QUAST: quality assessment tool for genome assemblies.

PubMed

Gurevich, Alexey; Saveliev, Vladislav; Vyahhi, Nikolay; Tesler, Glenn

2013-04-15

Limitations of genome sequencing techniques have led to dozens of assembly algorithms, none of which is perfect. A number of methods for comparing assemblers have been developed, but none is yet a recognized benchmark. Further, most existing methods for comparing assemblies are only applicable to new assemblies of finished genomes; the problem of evaluating assemblies of previously unsequenced species has not been adequately considered. Here, we present QUAST-a quality assessment tool for evaluating and comparing genome assemblies. This tool improves on leading assembly comparison software with new ideas and quality metrics. QUAST can evaluate assemblies both with a reference genome, as well as without a reference. QUAST produces many reports, summary tables and plots to help scientists in their research and in their publications. In this study, we used QUAST to compare several genome assemblers on three datasets. QUAST tables and plots for all of them are available in the Supplementary Material, and interactive versions of these reports are on the QUAST website. http://bioinf.spbau.ru/quast . Supplementary data are available at Bioinformatics online.

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.

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly

PubMed Central

Schneider, Valerie A.; Graves-Lindsay, Tina; Howe, Kerstin; Bouk, Nathan; Chen, Hsiu-Chuan; Kitts, Paul A.; Murphy, Terence D.; Pruitt, Kim D.; Thibaud-Nissen, Françoise; Albracht, Derek; Fulton, Robert S.; Kremitzki, Milinn; Magrini, Vincent; Markovic, Chris; McGrath, Sean; Steinberg, Karyn Meltz; Auger, Kate; Chow, William; Collins, Joanna; Harden, Glenn; Hubbard, Timothy; Pelan, Sarah; Simpson, Jared T.; Threadgold, Glen; Torrance, James; Wood, Jonathan M.; Clarke, Laura; Koren, Sergey; Boitano, Matthew; Peluso, Paul; Li, Heng; Chin, Chen-Shan; Phillippy, Adam M.; Durbin, Richard; Wilson, Richard K.; Flicek, Paul; Eichler, Evan E.; Church, Deanna M.

2017-01-01

The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health. PMID:28396521

Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing.

PubMed

Morse, Alison M; Calabro, Kaitlyn R; Fear, Justin M; Bloom, David C; McIntyre, Lauren M

2017-08-16

High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.

Genome sequencing of bacteria: sequencing, de novo assembly and rapid analysis using open source tools.

PubMed

Kisand, Veljo; Lettieri, Teresa

2013-04-01

De novo genome sequencing of previously uncharacterized microorganisms has the potential to open up new frontiers in microbial genomics by providing insight into both functional capabilities and biodiversity. Until recently, Roche 454 pyrosequencing was the NGS method of choice for de novo assembly because it generates hundreds of thousands of long reads (<450 bps), which are presumed to aid in the analysis of uncharacterized genomes. The array of tools for processing NGS data are increasingly free and open source and are often adopted for both their high quality and role in promoting academic freedom. The error rate of pyrosequencing the Alcanivorax borkumensis genome was such that thousands of insertions and deletions were artificially introduced into the finished genome. Despite a high coverage (~30 fold), it did not allow the reference genome to be fully mapped. Reads from regions with errors had low quality, low coverage, or were missing. The main defect of the reference mapping was the introduction of artificial indels into contigs through lower than 100% consensus and distracting gene calling due to artificial stop codons. No assembler was able to perform de novo assembly comparable to reference mapping. Automated annotation tools performed similarly on reference mapped and de novo draft genomes, and annotated most CDSs in the de novo assembled draft genomes. Free and open source software (FOSS) tools for assembly and annotation of NGS data are being developed rapidly to provide accurate results with less computational effort. Usability is not high priority and these tools currently do not allow the data to be processed without manual intervention. Despite this, genome assemblers now readily assemble medium short reads into long contigs (>97-98% genome coverage). A notable gap in pyrosequencing technology is the quality of base pair calling and conflicting base pairs between single reads at the same nucleotide position. Regardless, using draft whole genomes that are not finished and remain fragmented into tens of contigs allows one to characterize unknown bacteria with modest effort.

QUAST: quality assessment tool for genome assemblies

PubMed Central

Gurevich, Alexey; Saveliev, Vladislav; Vyahhi, Nikolay; Tesler, Glenn

2013-01-01

Summary: Limitations of genome sequencing techniques have led to dozens of assembly algorithms, none of which is perfect. A number of methods for comparing assemblers have been developed, but none is yet a recognized benchmark. Further, most existing methods for comparing assemblies are only applicable to new assemblies of finished genomes; the problem of evaluating assemblies of previously unsequenced species has not been adequately considered. Here, we present QUAST—a quality assessment tool for evaluating and comparing genome assemblies. This tool improves on leading assembly comparison software with new ideas and quality metrics. QUAST can evaluate assemblies both with a reference genome, as well as without a reference. QUAST produces many reports, summary tables and plots to help scientists in their research and in their publications. In this study, we used QUAST to compare several genome assemblers on three datasets. QUAST tables and plots for all of them are available in the Supplementary Material, and interactive versions of these reports are on the QUAST website. Availability: http://bioinf.spbau.ru/quast Contact: gurevich@bioinf.spbau.ru Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23422339

Construction of Red Fox Chromosomal Fragments from the Short-Read Genome Assembly.

PubMed

Rando, Halie M; Farré, Marta; Robson, Michael P; Won, Naomi B; Johnson, Jennifer L; Buch, Ronak; Bastounes, Estelle R; Xiang, Xueyan; Feng, Shaohong; Liu, Shiping; Xiong, Zijun; Kim, Jaebum; Zhang, Guojie; Trut, Lyudmila N; Larkin, Denis M; Kukekova, Anna V

2018-06-20

The genome of a red fox ( Vulpes vulpes ) was recently sequenced and assembled using next-generation sequencing (NGS). The assembly is of high quality, with 94X coverage and a scaffold N50 of 11.8 Mbp, but is split into 676,878 scaffolds, some of which are likely to contain assembly errors. Fragmentation and misassembly hinder accurate gene prediction and downstream analysis such as the identification of loci under selection. Therefore, assembly of the genome into chromosome-scale fragments was an important step towards developing this genomic model. Scaffolds from the assembly were aligned to the dog reference genome and compared to the alignment of an outgroup genome (cat) against the dog to identify syntenic sequences among species. The program Reference-Assisted Chromosome Assembly (RACA) then integrated the comparative alignment with the mapping of the raw sequencing reads generated during assembly against the fox scaffolds. The 128 sequence fragments RACA assembled were compared to the fox meiotic linkage map to guide the construction of 40 chromosomal fragments. This computational approach to assembly was facilitated by prior research in comparative mammalian genomics, and the continued improvement of the red fox genome can in turn offer insight into canid and carnivore chromosome evolution. This assembly is also necessary for advancing genetic research in foxes and other canids.

Using optical mapping data for the improvement of vertebrate genome assemblies.

PubMed

Howe, Kerstin; Wood, Jonathan M D

2015-01-01

Optical mapping is a technology that gathers long-range information on genome sequences similar to ordered restriction digest maps. Because it is not subject to cloning, amplification, hybridisation or sequencing bias, it is ideally suited to the improvement of fragmented genome assemblies that can no longer be improved by classical methods. In addition, its low cost and rapid turnaround make it equally useful during the scaffolding process of de novo assembly from high throughput sequencing reads. We describe how optical mapping has been used in practice to produce high quality vertebrate genome assemblies. In particular, we detail the efforts undertaken by the Genome Reference Consortium (GRC), which maintains the reference genomes for human, mouse, zebrafish and chicken, and uses different optical mapping platforms for genome curation.

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly.

PubMed

Schneider, Valerie A; Graves-Lindsay, Tina; Howe, Kerstin; Bouk, Nathan; Chen, Hsiu-Chuan; Kitts, Paul A; Murphy, Terence D; Pruitt, Kim D; Thibaud-Nissen, Françoise; Albracht, Derek; Fulton, Robert S; Kremitzki, Milinn; Magrini, Vincent; Markovic, Chris; McGrath, Sean; Steinberg, Karyn Meltz; Auger, Kate; Chow, William; Collins, Joanna; Harden, Glenn; Hubbard, Timothy; Pelan, Sarah; Simpson, Jared T; Threadgold, Glen; Torrance, James; Wood, Jonathan M; Clarke, Laura; Koren, Sergey; Boitano, Matthew; Peluso, Paul; Li, Heng; Chin, Chen-Shan; Phillippy, Adam M; Durbin, Richard; Wilson, Richard K; Flicek, Paul; Eichler, Evan E; Church, Deanna M

2017-05-01

The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health. © 2017 Schneider et al.; Published by Cold Spring Harbor Laboratory Press.

