2017-12-01

The increasing amount of sequencing data available for a wide variety of species can be theoretically used for detecting copy number variations (CNVs) at the population level. However, the growing sample sizes and the divergent complexity of nonhuman genomes challenge the efficiency and robustness of current human-oriented CNV detection methods. Here, we present CNVcaller, a read-depth method for discovering CNVs in population sequencing data. The computational speed of CNVcaller was 1-2 orders of magnitude faster than CNVnator and Genome STRiP for complex genomes with thousands of unmapped scaffolds. CNV detection of 232 goats required only 1.4 days on a single compute node. Additionally, the Mendelian consistency of sheep trios indicated that CNVcaller mitigated the influence of high proportions of gaps and misassembled duplications in the nonhuman reference genome assembly. Furthermore, multiple evaluations using real sheep and human data indicated that CNVcaller achieved the best accuracy and sensitivity for detecting duplications. The fast generalized detection algorithms included in CNVcaller overcome prior computational barriers for detecting CNVs in large-scale sequencing data with complex genomic structures. Therefore, CNVcaller promotes population genetic analyses of functional CNVs in more species. © The Authors 2017. Published by Oxford University Press.

CNVcaller: highly efficient and widely applicable software for detecting copy number variations in large populations

PubMed Central

Wang, Xihong; Zheng, Zhuqing; Cai, Yudong; Chen, Ting; Li, Chao; Fu, Weiwei

2017-01-01

Abstract Background The increasing amount of sequencing data available for a wide variety of species can be theoretically used for detecting copy number variations (CNVs) at the population level. However, the growing sample sizes and the divergent complexity of nonhuman genomes challenge the efficiency and robustness of current human-oriented CNV detection methods. Results Here, we present CNVcaller, a read-depth method for discovering CNVs in population sequencing data. The computational speed of CNVcaller was 1–2 orders of magnitude faster than CNVnator and Genome STRiP for complex genomes with thousands of unmapped scaffolds. CNV detection of 232 goats required only 1.4 days on a single compute node. Additionally, the Mendelian consistency of sheep trios indicated that CNVcaller mitigated the influence of high proportions of gaps and misassembled duplications in the nonhuman reference genome assembly. Furthermore, multiple evaluations using real sheep and human data indicated that CNVcaller achieved the best accuracy and sensitivity for detecting duplications. Conclusions The fast generalized detection algorithms included in CNVcaller overcome prior computational barriers for detecting CNVs in large-scale sequencing data with complex genomic structures. Therefore, CNVcaller promotes population genetic analyses of functional CNVs in more species. PMID:29220491

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.

Phylogenetic shadowing of primate sequences to find functional regions of the human genome.

PubMed

Boffelli, Dario; McAuliffe, Jon; Ovcharenko, Dmitriy; Lewis, Keith D; Ovcharenko, Ivan; Pachter, Lior; Rubin, Edward M

2003-02-28

Nonhuman primates represent the most relevant model organisms to understand the biology of Homo sapiens. The recent divergence and associated overall sequence conservation between individual members of this taxon have nonetheless largely precluded the use of primates in comparative sequence studies. We used sequence comparisons of an extensive set of Old World and New World monkeys and hominoids to identify functional regions in the human genome. Analysis of these data enabled the discovery of primate-specific gene regulatory elements and the demarcation of the exons of multiple genes. Much of the information content of the comprehensive primate sequence comparisons could be captured with a small subset of phylogenetically close primates. These results demonstrate the utility of intraprimate sequence comparisons to discover common mammalian as well as primate-specific functional elements in the human genome, which are unattainable through the evaluation of more evolutionarily distant species.

Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes.

PubMed

Owor, Betty E; Shepherd, Dionne N; Taylor, Nigel J; Edema, Richard; Monjane, Adérito L; Thomson, Jennifer A; Martin, Darren P; Varsani, Arvind

2007-03-01

Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers' fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage phi29 DNA polymerase using the TempliPhi system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the phi29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes.

A comparative genomics strategy for targeted discovery of single-nucleotide polymorphisms and conserved-noncoding sequences in orphan crops.

PubMed

Feltus, F A; Singh, H P; Lohithaswa, H C; Schulze, S R; Silva, T D; Paterson, A H

2006-04-01

Completed genome sequences provide templates for the design of genome analysis tools in orphan species lacking sequence information. To demonstrate this principle, we designed 384 PCR primer pairs to conserved exonic regions flanking introns, using Sorghum/Pennisetum expressed sequence tag alignments to the Oryza genome. Conserved-intron scanning primers (CISPs) amplified single-copy loci at 37% to 80% success rates in taxa that sample much of the approximately 50-million years of Poaceae divergence. While the conserved nature of exons fostered cross-taxon amplification, the lesser evolutionary constraints on introns enhanced single-nucleotide polymorphism detection. For example, in eight rice (Oryza sativa) genotypes, polymorphism averaged 12.1 per kb in introns but only 3.6 per kb in exons. Curiously, among 124 CISPs evaluated across Oryza, Sorghum, Pennisetum, Cynodon, Eragrostis, Zea, Triticum, and Hordeum, 23 (18.5%) seemed to be subject to rigid intron size constraints that were independent of per-nucleotide DNA sequence variation. Furthermore, we identified 487 conserved-noncoding sequence motifs in 129 CISP loci. A large CISP set (6,062 primer pairs, amplifying introns from 1,676 genes) designed using an automated pipeline showed generally higher abundance in recombinogenic than in nonrecombinogenic regions of the rice genome, thus providing relatively even distribution along genetic maps. CISPs are an effective means to explore poorly characterized genomes for both DNA polymorphism and noncoding sequence conservation on a genome-wide or candidate gene basis, and also provide anchor points for comparative genomics across a diverse range of species.

A Comparative Genomics Strategy for Targeted Discovery of Single-Nucleotide Polymorphisms and Conserved-Noncoding Sequences in Orphan Crops1[W

PubMed Central

Feltus, F.A.; Singh, H.P.; Lohithaswa, H.C.; Schulze, S.R.; Silva, T.D.; Paterson, A.H.

2006-01-01

Completed genome sequences provide templates for the design of genome analysis tools in orphan species lacking sequence information. To demonstrate this principle, we designed 384 PCR primer pairs to conserved exonic regions flanking introns, using Sorghum/Pennisetum expressed sequence tag alignments to the Oryza genome. Conserved-intron scanning primers (CISPs) amplified single-copy loci at 37% to 80% success rates in taxa that sample much of the approximately 50-million years of Poaceae divergence. While the conserved nature of exons fostered cross-taxon amplification, the lesser evolutionary constraints on introns enhanced single-nucleotide polymorphism detection. For example, in eight rice (Oryza sativa) genotypes, polymorphism averaged 12.1 per kb in introns but only 3.6 per kb in exons. Curiously, among 124 CISPs evaluated across Oryza, Sorghum, Pennisetum, Cynodon, Eragrostis, Zea, Triticum, and Hordeum, 23 (18.5%) seemed to be subject to rigid intron size constraints that were independent of per-nucleotide DNA sequence variation. Furthermore, we identified 487 conserved-noncoding sequence motifs in 129 CISP loci. A large CISP set (6,062 primer pairs, amplifying introns from 1,676 genes) designed using an automated pipeline showed generally higher abundance in recombinogenic than in nonrecombinogenic regions of the rice genome, thus providing relatively even distribution along genetic maps. CISPs are an effective means to explore poorly characterized genomes for both DNA polymorphism and noncoding sequence conservation on a genome-wide or candidate gene basis, and also provide anchor points for comparative genomics across a diverse range of species. PMID:16607031

Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar Micro-Tom, a reference system for the Solanaceae genomics.