Effective de novo assembly of fish genome using haploid larvae.

PubMed

Iwasaki, Yuki; Nishiki, Issei; Nakamura, Yoji; Yasuike, Motoshige; Kai, Wataru; Nomura, Kazuharu; Yoshida, Kazunori; Nomura, Yousuke; Fujiwara, Atushi; Kobayashi, Takanori; Ototake, Mitsuru

2016-02-01

Recent improvements in next-generation sequencing technology have made it possible to do whole genome sequencing, on even non-model eukaryote species with no available reference genomes. However, de novo assembly of diploid genomes is still a big challenge because of allelic variation. The aim of this study was to determine the feasibility of utilizing the genome of haploid fish larvae for de novo assembly of whole-genome sequences. We compared the efficiency of assembly using the haploid genome of yellowtail (Seriola quinqueradiata) with that using the diploid genome obtained from the dam. De novo assembly from the haploid and the diploid sequence reads (100 million reads per each datasets) generated by the Ion Proton sequencer (200 bp) was done under two different assembly algorithms, namely overlap-layout-consensus (OLC) and de Bruijn graph (DBG). This revealed that the assembly of the haploid genome significantly reduced (approximately 22% for OLC, 9% for DBG) the total number of contigs (with longer average and N50 contig lengths) when compared to the diploid genome assembly. The haploid assembly also improved the quality of the scaffolds by reducing the number of regions with unassigned nucleotides (Ns) (total length of Ns; 45,331,916 bp for haploids and 67,724,360 bp for diploids) in OLC-based assemblies. It appears clear that the haploid genome assembly is better because the allelic variation in the diploid genome disrupts the extension of contigs during the assembly process. Our results indicate that utilizing the genome of haploid larvae leads to a significant improvement in the de novo assembly process, thus providing a novel strategy for the construction of reference genomes from non-model diploid organisms such as fish. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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

Improved maize reference genome with single-molecule technologies.

PubMed

Jiao, Yinping; Peluso, Paul; Shi, Jinghua; Liang, Tiffany; Stitzer, Michelle C; Wang, Bo; Campbell, Michael S; Stein, Joshua C; Wei, Xuehong; Chin, Chen-Shan; Guill, Katherine; Regulski, Michael; Kumari, Sunita; Olson, Andrew; Gent, Jonathan; Schneider, Kevin L; Wolfgruber, Thomas K; May, Michael R; Springer, Nathan M; Antoniou, Eric; McCombie, W Richard; Presting, Gernot G; McMullen, Michael; Ross-Ibarra, Jeffrey; Dawe, R Kelly; Hastie, Alex; Rank, David R; Ware, Doreen

2017-06-22

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

The value of new genome references.

PubMed

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

2017-09-15

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

Assembly of highly repetitive genomes using short reads: the genome of discrete typing unit III Trypanosoma cruzi strain 231.

PubMed

Baptista, Rodrigo P; Reis-Cunha, Joao Luis; DeBarry, Jeremy D; Chiari, Egler; Kissinger, Jessica C; Bartholomeu, Daniella C; Macedo, Andrea M

2018-02-14

Next-generation sequencing (NGS) methods are low-cost high-throughput technologies that produce thousands to millions of sequence reads. Despite the high number of raw sequence reads, their short length, relative to Sanger, PacBio or Nanopore reads, complicates the assembly of genomic repeats. Many genome tools are available, but the assembly of highly repetitive genome sequences using only NGS short reads remains challenging. Genome assembly of organisms responsible for important neglected diseases such as Trypanosoma cruzi, the aetiological agent of Chagas disease, is known to be challenging because of their repetitive nature. Only three of six recognized discrete typing units (DTUs) of the parasite have their draft genomes published and therefore genome evolution analyses in the taxon are limited. In this study, we developed a computational workflow to assemble highly repetitive genomes via a combination of de novo and reference-based assembly strategies to better overcome the intrinsic limitations of each, based on Illumina reads. The highly repetitive genome of the human-infecting parasite T. cruzi 231 strain was used as a test subject. The combined-assembly approach shown in this study benefits from the reference-based assembly ability to resolve highly repetitive sequences and from the de novo capacity to assemble genome-specific regions, improving the quality of the assembly. The acceptable confidence obtained by analyzing our results showed that our combined approach is an attractive option to assemble highly repetitive genomes with NGS short reads. Phylogenomic analysis including the 231 strain, the first representative of DTU III whose genome was sequenced, was also performed and provides new insights into T. cruzi genome evolution.

ReprDB and panDB: minimalist databases with maximal microbial representation.

PubMed

Zhou, Wei; Gay, Nicole; Oh, Julia

2018-01-18

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

Long-read sequencing improves assembly of Trichinella genomes 10-fold, revealing substantial synteny between lineages diverged over 7 million years.

PubMed

Thompson, Peter C; Zarlenga, Dante S; Liu, Ming-Yuan; Rosenthal, Benjamin M

2017-09-01

Genome assemblies can form the basis of comparative analyses fostering insight into the evolutionary genetics of a parasite's pathogenicity, host-pathogen interactions, environmental constraints and invasion biology; however, the length and complexity of many parasite genomes has hampered the development of well-resolved assemblies. In order to improve Trichinella genome assemblies, the genome of the sylvatic encapsulated species Trichinella murrelli was sequenced using third-generation, long-read technology and, using syntenic comparisons, scaffolded to a reference genome assembly of Trichinella spiralis, markedly improving both. A high-quality draft assembly for T. murrelli was achieved that totalled 63·2 Mbp, half of which was condensed into 26 contigs each longer than 571 000 bp. When compared with previous assemblies for parasites in the genus, ours required 10-fold fewer contigs, which were five times longer, on average. Better assembly across repetitive regions also enabled resolution of 8 Mbp of previously indeterminate sequence. Furthermore, syntenic comparisons identified widespread scaffold misassemblies in the T. spiralis reference genome. The two new assemblies, organized for the first time into three chromosomal scaffolds, will be valuable resources for future studies linking phenotypic traits within each species to their underlying genetic bases.

Comprehensive Annotation of the Parastagonospora nodorum Reference Genome Using Next-Generation Genomics, Transcriptomics and Proteogenomics

PubMed Central

Dodhia, Kejal; Stoll, Thomas; Hastie, Marcus; Furuki, Eiko; Ellwood, Simon R.; Williams, Angela H.; Tan, Yew-Foon; Testa, Alison C.; Gorman, Jeffrey J.; Oliver, Richard P.

2016-01-01

Parastagonospora nodorum, the causal agent of Septoria nodorum blotch (SNB), is an economically important pathogen of wheat (Triticum spp.), and a model for the study of necrotrophic pathology and genome evolution. The reference P. nodorum strain SN15 was the first Dothideomycete with a published genome sequence, and has been used as the basis for comparison within and between species. Here we present an updated reference genome assembly with corrections of SNP and indel errors in the underlying genome assembly from deep resequencing data as well as extensive manual annotation of gene models using transcriptomic and proteomic sources of evidence (https://github.com/robsyme/Parastagonospora_nodorum_SN15). The updated assembly and annotation includes 8,366 genes with modified protein sequence and 866 new genes. This study shows the benefits of using a wide variety of experimental methods allied to expert curation to generate a reliable set of gene models. PMID:26840125

Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula.

PubMed

Moll, Karen M; Zhou, Peng; Ramaraj, Thiruvarangan; Fajardo, Diego; Devitt, Nicholas P; Sadowsky, Michael J; Stupar, Robert M; Tiffin, Peter; Miller, Jason R; Young, Nevin D; Silverstein, Kevin A T; Mudge, Joann

2017-08-04

Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner. Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also showed complementary gains in continuity and join numbers. Both technologies spanned repetitive regions that PacBio alone was unable to bridge. Combining technologies, particularly Dovetail followed by BioNano, resulted in notable improvements compared to Dovetail or BioNano alone. A combination of PacBio, Dovetail, and BioNano was used to generate a high quality draft assembly of R108, a M. truncatula accession widely used in studies of functional genomics. As a test for the usefulness of the resulting genome sequence, the new R108 assembly was used to pinpoint breakpoints and characterize flanking sequence of a previously identified translocation between chromosomes 4 and 8, identifying more than 22.7 Mb of novel sequence not present in the earlier A17 reference assembly. Adding Dovetail followed by BioNano data yielded complementary improvements in continuity over the original PacBio assembly. This strategy proved efficient and cost-effective for developing a quality draft assembly compared to traditional reference assemblies.

Alignment of 1000 Genomes Project reads to reference assembly GRCh38.