PubMed

Aoki, Koh; Yano, Kentaro; Suzuki, Ayako; Kawamura, Shingo; Sakurai, Nozomu; Suda, Kunihiro; Kurabayashi, Atsushi; Suzuki, Tatsuya; Tsugane, Taneaki; Watanabe, Manabu; Ooga, Kazuhide; Torii, Maiko; Narita, Takanori; Shin-I, Tadasu; Kohara, Yuji; Yamamoto, Naoki; Takahashi, Hideki; Watanabe, Yuichiro; Egusa, Mayumi; Kodama, Motoichiro; Ichinose, Yuki; Kikuchi, Mari; Fukushima, Sumire; Okabe, Akiko; Arie, Tsutomu; Sato, Yuko; Yazawa, Katsumi; Satoh, Shinobu; Omura, Toshikazu; Ezura, Hiroshi; Shibata, Daisuke

2010-03-30

The Solanaceae family includes several economically important vegetable crops. The tomato (Solanum lycopersicum) is regarded as a model plant of the Solanaceae family. Recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. In particular, a miniature cultivar, Micro-Tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the Micro-Tom-background, such as ESTs and mutagenized lines, have been established by an international alliance. To accelerate the progress in tomato genomics, we developed a collection of fully-sequenced 13,227 Micro-Tom full-length cDNAs. By checking redundant sequences, coding sequences, and chimeric sequences, a set of 11,502 non-redundant full-length cDNAs (nrFLcDNAs) was generated. Analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. Classification of functions of proteins predicted from the coding sequences demonstrated that nrFLcDNAs covered a broad range of functions. A comparison of nrFLcDNAs with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. Mapping of the nrFLcDNAs onto currently available tomato genome sequences facilitated prediction of exon-intron structure. Introns of tomato genes were longer than those of Arabidopsis and rice. According to a comparison of exon sequences between the nrFLcDNAs and the tomato genome sequences, the frequency of nucleotide mismatch in exons between Micro-Tom and the genome-sequencing cultivar (Heinz 1706) was estimated to be 0.061%. The collection of Micro-Tom nrFLcDNAs generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. The nrFLcDNA sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. Full-length cDNA sequences and their annotations are provided in the database KaFTom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the National Bioresource Project Tomato http://tomato.nbrp.jp.

AmphiBase: A new genomic resource for non-model amphibian species.

PubMed

Kwon, Taejoon

2017-01-01

More than five thousand genes annotated in the recently published Xenopus laevis and Xenopus tropicalis genomes do not have a candidate orthologous counterpart in other vertebrate species. To determine whether these sequences represent genuine amphibian-specific genes or annotation errors, it is necessary to analyze them alongside sequences from other amphibian species. However, due to large genome sizes and an abundance of repeat sequences, there are limited numbers of gene sequences available from amphibian species other than Xenopus. AmphiBase is a new genomic resource covering non-model amphibian species, based on public domain transcriptome data and computational methods developed during the X. laevis genome project. Here, I review the current status of AmphiBase, including amphibian species with available transcriptome data or biological samples, and describe the challenges of building a comprehensive amphibian genomic resource in the absence of genomes. This mini-review will be informative for researchers interested in functional genomic experiments using amphibian model organisms, such as Xenopus and axolotl, and will assist in interpretation of results implicating "orphan genes." Additionally, this study highlights an opportunity for researchers working on non-model amphibian species to collaborate in their future efforts and develop amphibian genomic resources as a community. © 2017 Wiley Periodicals, Inc.

Asymmetric histone modifications between the original and derived loci of human segmental duplications

PubMed Central

Zheng, Deyou

2008-01-01

Background Sequencing and annotation of several mammalian genomes have revealed that segmental duplications are a common architectural feature of primate genomes; in fact, about 5% of the human genome is composed of large blocks of interspersed segmental duplications. These segmental duplications have been implicated in genomic copy-number variation, gene novelty, and various genomic disorders. However, the molecular processes involved in the evolution and regulation of duplicated sequences remain largely unexplored. Results In this study, the profile of about 20 histone modifications within human segmental duplications was characterized using high-resolution, genome-wide data derived from a ChIP-Seq study. The analysis demonstrates that derivative loci of segmental duplications often differ significantly from the original with respect to many histone methylations. Further investigation showed that genes are present three times more frequently in the original than in the derivative, whereas pseudogenes exhibit the opposite trend. These asymmetries tend to increase with the age of segmental duplications. The uneven distribution of genes and pseudogenes does not, however, fully account for the asymmetry in the profile of histone modifications. Conclusion The first systematic analysis of histone modifications between segmental duplications demonstrates that two seemingly 'identical' genomic copies are distinct in their epigenomic properties. Results here suggest that local chromatin environments may be implicated in the discrimination of derived copies of segmental duplications from their originals, leading to a biased pseudogenization of the new duplicates. The data also indicate that further exploration of the interactions between histone modification and sequence degeneration is necessary in order to understand the divergence of duplicated sequences. PMID:18598352

Genomic diversity of necrotic enteritis-associated strains of Clostridium perfringens: a review.

PubMed

Lacey, Jake A; Johanesen, Priscilla A; Lyras, Dena; Moore, Robert J

2016-06-01

The investigation of genomic variation between Clostridium perfringens isolates from poultry has been an important tool to enhance our understanding of the genetic basis of strain pathogenicity and the epidemiology of virulent and avirulent strains within the context of necrotic enteritis (NE). The earliest studies used whole genome profiling techniques such as pulsed-field gel electrophoresis to differentiate isolates and determine their relative levels of relatedness. DNA sequencing has been used to investigate genetic variation in (a) individual genes, such as those encoding the alpha and NetB toxins; (b) panels of housekeeping genes for multi-locus sequence typing and (c) most recently whole genome sequencing to build a more complete picture of genomic differences between isolates. Conclusions drawn from these studies include: differential carriage of large conjugative plasmids accounts for a large proportion of inter-strain differences; plasmid-encoded genes are more highly conserved than chromosomal genes, perhaps indicating a relatively recent origin for the plasmids; isolates from NE-affected birds fall into three distinct sequence-based clades while non-pathogenic isolates from healthy birds tend to be more genomically diverse. Overall, the NE causing strains are closely related to C. perfringens isolates from other birds and other diseases whereas the non-pathogenic poultry strains are generally more remotely related to either the pathogenic strains or the strains from other birds. Genomic analysis has indicated that genes in addition to netB are associated with NE pathogenic isolates. Collectively, this work has resulted in a deeper understanding of the pathogenesis of this important poultry disease.

Natural product discovery: past, present, and future.

PubMed

Katz, Leonard; Baltz, Richard H

2016-03-01

Microorganisms have provided abundant sources of natural products which have been developed as commercial products for human medicine, animal health, and plant crop protection. In the early years of natural product discovery from microorganisms (The Golden Age), new antibiotics were found with relative ease from low-throughput fermentation and whole cell screening methods. Later, molecular genetic and medicinal chemistry approaches were applied to modify and improve the activities of important chemical scaffolds, and more sophisticated screening methods were directed at target disease states. In the 1990s, the pharmaceutical industry moved to high-throughput screening of synthetic chemical libraries against many potential therapeutic targets, including new targets identified from the human genome sequencing project, largely to the exclusion of natural products, and discovery rates dropped dramatically. Nonetheless, natural products continued to provide key scaffolds for drug development. In the current millennium, it was discovered from genome sequencing that microbes with large genomes have the capacity to produce about ten times as many secondary metabolites as was previously recognized. Indeed, the most gifted actinomycetes have the capacity to produce around 30-50 secondary metabolites. With the precipitous drop in cost for genome sequencing, it is now feasible to sequence thousands of actinomycete genomes to identify the "biosynthetic dark matter" as sources for the discovery of new and novel secondary metabolites. Advances in bioinformatics, mass spectrometry, proteomics, transcriptomics, metabolomics and gene expression are driving the new field of microbial genome mining for applications in natural product discovery and development.

Haplotag: Software for Haplotype-Based Genotyping-by-Sequencing Analysis

PubMed Central

Tinker, Nicholas A.; Bekele, Wubishet A.; Hattori, Jiro

2016-01-01

Genotyping-by-sequencing (GBS), and related methods, are based on high-throughput short-read sequencing of genomic complexity reductions followed by discovery of single nucleotide polymorphisms (SNPs) within sequence tags. This provides a powerful and economical approach to whole-genome genotyping, facilitating applications in genomics, diversity analysis, and molecular breeding. However, due to the complexity of analyzing large data sets, applications of GBS may require substantial time, expertise, and computational resources. Haplotag, the novel GBS software described here, is freely available, and operates with minimal user-investment on widely available computer platforms. Haplotag is unique in fulfilling the following set of criteria: (1) operates without a reference genome; (2) can be used in a polyploid species; (3) provides a discovery mode, and a production mode; (4) discovers polymorphisms based on a model of tag-level haplotypes within sequenced tags; (5) reports SNPs as well as haplotype-based genotypes; and (6) provides an intuitive visual “passport” for each inferred locus. Haplotag is optimized for use in a self-pollinating plant species. PMID:26818073

Complete annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono).

PubMed

Miyoshi-Akiyama, Tohru; Satou, Kazuhito; Kato, Masako; Shiroma, Akino; Matsumura, Kazunori; Tamotsu, Hinako; Iwai, Hiroki; Teruya, Kuniko; Funatogawa, Keiji; Hirano, Takashi; Kirikae, Teruo

2015-01-01

We report the completely annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono), which is a used for virulence and/or immunization studies. The complete genome sequence of M. tuberculosis Kurono was determined with a length of 4,415,078 bp and a G+C content of 65.60%. The chromosome was shown to contain a total of 4,340 protein-coding genes, 53 tRNA genes, one transfer messenger RNA for all amino acids, and 1 rrn operon. Lineage analysis based on large sequence polymorphisms indicated that M. tuberculosis Kurono belongs to the Euro-American lineage (lineage 4). Phylogenetic analysis using whole genome sequences of M. tuberculosis Kurono in addition to 22 M. tuberculosis complex strains indicated that H37Rv is the closest relative of Kurono based on the results of phylogenetic analysis. These findings provide a basis for research using M. tuberculosis Kurono, especially in animal models. Copyright © 2014 Elsevier Ltd. All rights reserved.

Well-characterized sequence features of eukaryote genomes and implications for ab initio gene prediction.

PubMed

Huang, Ying; Chen, Shi-Yi; Deng, Feilong

2016-01-01

In silico analysis of DNA sequences is an important area of computational biology in the post-genomic era. Over the past two decades, computational approaches for ab initio prediction of gene structure from genome sequence alone have largely facilitated our understanding on a variety of biological questions. Although the computational prediction of protein-coding genes has already been well-established, we are also facing challenges to robustly find the non-coding RNA genes, such as miRNA and lncRNA. Two main aspects of ab initio gene prediction include the computed values for describing sequence features and used algorithm for training the discriminant function, and by which different combinations are employed into various bioinformatic tools. Herein, we briefly review these well-characterized sequence features in eukaryote genomes and applications to ab initio gene prediction. The main purpose of this article is to provide an overview to beginners who aim to develop the related bioinformatic tools.

Breaking the computational barriers of pairwise genome comparison.

PubMed

Torreno, Oscar; Trelles, Oswaldo

2015-08-11

Conventional pairwise sequence comparison software algorithms are being used to process much larger datasets than they were originally designed for. This can result in processing bottlenecks that limit software capabilities or prevent full use of the available hardware resources. Overcoming the barriers that limit the efficient computational analysis of large biological sequence datasets by retrofitting existing algorithms or by creating new applications represents a major challenge for the bioinformatics community. We have developed C libraries for pairwise sequence comparison within diverse architectures, ranging from commodity systems to high performance and cloud computing environments. Exhaustive tests were performed using different datasets of closely- and distantly-related sequences that span from small viral genomes to large mammalian chromosomes. The tests demonstrated that our solution is capable of generating high quality results with a linear-time response and controlled memory consumption, being comparable or faster than the current state-of-the-art methods. We have addressed the problem of pairwise and all-versus-all comparison of large sequences in general, greatly increasing the limits on input data size. The approach described here is based on a modular out-of-core strategy that uses secondary storage to avoid reaching memory limits during the identification of High-scoring Segment Pairs (HSPs) between the sequences under comparison. Software engineering concepts were applied to avoid intermediate result re-calculation, to minimise the performance impact of input/output (I/O) operations and to modularise the process, thus enhancing application flexibility and extendibility. Our computationally-efficient approach allows tasks such as the massive comparison of complete genomes, evolutionary event detection, the identification of conserved synteny blocks and inter-genome distance calculations to be performed more effectively.

The complete chloroplast genome of a medicinal plant Epimedium koreanum Nakai (Berberidaceae).

PubMed

Lee, Jung-Hoon; Kim, Kyunghee; Kim, Na-Rae; Lee, Sang-Choon; Yang, Tae-Jin; Kim, Young-Dong

2016-11-01

Epimedium koreanum is a perennial medicinal plant distributed in Eastern Asia. The complete chloroplast genome sequences of E. koreanum was obtained by de novo assembly using whole genome next-generation sequences. The chloroplast genome of E. koreanum was 157 218 bp in length and separated into four distinct regions such as large single copy region (89 600 bp), small single copy region (17 222 bp) and a pair of inverted repeat regions (25 198 bp). The genome contained a total of 112 genes including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that E. koreanum is most closely related to Berberis bealei, a traditional medicinal plant in the Berberidaceae family.

Divergent copies of the large inverted repeat in the chloroplast genomes of ulvophycean green algae.

PubMed

Turmel, Monique; Otis, Christian; Lemieux, Claude

2017-04-20

The chloroplast genomes of many algae and almost all land plants carry two identical copies of a large inverted repeat (IR) sequence that can pair for flip-flop recombination and undergo expansion/contraction. Although the IR has been lost multiple times during the evolution of the green algae, the underlying mechanisms are still largely unknown. A recent comparison of IR-lacking and IR-containing chloroplast genomes of chlorophytes from the Ulvophyceae (Ulotrichales) suggested that differential elimination of genes from the IR copies might lead to IR loss. To gain deeper insights into the evolutionary history of the chloroplast genome in the Ulvophyceae, we analyzed the genomes of Ignatius tetrasporus and Pseudocharacium americanum (Ignatiales, an order not previously sampled), Dangemannia microcystis (Oltmannsiellopsidales), Pseudoneochloris marina (Ulvales) and also Chamaetrichon capsulatum and Trichosarcina mucosa (Ulotrichales). Our comparison of these six chloroplast genomes with those previously reported for nine ulvophyceans revealed unsuspected variability. All newly examined genomes feature an IR, but remarkably, the copies of the IR present in the Ignatiales, Pseudoneochloris, and Chamaetrichon diverge in sequence, with the tRNA genes from the rRNA operon missing in one IR copy. The implications of this unprecedented finding for the mechanism of IR loss and flip-flop recombination are discussed.