PubMed

Zheng-Bradley, Xiangqun; Streeter, Ian; Fairley, Susan; Richardson, David; Clarke, Laura; Flicek, Paul

2017-07-01

The 1000 Genomes Project produced more than 100 trillion basepairs of short read sequence from more than 2600 samples in 26 populations over a period of five years. In its final phase, the project released over 85 million genotyped and phased variants on human reference genome assembly GRCh37. An updated reference assembly, GRCh38, was released in late 2013, but there was insufficient time for the final phase of the project analysis to change to the new assembly. Although it is possible to lift the coordinates of the 1000 Genomes Project variants to the new assembly, this is a potentially error-prone process as coordinate remapping is most appropriate only for non-repetitive regions of the genome and those that did not see significant change between the two assemblies. It will also miss variants in any region that was newly added to GRCh38. Thus, to produce the highest quality variants and genotypes on GRCh38, the best strategy is to realign the reads and recall the variants based on the new alignment. As the first step of variant calling for the 1000 Genomes Project data, we have finished remapping all of the 1000 Genomes sequence reads to GRCh38 with alternative scaffold-aware BWA-MEM. The resulting alignments are available as CRAM, a reference-based sequence compression format. The data have been released on our FTP site and are also available from European Nucleotide Archive to facilitate researchers discovering variants on the primary sequences and alternative contigs of GRCh38. © The Authors 2017. Published by Oxford University Press.

Potential benefits from using a new reference map in genomic prediction

USDA-ARS?s Scientific Manuscript database

Many genomic studies in cattle have used the 2009 reference assembly from the University of Maryland (UMD3.1). A new USDA Agricultural Research Service-University of California, Davis (ARS-UCD) assembly based on longer DNA reads from the same cow (Dominette) should improve sequence alignment, imputa...

Choice of Reference Sequence and Assembler for Alignment of Listeria monocytogenes Short-Read Sequence Data Greatly Influences Rates of Error in SNP Analyses

PubMed Central

Pightling, Arthur W.; Petronella, Nicholas; Pagotto, Franco

2014-01-01

The wide availability of whole-genome sequencing (WGS) and an abundance of open-source software have made detection of single-nucleotide polymorphisms (SNPs) in bacterial genomes an increasingly accessible and effective tool for comparative analyses. Thus, ensuring that real nucleotide differences between genomes (i.e., true SNPs) are detected at high rates and that the influences of errors (such as false positive SNPs, ambiguously called sites, and gaps) are mitigated is of utmost importance. The choices researchers make regarding the generation and analysis of WGS data can greatly influence the accuracy of short-read sequence alignments and, therefore, the efficacy of such experiments. We studied the effects of some of these choices, including: i) depth of sequencing coverage, ii) choice of reference-guided short-read sequence assembler, iii) choice of reference genome, and iv) whether to perform read-quality filtering and trimming, on our ability to detect true SNPs and on the frequencies of errors. We performed benchmarking experiments, during which we assembled simulated and real Listeria monocytogenes strain 08-5578 short-read sequence datasets of varying quality with four commonly used assemblers (BWA, MOSAIK, Novoalign, and SMALT), using reference genomes of varying genetic distances, and with or without read pre-processing (i.e., quality filtering and trimming). We found that assemblies of at least 50-fold coverage provided the most accurate results. In addition, MOSAIK yielded the fewest errors when reads were aligned to a nearly identical reference genome, while using SMALT to align reads against a reference sequence that is ∼0.82% distant from 08-5578 at the nucleotide level resulted in the detection of the greatest numbers of true SNPs and the fewest errors. Finally, we show that whether read pre-processing improves SNP detection depends upon the choice of reference sequence and assembler. In total, this study demonstrates that researchers should test a variety of conditions to achieve optimal results. PMID:25144537

Comparison of three assembly strategies for a heterozygous seedless grapevine genome assembly.

PubMed

Patel, Sagar; Lu, Zhixiu; Jin, Xiaozhu; Swaminathan, Padmapriya; Zeng, Erliang; Fennell, Anne Y

2018-01-17

De novo heterozygous assembly is an ongoing challenge requiring improved assembly approaches. In this study, three strategies were used to develop de novo Vitis vinifera 'Sultanina' genome assemblies for comparison with the inbred V. vinifera (PN40024 12X.v2) reference genome and a published Sultanina ALLPATHS-LG assembly (AP). The strategies were: 1) a default PLATANUS assembly (PLAT_d) for direct comparison with AP assembly, 2) an iterative merging strategy using METASSEMBLER to combine PLAT_d and AP assemblies (MERGE) and 3) PLATANUS parameter modifications plus GapCloser (PLAT*_GC). The three new assemblies were greater in size than the AP assembly. PLAT*_GC had the greatest number of scaffolds aligning with a minimum of 95% identity and ≥1000 bp alignment length to V. vinifera (PN40024 12X.v2) reference genome. SNP analysis also identified additional high quality SNPs. A greater number of sequence reads mapped back with zero-mismatch to the PLAT_d, MERGE, and PLAT*_GC (>94%) than was found in the AP assembly (87%) indicating a greater fidelity to the original sequence data in the new assemblies than in AP assembly. A de novo gene prediction conducted using seedless RNA-seq data predicted > 30,000 coding sequences for the three new de novo assemblies, with the greatest number (30,544) in PLAT*_GC and only 26,515 for the AP assembly. Transcription factor analysis indicated good family coverage, but some genes found in the VCOST.v3 annotation were not identified in any of the de novo assemblies, particularly some from  the MYB and ERF families. The PLAT_d and PLAT*_GC had a greater number of synteny blocks with the V. vinifera (PN40024 12X.v2) reference genome than AP or MERGE. PLAT*_GC provided the most contiguous assembly with only 1.2% scaffold N, in contrast to AP (10.7% N), PLAT_d (6.6% N) and Merge (6.4% N). A PLAT*_GC pseudo-chromosome assembly with chromosome alignment to the reference genome V. vinifera, (PN40024 12X.v2) provides new information for use in seedless grape genetic mapping studies. An annotated de novo gene prediction for the PLAT*_GC assembly, aligned with VitisNet pathways provides new seedless grapevine specific transcriptomic resource that has excellent fidelity with the seedless short read sequence data.

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

PubMed

Nielsen, H Bjørn; Almeida, Mathieu; Juncker, Agnieszka Sierakowska; Rasmussen, Simon; Li, Junhua; Sunagawa, Shinichi; Plichta, Damian R; Gautier, Laurent; Pedersen, Anders G; Le Chatelier, Emmanuelle; Pelletier, Eric; Bonde, Ida; Nielsen, Trine; Manichanh, Chaysavanh; Arumugam, Manimozhiyan; Batto, Jean-Michel; Quintanilha Dos Santos, Marcelo B; Blom, Nikolaj; Borruel, Natalia; Burgdorf, Kristoffer S; Boumezbeur, Fouad; Casellas, Francesc; Doré, Joël; Dworzynski, Piotr; Guarner, Francisco; Hansen, Torben; Hildebrand, Falk; Kaas, Rolf S; Kennedy, Sean; Kristiansen, Karsten; Kultima, Jens Roat; Léonard, Pierre; Levenez, Florence; Lund, Ole; Moumen, Bouziane; Le Paslier, Denis; Pons, Nicolas; Pedersen, Oluf; Prifti, Edi; Qin, Junjie; Raes, Jeroen; Sørensen, Søren; Tap, Julien; Tims, Sebastian; Ussery, David W; Yamada, Takuji; Renault, Pierre; Sicheritz-Ponten, Thomas; Bork, Peer; Wang, Jun; Brunak, Søren; Ehrlich, S Dusko

2014-08-01

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

Coordinates and intervals in graph-based reference genomes.

PubMed

Rand, Knut D; Grytten, Ivar; Nederbragt, Alexander J; Storvik, Geir O; Glad, Ingrid K; Sandve, Geir K

2017-05-18

It has been proposed that future reference genomes should be graph structures in order to better represent the sequence diversity present in a species. However, there is currently no standard method to represent genomic intervals, such as the positions of genes or transcription factor binding sites, on graph-based reference genomes. We formalize offset-based coordinate systems on graph-based reference genomes and introduce methods for representing intervals on these reference structures. We show the advantage of our methods by representing genes on a graph-based representation of the newest assembly of the human genome (GRCh38) and its alternative loci for regions that are highly variable. More complex reference genomes, containing alternative loci, require methods to represent genomic data on these structures. Our proposed notation for genomic intervals makes it possible to fully utilize the alternative loci of the GRCh38 assembly and potential future graph-based reference genomes. We have made a Python package for representing such intervals on offset-based coordinate systems, available at https://github.com/uio-cels/offsetbasedgraph . An interactive web-tool using this Python package to visualize genes on a graph created from GRCh38 is available at https://github.com/uio-cels/genomicgraphcoords .

SCARF: maximizing next-generation EST assemblies for evolutionary and population genomic analyses.