[Efficient genome editing in human pluripotent stem cells through CRISPR/Cas9].

PubMed

Liu, Gai-gai; Li, Shuang; Wei, Yu-da; Zhang, Yong-xian; Ding, Qiu-rong

2015-11-01

The RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has offered a new platform for genome editing with high efficiency. Here, we report the use of CRISPR/Cas9 technology to target a specific genomic region in human pluripotent stem cells. We show that CRISPR/Cas9 can be used to disrupt a gene by introducing frameshift mutations to gene coding region; to knock in specific sequences (e.g. FLAG tag DNA sequence) to targeted genomic locus via homology directed repair; to induce large genomic deletion through dual-guide multiplex. Our results demonstrate the versatile application of CRISPR/Cas9 in stem cell genome editing, which can be widely utilized for functional studies of genes or genome loci in human pluripotent stem cells.

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

Genomewide Function Conservation and Phylogeny in the Herpesviridae

PubMed Central

Albà, M. Mar; Das, Rhiju; Orengo, Christine A.; Kellam, Paul

2001-01-01

The Herpesviridae are a large group of well-characterized double-stranded DNA viruses for which many complete genome sequences have been determined. We have extracted protein sequences from all predicted open reading frames of 19 herpesvirus genomes. Sequence comparison and protein sequence clustering methods have been used to construct herpesvirus protein homologous families. This resulted in 1692 proteins being clustered into 243 multiprotein families and 196 singleton proteins. Predicted functions were assigned to each homologous family based on genome annotation and published data and each family classified into seven broad functional groups. Phylogenetic profiles were constructed for each herpesvirus from the homologous protein families and used to determine conserved functions and genomewide phylogenetic trees. These trees agreed with molecular-sequence-derived trees and allowed greater insight into the phylogeny of ungulate and murine gammaherpesviruses. PMID:11156614

Molecular epidemiology of infectious laryngotracheitis: a review

USDA-ARS?s Scientific Manuscript database

Falconid herpesvirus type 1 (FHV-1) is the causative agent of falcon inclusion body disease, an acute, highly contagious disease of raptors. The complete nucleotide sequence of the genome of FHV-1 has been determined. The genome is arranged as a D-type genome with large inverted repeats flanking a ...

Whole genome resequencing of a laboratory-adapted Drosophila melanogaster population sample

PubMed Central

Gilks, William P.; Pennell, Tanya M.; Flis, Ilona; Webster, Matthew T.; Morrow, Edward H.

2016-01-01

As part of a study into the molecular genetics of sexually dimorphic complex traits, we used high-throughput sequencing to obtain data on genomic variation in an outbred laboratory-adapted fruit fly ( Drosophila melanogaster) population. We successfully resequenced the whole genome of 220 hemiclonal females that were heterozygous for the same Berkeley reference line genome (BDGP6/dm6), and a unique haplotype from the outbred base population (LH M). The use of a static and known genetic background enabled us to obtain sequences from whole-genome phased haplotypes. We used a BWA-Picard-GATK pipeline for mapping sequence reads to the dm6 reference genome assembly, at a median depth-of coverage of 31X, and have made the resulting data publicly-available in the NCBI Short Read Archive (Accession number SRP058502). We used Haplotype Caller to discover and genotype 1,726,931 small genomic variants (SNPs and indels, <200bp). Additionally we detected and genotyped 167 large structural variants (1-100Kb in size) using GenomeStrip/2.0. Sequence and genotype data are publicly-available at the corresponding NCBI databases: Short Read Archive, dbSNP and dbVar (BioProject PRJNA282591). We have also released the unfiltered genotype data, and the code and logs for data processing and summary statistics ( https://zenodo.org/communities/sussex_drosophila_sequencing/). PMID:27928499

Characterization of mango (Mangifera indica L.) transcriptome and chloroplast genome.

PubMed

Azim, M Kamran; Khan, Ishtaiq A; Zhang, Yong

2014-05-01

We characterized mango leaf transcriptome and chloroplast genome using next generation DNA sequencing. The RNA-seq output of mango transcriptome generated >12 million reads (total nucleotides sequenced >1 Gb). De novo transcriptome assembly generated 30,509 unigenes with lengths in the range of 300 to ≥3,000 nt and 67× depth of coverage. Blast searching against nonredundant nucleotide databases and several Viridiplantae genomic datasets annotated 24,593 mango unigenes (80% of total) and identified Citrus sinensis as closest neighbor of mango with 9,141 (37%) matched sequences. The annotation with gene ontology and Clusters of Orthologous Group terms categorized unigene sequences into 57 and 25 classes, respectively. More than 13,500 unigenes were assigned to 293 KEGG pathways. Besides major plant biology related pathways, KEGG based gene annotation pointed out active presence of an array of biochemical pathways involved in (a) biosynthesis of bioactive flavonoids, flavones and flavonols, (b) biosynthesis of terpenoids and lignins and (c) plant hormone signal transduction. The mango transcriptome sequences revealed 235 proteases belonging to five catalytic classes of proteolytic enzymes. The draft genome of mango chloroplast (cp) was obtained by a combination of Sanger and next generation sequencing. The draft mango cp genome size is 151,173 bp with a pair of inverted repeats of 27,093 bp separated by small and large single copy regions, respectively. Out of 139 genes in mango cp genome, 91 found to be protein coding. Sequence analysis revealed cp genome of C. sinensis as closest neighbor of mango. We found 51 short repeats in mango cp genome supposed to be associated with extensive rearrangements. This is the first report of transcriptome and chloroplast genome analysis of any Anacardiaceae family member.

The complete mitochondrial genome sequence of Eimeria innocua (Eimeriidae, Coccidia, Apicomplexa).

PubMed

Hafeez, Mian Abdul; Vrba, Vladimir; Barta, John Robert

2016-07-01

The complete mitochondrial genome of Eimeria innocua KR strain (Eimeriidae, Coccidia, Apicomplexa) was sequenced. This coccidium infects turkeys (Meleagris gallopavo), Bobwhite quails (Colinus virginianus), and Grey partridges (Perdix perdix). Genome organization and gene contents were comparable with other Eimeria spp. infecting galliform birds. The circular-mapping mt genome of E. innocua is 6247 bp in length with three protein-coding genes (cox1, cox3, and cytb), 19 gene fragments encoding large subunit (LSU) rRNA and 14 gene fragments encoding small subunit (SSU) rRNA. Like other Apicomplexa, no tRNA was encoded. The mitochondrial genome of E. innocua confirms its close phylogenetic affinities to Eimeria dispersa.

Continuous Influx of Genetic Material from Host to Virus Populations

PubMed Central

Gilbert, Clément; Peccoud, Jean; Chateigner, Aurélien; Moumen, Bouziane

2016-01-01

Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT) of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86) can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69) belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors. PMID:26829124

Continuous Influx of Genetic Material from Host to Virus Populations.

PubMed

Gilbert, Clément; Peccoud, Jean; Chateigner, Aurélien; Moumen, Bouziane; Cordaux, Richard; Herniou, Elisabeth A

2016-02-01

Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT) of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86) can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69) belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors.

Sequence analysis reveals genomic factors affecting EST-SSR primer performance and polymorphism

USDA-ARS?s Scientific Manuscript database

Search for simple sequence repeat (SSR) motifs and design of flanking primers in expressed sequence tag (EST) sequences can be easily done at a large scale using bioinformatics programs. However, failed amplification and/or detection, along with lack of polymorphism, is often seen among randomly sel...