PubMed

Barker, Michael S; Dlugosch, Katrina M; Reddy, A Chaitanya C; Amyotte, Sarah N; Rieseberg, Loren H

2009-02-15

Scaffolded and Corrected Assembly of Roche 454 (SCARF) is a next-generation sequence assembly tool for evolutionary genomics that is designed especially for assembling 454 EST sequences against high-quality reference sequences from related species. The program was created to knit together 454 contigs that do not assemble during traditional de novo assembly, using a reference sequence library to orient the 454 sequences. SCARF is freely available at http://msbarker.com/software.htm, and is released under the open source GPLv3 license (http://www.opensource.org/licenses/gpl-3.0.html.

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.

A new rainbow trout (Oncorhynchus mykiss) reference genome assembly

USDA-ARS?s Scientific Manuscript database

In an effort to improve the rainbow trout reference genome assembly, we have re-sequenced the doubled-haploid Swanson line using the longest available reads from the Illumina technology. Overall we generated over 510 million 260nt paired-end shotgun reads, and 1 billion 160nt mate-pair reads from f...

Reference quality assembly of the 3.5-Gb genome of Capsicum annuum from a single linked-read library.

PubMed

Hulse-Kemp, Amanda M; Maheshwari, Shamoni; Stoffel, Kevin; Hill, Theresa A; Jaffe, David; Williams, Stephen R; Weisenfeld, Neil; Ramakrishnan, Srividya; Kumar, Vijay; Shah, Preyas; Schatz, Michael C; Church, Deanna M; Van Deynze, Allen

2018-01-01

Linked-Read sequencing technology has recently been employed successfully for de novo assembly of human genomes, however, the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5-gigabase (Gb) diploid pepper ( Capsicum annuum ) genome with a single Linked-Read library. Plant genomes, including pepper, are characterized by long, highly similar repetitive sequences. Accordingly, significant effort is used to ensure that the sequenced plant is highly homozygous and the resulting assembly is a haploid consensus. With a phased assembly approach, we targeted a heterozygous F 1 derived from a wide cross to assess the ability to derive both haplotypes and characterize a pungency gene with a large insertion/deletion. The Supernova software generated a highly ordered, more contiguous sequence assembly than all currently available C. annuum reference genomes. Over 83% of the final assembly was anchored and oriented using four publicly available  de novo linkage maps. A comparison of the annotation of conserved eukaryotic genes indicated the completeness of assembly. The validity of the phased assembly is further demonstrated with the complete recovery of both 2.5-Kb insertion/deletion haplotypes of the PUN1 locus in the F 1 sample that represents pungent and nonpungent peppers, as well as nearly full recovery of the BUSCO2 gene set within each of the two haplotypes. The most contiguous pepper genome assembly to date has been generated which demonstrates that Linked-Read library technology provides a tool to de novo assemble complex highly repetitive heterozygous plant genomes. This technology can provide an opportunity to cost-effectively develop high-quality genome assemblies for other complex plants and compare structural and gene differences through accurate haplotype reconstruction.

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

PubMed

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

2016-03-16

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

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

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

PubMed

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

2016-08-08

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

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...

A New and Improved Rainbow Trout (Oncorhynchus mykiss) Reference Genome Assembly

USDA-ARS?s Scientific Manuscript database

In an effort to improve the rainbow trout reference genome assembly, we re-sequenced the doubled-haploid Swanson line using the longest available reads from the Illumina technology; generating over 510 million paired-end shotgun reads (2x260nt), and 1 billion mate-pair reads (2x160nt) from four sequ...

KAT: a K-mer analysis toolkit to quality control NGS datasets and genome assemblies.

PubMed

Mapleson, Daniel; Garcia Accinelli, Gonzalo; Kettleborough, George; Wright, Jonathan; Clavijo, Bernardo J

2017-02-15

De novo assembly of whole genome shotgun (WGS) next-generation sequencing (NGS) data benefits from high-quality input with high coverage. However, in practice, determining the quality and quantity of useful reads quickly and in a reference-free manner is not trivial. Gaining a better understanding of the WGS data, and how that data is utilized by assemblers, provides useful insights that can inform the assembly process and result in better assemblies. We present the K-mer Analysis Toolkit (KAT): a multi-purpose software toolkit for reference-free quality control (QC) of WGS reads and de novo genome assemblies, primarily via their k-mer frequencies and GC composition. KAT enables users to assess levels of errors, bias and contamination at various stages of the assembly process. In this paper we highlight KAT's ability to provide valuable insights into assembly composition and quality of genome assemblies through pairwise comparison of k-mers present in both input reads and the assemblies. KAT is available under the GPLv3 license at: https://github.com/TGAC/KAT . bernardo.clavijo@earlham.ac.uk. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

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.

Leishmania naiffi and Leishmania guyanensis reference genomes highlight genome structure and gene evolution in the Viannia subgenus

PubMed Central

Coughlan, Simone; Taylor, Ali Shirley; Feane, Eoghan; Sanders, Mandy; Schonian, Gabriele; Cotton, James A.

2018-01-01

The unicellular protozoan parasite Leishmania causes the neglected tropical disease leishmaniasis, affecting 12 million people in 98 countries. In South America, where the Viannia subgenus predominates, so far only L. (Viannia) braziliensis and L. (V.) panamensis have been sequenced, assembled and annotated as reference genomes. Addressing this deficit in molecular information can inform species typing, epidemiological monitoring and clinical treatment. Here, L. (V.) naiffi and L. (V.) guyanensis genomic DNA was sequenced to assemble these two genomes as draft references from short sequence reads. The methods used were tested using short sequence reads for L. braziliensis M2904 against its published reference as a comparison. This assembly and annotation pipeline identified 70 additional genes not annotated on the original M2904 reference. Phylogenetic and evolutionary comparisons of L. guyanensis and L. naiffi with 10 other Viannia genomes revealed four traits common to all Viannia: aneuploidy, 22 orthologous groups of genes absent in other Leishmania subgenera, elevated TATE transposon copies and a high NADH-dependent fumarate reductase gene copy number. Within the Viannia, there were limited structural changes in genome architecture specific to individual species: a 45 Kb amplification on chromosome 34 was present in all bar L. lainsoni, L. naiffi had a higher copy number of the virulence factor leishmanolysin, and laboratory isolate L. shawi M8408 had a possible minichromosome derived from the 3’ end of chromosome 34. This combination of genome assembly, phylogenetics and comparative analysis across an extended panel of diverse Viannia has uncovered new insights into the origin and evolution of this subgenus and can help improve diagnostics for leishmaniasis surveillance. PMID:29765675

Comparison of the Equine Reference Sequence with Its Sanger Source Data and New Illumina Reads

PubMed Central

Rebolledo-Mendez, Jovan; Hestand, Matthew S.; Coleman, Stephen J.; Zeng, Zheng; Orlando, Ludovic; MacLeod, James N.; Kalbfleisch, Ted

2015-01-01

The reference assembly for the domestic horse, EquCab2, published in 2009, was built using approximately 30 million Sanger reads from a Thoroughbred mare named Twilight. Contiguity in the assembly was facilitated using nearly 315 thousand BAC end sequences from Twilight’s half brother Bravo. Since then, it has served as the foundation for many genome-wide analyses that include not only the modern horse, but ancient horses and other equid species as well. As data mapped to this reference has accumulated, consistent variation between mapped datasets and the reference, in terms of regions with no read coverage, single nucleotide variants, and small insertions/deletions have become apparent. In many cases, it is not clear whether these differences are the result of true sequence variation between the research subjects’ and Twilight’s genome or due to errors in the reference. EquCab2 is regarded as “The Twilight Assembly.” The objective of this study was to identify inconsistencies between the EquCab2 assembly and the source Twilight Sanger data used to build it. To that end, the original Sanger and BAC end reads have been mapped back to this equine reference and assessed with the addition of approximately 40X coverage of new Illumina Paired-End sequence data. The resulting mapped datasets identify those regions with low Sanger read coverage, as well as variation in genomic content that is not consistent with either the original Twilight Sanger data or the new genomic sequence data generated from Twilight on the Illumina platform. As the haploid EquCab2 reference assembly was created using Sanger reads derived largely from a single individual, the vast majority of variation detected in a mapped dataset comprised of those same Sanger reads should be heterozygous. In contrast, homozygous variations would represent either errors in the reference or contributions from Bravo's BAC end sequences. Our analysis identifies 720,843 homozygous discrepancies between new, high throughput genomic sequence data generated for Twilight and the EquCab2 reference assembly. Most of these represent errors in the assembly, while approximately 10,000 are demonstrated to be contributions from another horse. Other results are presented that include the binary alignment map file of the mapped Sanger reads, a list of variants identified as discrepancies between the source data and resulting reference, and a BED annotation file that lists the regions of the genome whose consensus was likely derived from low coverage alignments. PMID:26107638

The Douglas-fir genome sequence reveals specialization of the photosynthetic apparatus in Pinaceae

Treesearch

David B. Neale; Patrick E. McGuire; Nicholas C. Wheeler; Kristian A. Stevens; Marc W. Crepeau; Charis Cardeno; Aleksey V. Zimin; Daniela Puiu; Geo M. Pertea; U. Uzay Sezen; Claudio Casola; Tomasz E. Koralewski; Robin Paul; Daniel Gonzalez-Ibeas; Sumaira Zaman; Richard Cronn; Mark Yandell; Carson Holt; Charles H. Langley; James A. Yorke; Steven L. Salzberg; Jill L. Wegrzyn

2017-01-01

A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb.) Franco (Coastal Douglas-fir) is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50...