Complete chloroplast genome sequence and comparative analysis of loblolly pine (Pinus taeda L.) with related species

PubMed Central

Khan, Abdul Latif; Khan, Muhammad Aaqil; Shahzad, Raheem; Lubna; Kang, Sang Mo; Al-Harrasi, Ahmed; Al-Rawahi, Ahmed; Lee, In-Jung

2018-01-01

Pinaceae, the largest family of conifers, has a diversified organization of chloroplast (cp) genomes with two typical highly reduced inverted repeats (IRs). In the current study, we determined the complete sequence of the cp genome of an economically and ecologically important conifer tree, the loblolly pine (Pinus taeda L.), using Illumina paired-end sequencing and compared the sequence with those of other pine species. The results revealed a genome size of 121,531 base pairs (bp) containing a pair of 830-bp IR regions, distinguished by a small single copy (42,258 bp) and large single copy (77,614 bp) region. The chloroplast genome of P. taeda encodes 120 genes, comprising 81 protein-coding genes, four ribosomal RNA genes, and 35 tRNA genes, with 151 randomly distributed microsatellites. Approximately 6 palindromic, 34 forward, and 22 tandem repeats were found in the P. taeda cp genome. Whole cp genome comparison with those of other Pinus species exhibited an overall high degree of sequence similarity, with some divergence in intergenic spacers. Higher and lower numbers of indels and single-nucleotide polymorphism substitutions were observed relative to P. contorta and P. monophylla, respectively. Phylogenomic analyses based on the complete genome sequence revealed that 60 shared genes generated trees with the same topologies, and P. taeda was closely related to P. contorta in the subgenus Pinus. Thus, the complete P. taeda genome provided valuable resources for population and evolutionary studies of gymnosperms and can be used to identify related species. PMID:29596414

Genotyping by Sequencing Using Specific Allelic Capture to Build a High-Density Genetic Map of Durum Wheat

PubMed Central

Holtz, Yan; Ardisson, Morgane; Ranwez, Vincent; Besnard, Alban; Leroy, Philippe; Poux, Gérard; Roumet, Pierre; Viader, Véronique; Santoni, Sylvain; David, Jacques

2016-01-01

Targeted sequence capture is a promising technology which helps reduce costs for sequencing and genotyping numerous genomic regions in large sets of individuals. Bait sequences are designed to capture specific alleles previously discovered in parents or reference populations. We studied a set of 135 RILs originating from a cross between an emmer cultivar (Dic2) and a recent durum elite cultivar (Silur). Six thousand sequence baits were designed to target Dic2 vs. Silur polymorphisms discovered in a previous RNAseq study. These baits were exposed to genomic DNA of the RIL population. Eighty percent of the targeted SNPs were recovered, 65% of which were of high quality and coverage. The final high density genetic map consisted of more than 3,000 markers, whose genetic and physical mapping were consistent with those obtained with large arrays. PMID:27171472

A bacterial genome in transition - an exceptional enrichment of IS elements but lack of evidence for recent transposition in the symbiont Amoebophilus asiaticus

PubMed Central

2011-01-01

Background Insertion sequence (IS) elements are important mediators of genome plasticity and are widespread among bacterial and archaeal genomes. The 1.88 Mbp genome of the obligate intracellular amoeba symbiont Amoebophilus asiaticus contains an unusually large number of transposase genes (n = 354; 23% of all genes). Results The transposase genes in the A. asiaticus genome can be assigned to 16 different IS elements termed ISCaa1 to ISCaa16, which are represented by 2 to 24 full-length copies, respectively. Despite this high IS element load, the A. asiaticus genome displays a GC skew pattern typical for most bacterial genomes, indicating that no major rearrangements have occurred recently. Additionally, the high sequence divergence of some IS elements, the high number of truncated IS element copies (n = 143), as well as the absence of direct repeats in most IS elements suggest that the IS elements of A. asiaticus are transpositionally inactive. Although we could show transcription of 13 IS elements, we did not find experimental evidence for transpositional activity, corroborating our results from sequence analyses. However, we detected contiguous transcripts between IS elements and their downstream genes at nine loci in the A. asiaticus genome, indicating that some IS elements influence the transcription of downstream genes, some of which might be important for host cell interaction. Conclusions Taken together, the IS elements in the A. asiaticus genome are currently in the process of degradation and largely represent reflections of the evolutionary past of A. asiaticus in which its genome was shaped by their activity. PMID:21943072

Complete Sequence and Analysis of Coconut Palm (Cocos nucifera) Mitochondrial Genome.

PubMed

Aljohi, Hasan Awad; Liu, Wanfei; Lin, Qiang; Zhao, Yuhui; Zeng, Jingyao; Alamer, Ali; Alanazi, Ibrahim O; Alawad, Abdullah O; Al-Sadi, Abdullah M; Hu, Songnian; Yu, Jun

2016-01-01

Coconut (Cocos nucifera L.), a member of the palm family (Arecaceae), is one of the most economically important crops in tropics, serving as an important source of food, drink, fuel, medicine, and construction material. Here we report an assembly of the coconut (C. nucifera, Oman local Tall cultivar) mitochondrial (mt) genome based on next-generation sequencing data. This genome, 678,653bp in length and 45.5% in GC content, encodes 72 proteins, 9 pseudogenes, 23 tRNAs, and 3 ribosomal RNAs. Within the assembly, we find that the chloroplast (cp) derived regions account for 5.07% of the total assembly length, including 13 proteins, 2 pseudogenes, and 11 tRNAs. The mt genome has a relatively large fraction of repeat content (17.26%), including both forward (tandem) and inverted (palindromic) repeats. Sequence variation analysis shows that the Ti/Tv ratio of the mt genome is lower as compared to that of the nuclear genome and neutral expectation. By combining public RNA-Seq data for coconut, we identify 734 RNA editing sites supported by at least two datasets. In summary, our data provides the second complete mt genome sequence in the family Arecaceae, essential for further investigations on mitochondrial biology of seed plants.

Dfam: a database of repetitive DNA based on profile hidden Markov models.

PubMed

Wheeler, Travis J; Clements, Jody; Eddy, Sean R; Hubley, Robert; Jones, Thomas A; Jurka, Jerzy; Smit, Arian F A; Finn, Robert D

2013-01-01

We present a database of repetitive DNA elements, called Dfam (http://dfam.janelia.org). Many genomes contain a large fraction of repetitive DNA, much of which is made up of remnants of transposable elements (TEs). Accurate annotation of TEs enables research into their biology and can shed light on the evolutionary processes that shape genomes. Identification and masking of TEs can also greatly simplify many downstream genome annotation and sequence analysis tasks. The commonly used TE annotation tools RepeatMasker and Censor depend on sequence homology search tools such as cross_match and BLAST variants, as well as Repbase, a collection of known TE families each represented by a single consensus sequence. Dfam contains entries corresponding to all Repbase TE entries for which instances have been found in the human genome. Each Dfam entry is represented by a profile hidden Markov model, built from alignments generated using RepeatMasker and Repbase. When used in conjunction with the hidden Markov model search tool nhmmer, Dfam produces a 2.9% increase in coverage over consensus sequence search methods on a large human benchmark, while maintaining low false discovery rates, and coverage of the full human genome is 54.5%. The website provides a collection of tools and data views to support improved TE curation and annotation efforts. Dfam is also available for download in flat file format or in the form of MySQL table dumps.