The wolf reference genome sequence (Canis lupus lupus) and its implications for Canis spp. population genomics.

PubMed

Gopalakrishnan, Shyam; Samaniego Castruita, Jose A; Sinding, Mikkel-Holger S; Kuderna, Lukas F K; Räikkönen, Jannikke; Petersen, Bent; Sicheritz-Ponten, Thomas; Larson, Greger; Orlando, Ludovic; Marques-Bonet, Tomas; Hansen, Anders J; Dalén, Love; Gilbert, M Thomas P

2017-06-29

An increasing number of studies are addressing the evolutionary genomics of dog domestication, principally through resequencing dog, wolf and related canid genomes. There is, however, only one de novo assembled canid genome currently available against which to map such data - that of a boxer dog (Canis lupus familiaris). We generated the first de novo wolf genome (Canis lupus lupus) as an additional choice of reference, and explored what implications may arise when previously published dog and wolf resequencing data are remapped to this reference. Reassuringly, we find that regardless of the reference genome choice, most evolutionary genomic analyses yield qualitatively similar results, including those exploring the structure between the wolves and dogs using admixture and principal component analysis. However, we do observe differences in the genomic coverage of re-mapped samples, the number of variants discovered, and heterozygosity estimates of the samples. In conclusion, the choice of reference is dictated by the aims of the study being undertaken; if the study focuses on the differences between the different dog breeds or the fine structure among dogs, then using the boxer reference genome is appropriate, but if the aim of the study is to look at the variation within wolves and their relationships to dogs, then there are clear benefits to using the de novo assembled wolf reference genome.

Improved Genome Assembly and Annotation for the Rock Pigeon (Columba livia)

PubMed Central

Holt, Carson; Campbell, Michael; Keays, David A.; Edelman, Nathaniel; Kapusta, Aurélie; Maclary, Emily; T. Domyan, Eric; Suh, Alexander; Warren, Wesley C.; Yandell, Mark; Gilbert, M. Thomas P.; Shapiro, Michael D.

2018-01-01

The domestic rock pigeon (Columba livia) is among the most widely distributed and phenotypically diverse avian species. C. livia is broadly studied in ecology, genetics, physiology, behavior, and evolutionary biology, and has recently emerged as a model for understanding the molecular basis of anatomical diversity, the magnetic sense, and other key aspects of avian biology. Here we report an update to the C. livia genome reference assembly and gene annotation dataset. Greatly increased scaffold lengths in the updated reference assembly, along with an updated annotation set, provide improved tools for evolutionary and functional genetic studies of the pigeon, and for comparative avian genomics in general. PMID:29519939

Genome assemblies for 11 Yersinia pestis strains isolated in the Caucasus region

DOE PAGES

Zhgenti, Ekaterine; Johnson, Shannon L.; Davenport, Karen W.; ...

2015-09-17

Yersinia pestis, the causative agent of plague, is endemic to the Caucasus region but few reference strain genome sequences from that region are available. We present the improved draft or finished assembled genomes from 11 strains isolated in the nation of Georgia and surrounding countries.

Construction and Annotation of a High Density SNP Linkage Map of the Atlantic Salmon (Salmo salar) Genome.

PubMed

Tsai, Hsin Y; Robledo, Diego; Lowe, Natalie R; Bekaert, Michael; Taggart, John B; Bron, James E; Houston, Ross D

2016-07-07

High density linkage maps are useful tools for fine-scale mapping of quantitative trait loci, and characterization of the recombination landscape of a species' genome. Genomic resources for Atlantic salmon (Salmo salar) include a well-assembled reference genome, and high density single nucleotide polymorphism (SNP) arrays. Our aim was to create a high density linkage map, and to align it with the reference genome assembly. Over 96,000 SNPs were mapped and ordered on the 29 salmon linkage groups using a pedigreed population comprising 622 fish from 60 nuclear families, all genotyped with the 'ssalar01' high density SNP array. The number of SNPs per group showed a high positive correlation with physical chromosome length (r = 0.95). While the order of markers on the genetic and physical maps was generally consistent, areas of discrepancy were identified. Approximately 6.5% of the previously unmapped reference genome sequence was assigned to chromosomes using the linkage map. Male recombination rate was lower than females across the vast majority of the genome, but with a notable peak in subtelomeric regions. Finally, using RNA-Seq data to annotate the reference genome, the mapped SNPs were categorized according to their predicted function, including annotation of ∼2500 putative nonsynonymous variants. The highest density SNP linkage map for any salmonid species has been created, annotated, and integrated with the Atlantic salmon reference genome assembly. This map highlights the marked heterochiasmy of salmon, and provides a useful resource for salmonid genetics and genomics research. Copyright © 2016 Tsai et al.

MetaSort untangles metagenome assembly by reducing microbial community complexity

PubMed Central

Ji, Peifeng; Zhang, Yanming; Wang, Jinfeng; Zhao, Fangqing

2017-01-01

Most current approaches to analyse metagenomic data rely on reference genomes. Novel microbial communities extend far beyond the coverage of reference databases and de novo metagenome assembly from complex microbial communities remains a great challenge. Here we present a novel experimental and bioinformatic framework, metaSort, for effective construction of bacterial genomes from metagenomic samples. MetaSort provides a sorted mini-metagenome approach based on flow cytometry and single-cell sequencing methodologies, and employs new computational algorithms to efficiently recover high-quality genomes from the sorted mini-metagenome by the complementary of the original metagenome. Through extensive evaluations, we demonstrated that metaSort has an excellent and unbiased performance on genome recovery and assembly. Furthermore, we applied metaSort to an unexplored microflora colonized on the surface of marine kelp and successfully recovered 75 high-quality genomes at one time. This approach will greatly improve access to microbial genomes from complex or novel communities. PMID:28112173

Reference genome sequence of the model plant Setaria

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

Bennetzen, Jeffrey L; Schmutz, Jeremy; Wang, Hao

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ~400-Mb assembly covers ~80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species thatmore » demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).« less

Reference genome sequence of the model plant Setaria.

PubMed

Bennetzen, Jeffrey L; Schmutz, Jeremy; Wang, Hao; Percifield, Ryan; Hawkins, Jennifer; Pontaroli, Ana C; Estep, Matt; Feng, Liang; Vaughn, Justin N; Grimwood, Jane; Jenkins, Jerry; Barry, Kerrie; Lindquist, Erika; Hellsten, Uffe; Deshpande, Shweta; Wang, Xuewen; Wu, Xiaomei; Mitros, Therese; Triplett, Jimmy; Yang, Xiaohan; Ye, Chu-Yu; Mauro-Herrera, Margarita; Wang, Lin; Li, Pinghua; Sharma, Manoj; Sharma, Rita; Ronald, Pamela C; Panaud, Olivier; Kellogg, Elizabeth A; Brutnell, Thomas P; Doust, Andrew N; Tuskan, Gerald A; Rokhsar, Daniel; Devos, Katrien M

2012-05-13

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The ∼400-Mb assembly covers ∼80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species that demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).

ABACAS: algorithm-based automatic contiguation of assembled sequences

PubMed Central

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

2009-01-01

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

De novo transciptome assembly in polyploid species

USDA-ARS?s Scientific Manuscript database

In the absence of a reference genome, the ultimate goal of a de novo transcriptome assembly is to accurately and comprehensively reconstruct the set of messenger RNA transcripts represented in the sample. Non-reference assembly of the transcriptome of polyploid species poses a particular challenge b...

Protein domain analysis of genomic sequence data reveals regulation of LRR related domains in plant transpiration in Ficus.

PubMed

Lang, Tiange; Yin, Kangquan; Liu, Jinyu; Cao, Kunfang; Cannon, Charles H; Du, Fang K

2014-01-01

Predicting protein domains is essential for understanding a protein's function at the molecular level. However, up till now, there has been no direct and straightforward method for predicting protein domains in species without a reference genome sequence. In this study, we developed a functionality with a set of programs that can predict protein domains directly from genomic sequence data without a reference genome. Using whole genome sequence data, the programming functionality mainly comprised DNA assembly in combination with next-generation sequencing (NGS) assembly methods and traditional methods, peptide prediction and protein domain prediction. The proposed new functionality avoids problems associated with de novo assembly due to micro reads and small single repeats. Furthermore, we applied our functionality for the prediction of leucine rich repeat (LRR) domains in four species of Ficus with no reference genome, based on NGS genomic data. We found that the LRRNT_2 and LRR_8 domains are related to plant transpiration efficiency, as indicated by the stomata index, in the four species of Ficus. The programming functionality established in this study provides new insights for protein domain prediction, which is particularly timely in the current age of NGS data expansion.

Single-molecule sequencing and conformational capture enable de novo mammalian reference genomes

USDA-ARS?s Scientific Manuscript database

Genome assemblies have been produced for numerous species as a result of advances in sequencing technologies. However, many of the assemblies are fragmented, with many gaps, ambiguities, and errors. We use the genome of the domestic goat (Capra hircus) to demonstrate current state of the art for ef...

MetaQUAST: evaluation of metagenome assemblies.

PubMed

Mikheenko, Alla; Saveliev, Vladislav; Gurevich, Alexey

2016-04-01

During the past years we have witnessed the rapid development of new metagenome assembly methods. Although there are many benchmark utilities designed for single-genome assemblies, there is no well-recognized evaluation and comparison tool for metagenomic-specific analogues. In this article, we present MetaQUAST, a modification of QUAST, the state-of-the-art tool for genome assembly evaluation based on alignment of contigs to a reference. MetaQUAST addresses such metagenome datasets features as (i) unknown species content by detecting and downloading reference sequences, (ii) huge diversity by giving comprehensive reports for multiple genomes and (iii) presence of highly relative species by detecting chimeric contigs. We demonstrate MetaQUAST performance by comparing several leading assemblers on one simulated and two real datasets. http://bioinf.spbau.ru/metaquast aleksey.gurevich@spbu.ru Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Complete Taiwanese Macaque (Macaca cyclopis) Mitochondrial Genome: Reference-Assisted de novo Assembly with Multiple k-mer Strategy.

PubMed

Huang, Yu-Feng; Midha, Mohit; Chen, Tzu-Han; Wang, Yu-Tai; Smith, David Glenn; Pei, Kurtis Jai-Chyi; Chiu, Kuo Ping

2015-01-01

The Taiwanese (Formosan) macaque (Macaca cyclopis) is the only nonhuman primate endemic to Taiwan. This primate species is valuable for evolutionary studies and as subjects in medical research. However, only partial fragments of the mitochondrial genome (mitogenome) of this primate species have been sequenced, not mentioning its nuclear genome. We employed next-generation sequencing to generate 2 x 90 bp paired-end reads, followed by reference-assisted de novo assembly with multiple k-mer strategy to characterize the M. cyclopis mitogenome. We compared the assembled mitogenome with that of other macaque species for phylogenetic analysis. Our results show that, the M. cyclopis mitogenome consists of 16,563 nucleotides encoding for 13 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNAs. Phylogenetic analysis indicates that M. cyclopis is most closely related to M. mulatta lasiota (Chinese rhesus macaque), supporting the notion of Asia-continental origin of M. cyclopis proposed in previous studies based on partial mitochondrial sequences. Our work presents a novel approach for assembling a mitogenome that utilizes the capabilities of de novo genome assembly with assistance of a reference genome. The availability of the complete Taiwanese macaque mitogenome will facilitate the study of primate evolution and the characterization of genetic variations for the potential usage of this species as a non-human primate model for medical research.

Improved Genome Assembly and Annotation for the Rock Pigeon (Columba livia).

PubMed

Holt, Carson; Campbell, Michael; Keays, David A; Edelman, Nathaniel; Kapusta, Aurélie; Maclary, Emily; T Domyan, Eric; Suh, Alexander; Warren, Wesley C; Yandell, Mark; Gilbert, M Thomas P; Shapiro, Michael D

2018-05-04

The domestic rock pigeon ( Columba livia ) is among the most widely distributed and phenotypically diverse avian species. C. livia is broadly studied in ecology, genetics, physiology, behavior, and evolutionary biology, and has recently emerged as a model for understanding the molecular basis of anatomical diversity, the magnetic sense, and other key aspects of avian biology. Here we report an update to the C. livia genome reference assembly and gene annotation dataset. Greatly increased scaffold lengths in the updated reference assembly, along with an updated annotation set, provide improved tools for evolutionary and functional genetic studies of the pigeon, and for comparative avian genomics in general. Copyright © 2018 Holt et al.

BAIT: Organizing genomes and mapping rearrangements in single cells.

PubMed

Hills, Mark; O'Neill, Kieran; Falconer, Ester; Brinkman, Ryan; Lansdorp, Peter M

2013-01-01

Strand-seq is a single-cell sequencing technique to finely map sister chromatid exchanges (SCEs) and other rearrangements. To analyze these data, we introduce BAIT, software which assigns templates and identifies and localizes SCEs. We demonstrate BAIT can refine completed reference assemblies, identifying approximately 21 Mb of incorrectly oriented fragments and placing over half (2.6 Mb) of the orphan fragments in mm10/GRCm38. BAIT also stratifies scaffold-stage assemblies, potentially accelerating the assembling and finishing of reference genomes. BAIT is available at http://sourceforge.net/projects/bait/.

Positional bias in variant calls against draft reference assemblies.

PubMed

Briskine, Roman V; Shimizu, Kentaro K

2017-03-28

Whole genome resequencing projects may implement variant calling using draft reference genomes assembled de novo from short-read libraries. Despite lower quality of such assemblies, they allowed researchers to extend a wide range of population genetic and genome-wide association analyses to non-model species. As the variant calling pipelines are complex and involve many software packages, it is important to understand inherent biases and limitations at each step of the analysis. In this article, we report a positional bias present in variant calling performed against draft reference assemblies constructed from de Bruijn or string overlap graphs. We assessed how frequently variants appeared at each position counted from ends of a contig or scaffold sequence, and discovered unexpectedly high number of variants at the positions related to the length of either k-mers or reads used for the assembly. We detected the bias in both publicly available draft assemblies from Assemblathon 2 competition as well as in the assemblies we generated from our simulated short-read data. Simulations confirmed that the bias causing variants are predominantly false positives induced by reads from spatially distant repeated sequences. The bias is particularly strong in contig assemblies. Scaffolding does not eliminate the bias but tends to mitigate it because of the changes in variants' relative positions and alterations in read alignments. The bias can be effectively reduced by filtering out the variants that reside in repetitive elements. Draft genome sequences generated by several popular assemblers appear to be susceptible to the positional bias potentially affecting many resequencing projects in non-model species. The bias is inherent to the assembly algorithms and arises from their particular handling of repeated sequences. It is recommended to reduce the bias by filtering especially if higher-quality genome assembly cannot be achieved. Our findings can help other researchers to improve the quality of their variant data sets and reduce artefactual findings in downstream analyses.

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.

A vertebrate case study of the quality of assemblies derived from next-generation sequences

PubMed Central

2011-01-01

The unparalleled efficiency of next-generation sequencing (NGS) has prompted widespread adoption, but significant problems remain in the use of NGS data for whole genome assembly. We explore the advantages and disadvantages of chicken genome assemblies generated using a variety of sequencing and assembly methodologies. NGS assemblies are equivalent in some ways to a Sanger-based assembly yet deficient in others. Nonetheless, these assemblies are sufficient for the identification of the majority of genes and can reveal novel sequences when compared to existing assembly references. PMID:21453517

Reference quality assembly of the 3.5 Gb genome of Capsicum annuum form a single linked-read library

USDA-ARS?s Scientific Manuscript database

Linked-Read sequencing technology has recently been employed successfully for de novo assembly of multiple human genomes, however the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5 gigabase (Gb) diploid pepper (Cap...

The Douglas-Fir Genome Sequence Reveals Specialization of the Photosynthetic Apparatus in Pinaceae

PubMed Central

Neale, David B.; McGuire, Patrick E.; Wheeler, Nicholas C.; Stevens, Kristian A.; Crepeau, Marc W.; Cardeno, Charis; Zimin, Aleksey V.; Puiu, Daniela; Pertea, Geo M.; Sezen, U. Uzay; Casola, Claudio; Koralewski, Tomasz E.; Paul, Robin; Gonzalez-Ibeas, Daniel; Zaman, Sumaira; Cronn, Richard; Yandell, Mark; Holt, Carson; Langley, Charles H.; Yorke, James A.; Salzberg, Steven L.; Wegrzyn, Jill L.