CanvasDB: a local database infrastructure for analysis of targeted- and whole genome re-sequencing projects

PubMed Central

Ameur, Adam; Bunikis, Ignas; Enroth, Stefan; Gyllensten, Ulf

2014-01-01

CanvasDB is an infrastructure for management and analysis of genetic variants from massively parallel sequencing (MPS) projects. The system stores SNP and indel calls in a local database, designed to handle very large datasets, to allow for rapid analysis using simple commands in R. Functional annotations are included in the system, making it suitable for direct identification of disease-causing mutations in human exome- (WES) or whole-genome sequencing (WGS) projects. The system has a built-in filtering function implemented to simultaneously take into account variant calls from all individual samples. This enables advanced comparative analysis of variant distribution between groups of samples, including detection of candidate causative mutations within family structures and genome-wide association by sequencing. In most cases, these analyses are executed within just a matter of seconds, even when there are several hundreds of samples and millions of variants in the database. We demonstrate the scalability of canvasDB by importing the individual variant calls from all 1092 individuals present in the 1000 Genomes Project into the system, over 4.4 billion SNPs and indels in total. Our results show that canvasDB makes it possible to perform advanced analyses of large-scale WGS projects on a local server. Database URL: https://github.com/UppsalaGenomeCenter/CanvasDB PMID:25281234

CanvasDB: a local database infrastructure for analysis of targeted- and whole genome re-sequencing projects.

PubMed

Ameur, Adam; Bunikis, Ignas; Enroth, Stefan; Gyllensten, Ulf

2014-01-01

CanvasDB is an infrastructure for management and analysis of genetic variants from massively parallel sequencing (MPS) projects. The system stores SNP and indel calls in a local database, designed to handle very large datasets, to allow for rapid analysis using simple commands in R. Functional annotations are included in the system, making it suitable for direct identification of disease-causing mutations in human exome- (WES) or whole-genome sequencing (WGS) projects. The system has a built-in filtering function implemented to simultaneously take into account variant calls from all individual samples. This enables advanced comparative analysis of variant distribution between groups of samples, including detection of candidate causative mutations within family structures and genome-wide association by sequencing. In most cases, these analyses are executed within just a matter of seconds, even when there are several hundreds of samples and millions of variants in the database. We demonstrate the scalability of canvasDB by importing the individual variant calls from all 1092 individuals present in the 1000 Genomes Project into the system, over 4.4 billion SNPs and indels in total. Our results show that canvasDB makes it possible to perform advanced analyses of large-scale WGS projects on a local server. Database URL: https://github.com/UppsalaGenomeCenter/CanvasDB. © The Author(s) 2014. Published by Oxford University Press.

Developing eThread pipeline using SAGA-pilot abstraction for large-scale structural bioinformatics.

PubMed

Ragothaman, Anjani; Boddu, Sairam Chowdary; Kim, Nayong; Feinstein, Wei; Brylinski, Michal; Jha, Shantenu; Kim, Joohyun

2014-01-01

While most of computational annotation approaches are sequence-based, threading methods are becoming increasingly attractive because of predicted structural information that could uncover the underlying function. However, threading tools are generally compute-intensive and the number of protein sequences from even small genomes such as prokaryotes is large typically containing many thousands, prohibiting their application as a genome-wide structural systems biology tool. To leverage its utility, we have developed a pipeline for eThread--a meta-threading protein structure modeling tool, that can use computational resources efficiently and effectively. We employ a pilot-based approach that supports seamless data and task-level parallelism and manages large variation in workload and computational requirements. Our scalable pipeline is deployed on Amazon EC2 and can efficiently select resources based upon task requirements. We present runtime analysis to characterize computational complexity of eThread and EC2 infrastructure. Based on results, we suggest a pathway to an optimized solution with respect to metrics such as time-to-solution or cost-to-solution. Our eThread pipeline can scale to support a large number of sequences and is expected to be a viable solution for genome-scale structural bioinformatics and structure-based annotation, particularly, amenable for small genomes such as prokaryotes. The developed pipeline is easily extensible to other types of distributed cyberinfrastructure.

Developing eThread Pipeline Using SAGA-Pilot Abstraction for Large-Scale Structural Bioinformatics

PubMed Central

Ragothaman, Anjani; Feinstein, Wei; Jha, Shantenu; Kim, Joohyun

2014-01-01

While most of computational annotation approaches are sequence-based, threading methods are becoming increasingly attractive because of predicted structural information that could uncover the underlying function. However, threading tools are generally compute-intensive and the number of protein sequences from even small genomes such as prokaryotes is large typically containing many thousands, prohibiting their application as a genome-wide structural systems biology tool. To leverage its utility, we have developed a pipeline for eThread—a meta-threading protein structure modeling tool, that can use computational resources efficiently and effectively. We employ a pilot-based approach that supports seamless data and task-level parallelism and manages large variation in workload and computational requirements. Our scalable pipeline is deployed on Amazon EC2 and can efficiently select resources based upon task requirements. We present runtime analysis to characterize computational complexity of eThread and EC2 infrastructure. Based on results, we suggest a pathway to an optimized solution with respect to metrics such as time-to-solution or cost-to-solution. Our eThread pipeline can scale to support a large number of sequences and is expected to be a viable solution for genome-scale structural bioinformatics and structure-based annotation, particularly, amenable for small genomes such as prokaryotes. The developed pipeline is easily extensible to other types of distributed cyberinfrastructure. PMID:24995285

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

Next-generation sequencing of cancer genomes: back to the future

PubMed Central

Walter, Matthew J; Graubert, Timothy A; DiPersio, John F; Mardis, Elaine R; Wilson, Richard K; Ley, Timothy J

2010-01-01

The systematic karyotyping of bone marrow cells was the first genomic approach used to personalize therapy for patients with leukemia. The paradigm established by cytogenetic studies in leukemia (from gene discovery to therapeutic intervention) now has the potential to be rapidly extended with the use of whole-genome sequencing approaches for cancer, which are now possible. We are now entering a period of exponential growth in cancer gene discovery that will provide many novel therapeutic targets for a large number of cancer types. Establishing the pathogenetic relevance of individual mutations is a major challenge that must be solved. However, after thousands of cancer genomes have been sequenced, the genetic rules of cancer will become known and new approaches for diagnosis, risk stratification and individualized treatment of cancer patients will surely follow. PMID:20161678

How to kill the honey bee larva: genomic potential and virulence mechanisms of Paenibacillus larvae.

PubMed

Djukic, Marvin; Brzuszkiewicz, Elzbieta; Fünfhaus, Anne; Voss, Jörn; Gollnow, Kathleen; Poppinga, Lena; Liesegang, Heiko; Garcia-Gonzalez, Eva; Genersch, Elke; Daniel, Rolf

2014-01-01

Paenibacillus larvae, a Gram positive bacterial pathogen, causes American Foulbrood (AFB), which is the most serious infectious disease of honey bees. In order to investigate the genomic potential of P. larvae, two strains belonging to two different genotypes were sequenced and used for comparative genome analysis. The complete genome sequence of P. larvae strain DSM 25430 (genotype ERIC II) consisted of 4,056,006 bp and harbored 3,928 predicted protein-encoding genes. The draft genome sequence of P. larvae strain DSM 25719 (genotype ERIC I) comprised 4,579,589 bp and contained 4,868 protein-encoding genes. Both strains harbored a 9.7 kb plasmid and encoded a large number of virulence-associated proteins such as toxins and collagenases. In addition, genes encoding large multimodular enzymes producing nonribosomally peptides or polyketides were identified. In the genome of strain DSM 25719 seven toxin associated loci were identified and analyzed. Five of them encoded putatively functional toxins. The genome of strain DSM 25430 harbored several toxin loci that showed similarity to corresponding loci in the genome of strain DSM 25719, but were non-functional due to point mutations or disruption by transposases. Although both strains cause AFB, significant differences between the genomes were observed including genome size, number and composition of transposases, insertion elements, predicted phage regions, and strain-specific island-like regions. Transposases, integrases and recombinases are important drivers for genome plasticity. A total of 390 and 273 mobile elements were found in strain DSM 25430 and strain DSM 25719, respectively. Comparative genomics of both strains revealed acquisition of virulence factors by horizontal gene transfer and provided insights into evolution and pathogenicity.