2017-01-01

A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb.) Franco (Coastal Douglas-fir) is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50 = 340,704 bp). Incremental improvements in sequencing and assembly technologies are in part responsible for the higher quality reference genome, but it may also be due to a slightly lower exact repeat content in Douglas-fir vs. pine and spruce. Comparative genome annotation with angiosperm species reveals gene-family expansion and contraction in Douglas-fir and other conifers which may account for some of the major morphological and physiological differences between the two major plant groups. Notable differences in the size of the NDH-complex gene family and genes underlying the functional basis of shade tolerance/intolerance were observed. This reference genome sequence not only provides an important resource for Douglas-fir breeders and geneticists but also sheds additional light on the evolutionary processes that have led to the divergence of modern angiosperms from the more ancient gymnosperms. PMID:28751502

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50)

PubMed Central

Iamartino, Daniela; Pruitt, Kim D; Sonstegard, Tad; Smith, Timothy P L; Low, Wai Yee; Biagini, Tommaso; Bomba, Lorenzo; Capomaccio, Stefano; Castiglioni, Bianca; Coletta, Angelo; Corrado, Federica; Ferré, Fabrizio; Iannuzzi, Leopoldo; Lawley, Cynthia; Macciotta, Nicolò; McClure, Matthew; Mancini, Giordano; Matassino, Donato; Mazza, Raffaele; Milanesi, Marco; Moioli, Bianca; Morandi, Nicola; Ramunno, Luigi; Peretti, Vincenzo; Pilla, Fabio; Ramelli, Paola; Schroeder, Steven; Strozzi, Francesco; Thibaud-Nissen, Francoise; Zicarelli, Luigi; Ajmone-Marsan, Paolo; Valentini, Alessio; Chillemi, Giovanni; Zimin, Aleksey

2017-01-01

Abstract Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2n = 50) and the swamp (2n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1. PMID:29048578

Chromosome-scale assembly of the Monopterus genome.

PubMed

Zhao, Xueya; Luo, Majing; Li, Zhigang; Zhong, Pei; Cheng, Yibin; Lai, Fengling; Wang, Xin; Min, Jiumeng; Bai, Mingzhou; Yang, Yulan; Cheng, Hanhua; Zhou, Rongjia

2018-05-01

The teleost fish Monopterus albus is emerging as a new model for biological studies due to its natural sex transition and small genome, in addition to its enormous economic and potential medical value. However, no genomic information for the Monopterus is currently available. Here, we sequenced and de novo assembled the genome of M. albus and report the de novochromosome assembly by FISH walking assisted by conserved synteny (Cafs). Using Cafs, 328 scaffolds were assembled into 12 chromosomes, which covered genomic sequences of 555 Mb, accounting for 81.3% of the sequences assembled in scaffolds (∼689 Mb). A total of 18 ,660 genes were mapped on the chromosomes and showed a nonrandom distribution along chromosomes. We report the first reference genome of the Monopterus and provide an efficient Cafs strategy for a de novo chromosome-level assembly of the Monopterus genome, which provides a valuable resource, not only for further studies in genetics, evolution, and development, particularly sex determination, but also for breed improvement of the species.

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

PubMed Central

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

2018-01-01

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

Comparison of the cattle leukocyte receptor complex with related livestock species

USDA-ARS?s Scientific Manuscript database

The natural killer (NK) cell receptor gene complexes are highly variable between species, and their repetitive nature makes genomic assembly and characterization problematic. As a result, most reference genome assemblies are heavily fragmented and/or misassembled over these regions. However, new lon...

Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity.

PubMed

Edger, Patrick P; VanBuren, Robert; Colle, Marivi; Poorten, Thomas J; Wai, Ching Man; Niederhuth, Chad E; Alger, Elizabeth I; Ou, Shujun; Acharya, Charlotte B; Wang, Jie; Callow, Pete; McKain, Michael R; Shi, Jinghua; Collier, Chad; Xiong, Zhiyong; Mower, Jeffrey P; Slovin, Janet P; Hytönen, Timo; Jiang, Ning; Childs, Kevin L; Knapp, Steven J

2018-02-01

Although draft genomes are available for most agronomically important plant species, the majority are incomplete, highly fragmented, and often riddled with assembly and scaffolding errors. These assembly issues hinder advances in tool development for functional genomics and systems biology. Here we utilized a robust, cost-effective approach to produce high-quality reference genomes. We report a near-complete genome of diploid woodland strawberry (Fragaria vesca) using single-molecule real-time sequencing from Pacific Biosciences (PacBio). This assembly has a contig N50 length of ∼7.9 million base pairs (Mb), representing a ∼300-fold improvement of the previous version. The vast majority (>99.8%) of the assembly was anchored to 7 pseudomolecules using 2 sets of optical maps from Bionano Genomics. We obtained ∼24.96 Mb of sequence not present in the previous version of the F. vesca genome and produced an improved annotation that includes 1496 new genes. Comparative syntenic analyses uncovered numerous, large-scale scaffolding errors present in each chromosome in the previously published version of the F. vesca genome. Our results highlight the need to improve existing short-read based reference genomes. Furthermore, we demonstrate how genome quality impacts commonly used analyses for addressing both fundamental and applied biological questions. © The Authors 2017. Published by Oxford University Press.

Integrating genome assemblies with MAIA

PubMed Central

Nijkamp, Jurgen; Winterbach, Wynand; van den Broek, Marcel; Daran, Jean-Marc; Reinders, Marcel; de Ridder, Dick

2010-01-01

Motivation: De novo assembly of a eukaryotic genome with next-generation sequencing data is still a challenging task. Over the past few years several assemblers have been developed, often suitable for one specific type of sequencing data. The number of known genomes is expanding rapidly, therefore it becomes possible to use multiple reference genomes for assembly projects. We introduce an assembly integrator that makes use of all available data, i.e. multiple de novo assemblies and mappings against multiple related genomes, by optimizing a weighted combination of criteria. Results: The developed algorithm was applied on the de novo sequencing of the Saccharomyces cerevisiae CEN.PK 113-7D strain. Using Solexa and 454 read data, two de novo and three comparative assemblies were constructed and subsequently integrated, yielding 29 contigs, covering more than 12 Mbp; a drastic improvement compared with the single assemblies. Availability: MAIA is available as a Matlab package and can be downloaded from http://bioinformatics.tudelft.nl Contact: j.f.nijkamp@tudelft.nl PMID:20823304

OSLay: optimal syntenic layout of unfinished assemblies.

PubMed

Richter, Daniel C; Schuster, Stephan C; Huson, Daniel H

2007-07-01

The whole genome shotgun approach to genome sequencing results in a collection of contigs that must be ordered and oriented to facilitate efficient gap closure. We present a new tool OSLay that uses synteny between matching sequences in a target assembly and a reference assembly to layout the contigs (or scaffolds) in the target assembly. The underlying algorithm is based on maximum weight matching. The tool provides an interactive visualization of the computed layout and the result can be imported into the assembly editing tool Consed to support the design of primer pairs for gap closure. To enhance efficiency in the gap closure phase of a genome project it is crucial to know which contigs are adjacent in the target genome. Related genome sequences can be used to layout contigs in an assembly. OSLay is freely available from: http://www-ab.informatik.unituebingen.de/software/oslay.

Completed Genome Sequences of Strains from 36 Serotypes of Salmonella

PubMed Central

Robertson, James; Yoshida, Catherine; Gurnik, Simone; Rankin, Marisa

2018-01-01

ABSTRACT We report here the completed closed genome sequences of strains representing 36 serotypes of Salmonella. These genome sequences will provide useful references for understanding the genetic variation between serotypes, particularly as references for mapping of raw reads or to create assemblies of higher quality, as well as to aid in studies of comparative genomics of Salmonella. PMID:29348347

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

Sequencing and assembly of the 22-gb loblolly pine genome.

PubMed

Zimin, Aleksey; Stevens, Kristian A; Crepeau, Marc W; Holtz-Morris, Ann; Koriabine, Maxim; Marçais, Guillaume; Puiu, Daniela; Roberts, Michael; Wegrzyn, Jill L; de Jong, Pieter J; Neale, David B; Salzberg, Steven L; Yorke, James A; Langley, Charles H

2014-03-01

Conifers are the predominant gymnosperm. The size and complexity of their genomes has presented formidable technical challenges for whole-genome shotgun sequencing and assembly. We employed novel strategies that allowed us to determine the loblolly pine (Pinus taeda) reference genome sequence, the largest genome assembled to date. Most of the sequence data were derived from whole-genome shotgun sequencing of a single megagametophyte, the haploid tissue of a single pine seed. Although that constrained the quantity of available DNA, the resulting haploid sequence data were well-suited for assembly. The haploid sequence was augmented with multiple linking long-fragment mate pair libraries from the parental diploid DNA. For the longest fragments, we used novel fosmid DiTag libraries. Sequences from the linking libraries that did not match the megagametophyte were identified and removed. Assembly of the sequence data were aided by condensing the enormous number of paired-end reads into a much smaller set of longer "super-reads," rendering subsequent assembly with an overlap-based assembly algorithm computationally feasible. To further improve the contiguity and biological utility of the genome sequence, additional scaffolding methods utilizing independent genome and transcriptome assemblies were implemented. The combination of these strategies resulted in a draft genome sequence of 20.15 billion bases, with an N50 scaffold size of 66.9 kbp.