Next-Generation Sequencing: The Translational Medicine Approach from “Bench to Bedside to Population”

PubMed Central

Beigh, Mohammad Muzafar

2016-01-01

Humans have predicted the relationship between heredity and diseases for a long time. Only in the beginning of the last century, scientists begin to discover the connotations between different genes and disease phenotypes. Recent trends in next-generation sequencing (NGS) technologies have brought a great momentum in biomedical research that in turn has remarkably augmented our basic understanding of human biology and its associated diseases. State-of-the-art next generation biotechnologies have started making huge strides in our current understanding of mechanisms of various chronic illnesses like cancers, metabolic disorders, neurodegenerative anomalies, etc. We are experiencing a renaissance in biomedical research primarily driven by next generation biotechnologies like genomics, transcriptomics, proteomics, metabolomics, lipidomics etc. Although genomic discoveries are at the forefront of next generation omics technologies, however, their implementation into clinical arena had been painstakingly slow mainly because of high reaction costs and unavailability of requisite computational tools for large-scale data analysis. However rapid innovations and steadily lowering cost of sequence-based chemistries along with the development of advanced bioinformatics tools have lately prompted launching and implementation of large-scale massively parallel genome sequencing programs in different fields ranging from medical genetics, infectious biology, agriculture sciences etc. Recent advances in large-scale omics-technologies is bringing healthcare research beyond the traditional “bench to bedside” approach to more of a continuum that will include improvements, in public healthcare and will be primarily based on predictive, preventive, personalized, and participatory medicine approach (P4). Recent large-scale research projects in genetic and infectious disease biology have indicated that massively parallel whole-genome/whole-exome sequencing, transcriptome analysis, and other functional genomic tools can reveal large number of unique functional elements and/or markers that otherwise would be undetected by traditional sequencing methodologies. Therefore, latest trends in the biomedical research is giving birth to the new branch in medicine commonly referred to as personalized and/or precision medicine. Developments in the post-genomic era are believed to completely restructure the present clinical pattern of disease prevention and treatment as well as methods of diagnosis and prognosis. The next important step in the direction of the precision/personalized medicine approach should be its early adoption in clinics for future medical interventions. Consequently, in coming year’s next generation biotechnologies will reorient medical practice more towards disease prediction and prevention approaches rather than curing them at later stages of their development and progression, even at wider population level(s) for general public healthcare system. PMID:28930123

Large-Scale Genomic Analysis of Codon Usage in Dengue Virus and Evaluation of Its Phylogenetic Dependence

PubMed Central

Lara-Ramírez, Edgar E.; Salazar, Ma Isabel; López-López, María de Jesús; Salas-Benito, Juan Santiago; Sánchez-Varela, Alejandro

2014-01-01

The increasing number of dengue virus (DENV) genome sequences available allows identifying the contributing factors to DENV evolution. In the present study, the codon usage in serotypes 1–4 (DENV1–4) has been explored for 3047 sequenced genomes using different statistics methods. The correlation analysis of total GC content (GC) with GC content at the three nucleotide positions of codons (GC1, GC2, and GC3) as well as the effective number of codons (ENC, ENCp) versus GC3 plots revealed mutational bias and purifying selection pressures as the major forces influencing the codon usage, but with distinct pressure on specific nucleotide position in the codon. The correspondence analysis (CA) and clustering analysis on relative synonymous codon usage (RSCU) within each serotype showed similar clustering patterns to the phylogenetic analysis of nucleotide sequences for DENV1–4. These clustering patterns are strongly related to the virus geographic origin. The phylogenetic dependence analysis also suggests that stabilizing selection acts on the codon usage bias. Our analysis of a large scale reveals new feature on DENV genomic evolution. PMID:25136631

The History of Bordetella pertussis Genome Evolution Includes Structural Rearrangement

PubMed Central

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

2017-01-01

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

Complete genome sequence of Nocardiopsis dassonvillei type strain (IMRU 509T)

PubMed Central

Sun, Hui; Lapidus, Alla; Nolan, Matt; Lucas, Susan; Del Rio, Tijana Glavina; Tice, Hope; Cheng, Jan-Fang; Tapia, Roxane; Han, Cliff; Goodwin, Lynne; Pitluck, Sam; Pagani, Ioanna; Ivanova, Natalia; Mavromatis, Konstantinos; Mikhailova, Natalia; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Jeffries, Cynthia D.; Djao, Olivier Duplex Ngatchou; Rohde, Manfred; Sikorski, Johannes; Göker, Markus; Woyke, Tanja; Bristow, James; Eisen, Jonathan A.; Markowitz, Victor; Hugenholtz, Philip; Kyrpides, Nikos C.; Klenk, Hans-Peter

2010-01-01

Nocardiopsis dassonvillei (Brocq-Rousseau 1904) Meyer 1976 is the type species of the genus Nocardiopsis, which in turn is the type genus of the family Nocardiopsaceae. This species is of interest because of its ecological versatility. Members of N. dassonvillei have been isolated from a large variety of natural habitats such as soil and marine sediments, from different plant and animal materials as well as from human patients. Moreover, representatives of the genus Nocardiopsis participate actively in biopolymer degradation. This is the first complete genome sequence in the family Nocardiopsaceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,543,312 bp long genome consist of a 5.77 Mbp chromosome and a 0.78 Mbp plasmid and with its 5,570 protein-coding and 77 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project. PMID:21304737

Fine definition of the pedigree haplotypes of closely related rice cultivars by means of genome-wide discovery of single-nucleotide polymorphisms.

PubMed

Yamamoto, Toshio; Nagasaki, Hideki; Yonemaru, Jun-ichi; Ebana, Kaworu; Nakajima, Maiko; Shibaya, Taeko; Yano, Masahiro

2010-04-27

To create useful gene combinations in crop breeding, it is necessary to clarify the dynamics of the genome composition created by breeding practices. A large quantity of single-nucleotide polymorphism (SNP) data is required to permit discrimination of chromosome segments among modern cultivars, which are genetically related. Here, we used a high-throughput sequencer to conduct whole-genome sequencing of an elite Japanese rice cultivar, Koshihikari, which is closely related to Nipponbare, whose genome sequencing has been completed. Then we designed a high-throughput typing array based on the SNP information by comparison of the two sequences. Finally, we applied this array to analyze historical representative rice cultivars to understand the dynamics of their genome composition. The total 5.89-Gb sequence for Koshihikari, equivalent to 15.7 x the entire rice genome, was mapped using the Pseudomolecules 4.0 database for Nipponbare. The resultant Koshihikari genome sequence corresponded to 80.1% of the Nipponbare sequence and led to the identification of 67,051 SNPs. A high-throughput typing array consisting of 1917 SNP sites distributed throughout the genome was designed to genotype 151 representative Japanese cultivars that have been grown during the past 150 years. We could identify the ancestral origin of the pedigree haplotypes in 60.9% of the Koshihikari genome and 18 consensus haplotype blocks which are inherited from traditional landraces to current improved varieties. Moreover, it was predicted that modern breeding practices have generally decreased genetic diversity Detection of genome-wide SNPs by both high-throughput sequencer and typing array made it possible to evaluate genomic composition of genetically related rice varieties. With the aid of their pedigree information, we clarified the dynamics of chromosome recombination during the historical rice breeding process. We also found several genomic regions decreasing genetic diversity which might be caused by a recent human selection in rice breeding. The definition of pedigree haplotypes by means of genome-wide SNPs will facilitate next-generation breeding of rice and other crops.

Cloud-based adaptive exon prediction for DNA analysis.

PubMed

Putluri, Srinivasareddy; Zia Ur Rahman, Md; Fathima, Shaik Yasmeen

2018-02-01

Cloud computing offers significant research and economic benefits to healthcare organisations. Cloud services provide a safe place for storing and managing large amounts of such sensitive data. Under conventional flow of gene information, gene sequence laboratories send out raw and inferred information via Internet to several sequence libraries. DNA sequencing storage costs will be minimised by use of cloud service. In this study, the authors put forward a novel genomic informatics system using Amazon Cloud Services, where genomic sequence information is stored and accessed for processing. True identification of exon regions in a DNA sequence is a key task in bioinformatics, which helps in disease identification and design drugs. Three base periodicity property of exons forms the basis of all exon identification techniques. Adaptive signal processing techniques found to be promising in comparison with several other methods. Several adaptive exon predictors (AEPs) are developed using variable normalised least mean square and its maximum normalised variants to reduce computational complexity. Finally, performance evaluation of various AEPs is done based on measures such as sensitivity, specificity and precision using various standard genomic datasets taken from National Center for Biotechnology Information genomic sequence database.

[Comparative analysis of clustered regularly interspaced short palindromic repeats (CRISPRs) loci in the genomes of halophilic archaea].

PubMed

Zhang, Fan; Zhang, Bing; Xiang, Hua; Hu, Songnian

2009-11-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a widespread system that provides acquired resistance against phages in bacteria and archaea. Here we aim to genome-widely analyze the CRISPR in extreme halophilic archaea, of which the whole genome sequences are available at present time. We used bioinformatics methods including alignment, conservation analysis, GC content and RNA structure prediction to analyze the CRISPR structures of 7 haloarchaeal genomes. We identified the CRISPR structures in 5 halophilic archaea and revealed a conserved palindromic motif in the flanking regions of these CRISPR structures. In addition, we found that the repeat sequences of large CRISPR structures in halophilic archaea were greatly conserved, and two types of predicted RNA secondary structures derived from the repeat sequences were likely determined by the fourth base of the repeat sequence. Our results support the proposal that the leader sequence may function as recognition site by having palindromic structures in flanking regions, and the stem-loop secondary structure formed by repeat sequences may function in mediating the interaction between foreign genetic elements and CAS-encoded proteins.

Whole exome sequencing: a state-of-the-art approach for defining (and exploring!) genetic landscapes in pediatric nephrology.

PubMed

Gulati, Ashima; Somlo, Stefan

2018-05-01

The genesis of whole exome sequencing as a powerful tool for detailing the protein coding sequence of the human genome was conceptualized based on the availability of next-generation sequencing technology and knowledge of the human reference genome. The field of pediatric nephrology enriched with molecularly unsolved phenotypes is allowing the clinical and research application of whole exome sequencing to enable novel gene discovery and provide amendment of phenotypic misclassification. Recent studies in the field have informed us that newer high-throughput sequencing techniques are likely to be of high yield when applied in conjunction with conventional genomic approaches such as linkage analysis and other strategies used to focus subsequent analysis. They have also emphasized the need for the validation of novel genetic findings in large collaborative cohorts and the production of robust corroborative biological data. The well-structured application of comprehensive genomic testing in clinical and research arenas will hopefully continue to advance patient care and precision medicine, but does call for attention to be paid to its integrated challenges.

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

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

Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.

PubMed

Oyola, Samuel O; Otto, Thomas D; Gu, Yong; Maslen, Gareth; Manske, Magnus; Campino, Susana; Turner, Daniel J; Macinnis, Bronwyn; Kwiatkowski, Dominic P; Swerdlow, Harold P; Quail, Michael A

2012-01-03

Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

Using Full Genomic Information to Predict Disease: Breaking Down the Barriers Between Complex and Mendelian Diseases.

PubMed

Jordan, Daniel M; Do, Ron

2018-04-11

While sequence-based genetic tests have long been available for specific loci, especially for Mendelian disease, the rapidly falling costs of genome-wide genotyping arrays, whole-exome sequencing, and whole-genome sequencing are moving us toward a future where full genomic information might inform the prognosis and treatment of a variety of diseases, including complex disease. Similarly, the availability of large populations with full genomic information has enabled new insights about the etiology and genetic architecture of complex disease. Insights from the latest generation of genomic studies suggest that our categorization of diseases as complex may conceal a wide spectrum of genetic architectures and causal mechanisms that ranges from Mendelian forms of complex disease to complex regulatory structures underlying Mendelian disease. Here, we review these insights, along with advances in the prediction of disease risk and outcomes from full genomic information. Expected final online publication date for the Annual Review of Genomics and Human Genetics Volume 19 is August 31, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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

PubMed Central

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

2012-01-01

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

An Expressed Sequence Tag (EST)-enriched genetic map of turbot (Scophthalmus maximus): a useful framework for comparative genomics across model and farmed teleosts

PubMed Central

2012-01-01

Background The turbot (Scophthalmus maximus) is a relevant species in European aquaculture. The small turbot genome provides a source for genomics strategies to use in order to understand the genetic basis of productive traits, particularly those related to sex, growth and pathogen resistance. Genetic maps represent essential genomic screening tools allowing to localize quantitative trait loci (QTL) and to identify candidate genes through comparative mapping. This information is the backbone to develop marker-assisted selection (MAS) programs in aquaculture. Expressed sequenced tag (EST) resources have largely increased in turbot, thus supplying numerous type I markers suitable for extending the previous linkage map, which was mostly based on anonymous loci. The aim of this study was to construct a higher-resolution turbot genetic map using EST-linked markers, which will turn out to be useful for comparative mapping studies. Results A consensus gene-enriched genetic map of the turbot was constructed using 463 SNP and microsatellite markers in nine reference families. This map contains 438 markers, 180 EST-linked, clustered at 24 linkage groups. Linkage and comparative genomics evidences suggested additional linkage group fusions toward the consolidation of turbot map according to karyotype information. The linkage map showed a total length of 1402.7 cM with low average intermarker distance (3.7 cM; ~2 Mb). A global 1.6:1 female-to-male recombination frequency (RF) ratio was observed, although largely variable among linkage groups and chromosome regions. Comparative sequence analysis revealed large macrosyntenic patterns against model teleost genomes, significant hits decreasing from stickleback (54%) to zebrafish (20%). Comparative mapping supported particular chromosome rearrangements within Acanthopterygii and aided to assign unallocated markers to specific turbot linkage groups. Conclusions The new gene-enriched high-resolution turbot map represents a useful genomic tool for QTL identification, positional cloning strategies, and future genome assembling. This map showed large synteny conservation against model teleost genomes. Comparative genomics and data mining from landmarks will provide straightforward access to candidate genes, which will be the basis for genetic breeding programs and evolutionary studies in this species. PMID:22747677