A reference genome of the European beech (Fagus sylvatica L.).

PubMed

Mishra, Bagdevi; Gupta, Deepak K; Pfenninger, Markus; Hickler, Thomas; Langer, Ewald; Nam, Bora; Paule, Juraj; Sharma, Rahul; Ulaszewski, Bartosz; Warmbier, Joanna; Burczyk, Jaroslaw; Thines, Marco

2018-06-01

The European beech is arguably the most important climax broad-leaved tree species in Central Europe, widely planted for its valuable wood. Here, we report the 542 Mb draft genome sequence of an up to 300-year-old individual (Bhaga) from an undisturbed stand in the Kellerwald-Edersee National Park in central Germany. Using a hybrid assembly approach, Illumina reads with short- and long-insert libraries, coupled with long Pacific Biosciences reads, we obtained an assembled genome size of 542 Mb, in line with flow cytometric genome size estimation. The largest scaffold was of 1.15 Mb, the N50 length was 145 kb, and the L50 count was 983. The assembly contained 0.12% of Ns. A Benchmarking with Universal Single-Copy Orthologs (BUSCO) analysis retrieved 94% complete BUSCO genes, well in the range of other high-quality draft genomes of trees. A total of 62,012 protein-coding genes were predicted, assisted by transcriptome sequencing. In addition, we are reporting an efficient method for extracting high-molecular-weight DNA from dormant buds, by which contamination by environmental bacteria and fungi was kept at a minimum. The assembled genome will be a valuable resource and reference for future population genomics studies on the evolution and past climate change adaptation of beech and will be helpful for identifying genes, e.g., involved in drought tolerance, in order to select and breed individuals to adapt forestry to climate change in Europe. A continuously updated genome browser and download page can be accessed from beechgenome.net, which will include future genome versions of the reference individual Bhaga, as new sequencing approaches develop.

De novo assembly and phasing of a Korean human genome.

PubMed

Seo, Jeong-Sun; Rhie, Arang; Kim, Junsoo; Lee, Sangjin; Sohn, Min-Hwan; Kim, Chang-Uk; Hastie, Alex; Cao, Han; Yun, Ji-Young; Kim, Jihye; Kuk, Junho; Park, Gun Hwa; Kim, Juhyeok; Ryu, Hanna; Kim, Jongbum; Roh, Mira; Baek, Jeonghun; Hunkapiller, Michael W; Korlach, Jonas; Shin, Jong-Yeon; Kim, Changhoon

2016-10-13

Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9 Mb and a scaffold N50 size of 44.8 Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03 Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6 Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of unreported and Asian-specific structural variants, and high-quality haplotyping of clinically relevant alleles for precision medicine.

Sequencing and De novo Draft Assemblies of the Fathead Minnow (Pimphales promelas)Reference Genome

EPA Science Inventory

This study was undertaken to develop genome-scale resources for the fathead minnow (Pimphales promelas) an important model organism widely used in both aquatic ecotoxicology research and in regulatory toxicity testing. We report on the first sequencing and two draft assemblies fo...

Transcriptome assembly, gene annotation and tissue gene expression atlas of the rainbow trout

USDA-ARS?s Scientific Manuscript database

Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complimented by transcriptome information that will enhance genome assembly and annotation. Previously, we reported a transcriptome reference sequence using a 19X coverage of Sanger and 454-pyrosequencing dat...

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 .

New in-depth rainbow trout transcriptome reference and digital atlas of gene expression

USDA-ARS?s Scientific Manuscript database

Sequencing the rainbow trout genome is underway and a transcriptome reference sequence is required to help in genome assembly and gene discovery. Previously, we reported a transcriptome reference sequence using a 19X coverage of 454-pyrosequencing data. Although this work added a great wealth of ann...

A comprehensive profile of DNA copy number variations in a Korean population: identification of copy number invariant regions among Koreans.

PubMed

Jeon, Jae Pil; Shim, Sung Mi; Jung, Jong Sun; Nam, Hye Young; Lee, Hye Jin; Oh, Berm Seok; Kim, Kuchan; Kim, Hyung Lae; Han, Bok Ghee

2009-09-30

To examine copy number variations among the Korean population, we compared individual genomes with the Korean reference genome assembly using the publicly available Korean HapMap SNP 50 k chip data from 90 individuals. Korean individuals exhibited 123 copy number variation regions (CNVRs) covering 27.2 mb, equivalent to 1.0% of the genome in the copy number variation (CNV) analysis using the combined criteria of P value (P<0.01) and standard deviation of copy numbers (SD>or= 0.25) among study subjects. In contrast, when compared to the Affymetrix reference genome assembly from multiple ethnic groups, considerably more CNVRs (n=643) were detected in larger proportions (5.0%) of the genome covering 135.1 mb even by more stringent criteria (P<0.001 and SD>or=0.25), reflecting ethnic diversity of structural variations between Korean and other populations. Some CNVRs were validated by the quantitative multiplex PCR of short fluorescent fragment (QMPSF) method, and then copy number invariant regions were detected among the study subjects. These copy number invariant regions would be used as good internal controls for further CNV studies. Lastly, we demonstrated that the CNV information could stratify even a single ethnic population with a proper reference genome assembly from multiple heterogeneous populations.

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50).

PubMed

Williams, John L; Iamartino, Daniela; Pruitt, Kim D; Sonstegard, Tad; Smith, Timothy P L; Low, Wai Yee; Biagini, Tommaso; Bomba, Lorenzo; Capomaccio, Stefano; Castiglioni, Bianca; Coletta, Angelo; Corrado, Federica; Ferré, Fabrizio; Iannuzzi, Leopoldo; Lawley, Cynthia; Macciotta, Nicolò; McClure, Matthew; Mancini, Giordano; Matassino, Donato; Mazza, Raffaele; Milanesi, Marco; Moioli, Bianca; Morandi, Nicola; Ramunno, Luigi; Peretti, Vincenzo; Pilla, Fabio; Ramelli, Paola; Schroeder, Steven; Strozzi, Francesco; Thibaud-Nissen, Francoise; Zicarelli, Luigi; Ajmone-Marsan, Paolo; Valentini, Alessio; Chillemi, Giovanni; Zimin, Aleksey

2017-10-01

Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2 n = 50) and the swamp (2 n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1. © The Author 2017. Published by Oxford University Press.

Transcriptome Assembly, Gene Annotation and Tissue Gene Expression Atlas of the Rainbow Trout

PubMed Central

Salem, Mohamed; Paneru, Bam; Al-Tobasei, Rafet; Abdouni, Fatima; Thorgaard, Gary H.; Rexroad, Caird E.; Yao, Jianbo

2015-01-01

Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complemented by transcriptome information that will enhance genome assembly and annotation. Previously, transcriptome reference sequences were reported using data from different sources. Although the previous work added a great wealth of sequences, a complete and well-annotated transcriptome is still needed. In addition, gene expression in different tissues was not completely addressed in the previous studies. In this study, non-normalized cDNA libraries were sequenced from 13 different tissues of a single doubled haploid rainbow trout from the same source used for the rainbow trout genome sequence. A total of ~1.167 billion paired-end reads were de novo assembled using the Trinity RNA-Seq assembler yielding 474,524 contigs > 500 base-pairs. Of them, 287,593 had homologies to the NCBI non-redundant protein database. The longest contig of each cluster was selected as a reference, yielding 44,990 representative contigs. A total of 4,146 contigs (9.2%), including 710 full-length sequences, did not match any mRNA sequences in the current rainbow trout genome reference. Mapping reads to the reference genome identified an additional 11,843 transcripts not annotated in the genome. A digital gene expression atlas revealed 7,678 housekeeping and 4,021 tissue-specific genes. Expression of about 16,000–32,000 genes (35–71% of the identified genes) accounted for basic and specialized functions of each tissue. White muscle and stomach had the least complex transcriptomes, with high percentages of their total mRNA contributed by a small number of genes. Brain, testis and intestine, in contrast, had complex transcriptomes, with a large numbers of genes involved in their expression patterns. This study provides comprehensive de novo transcriptome information that is suitable for functional and comparative genomics studies in rainbow trout, including annotation of the genome. PMID:25793877

Fathead minnow genome sequencing and assembly

EPA Pesticide Factsheets

The dataset provides the URLs for accessing the genome sequence data and two draft assemblies as well as fathead minnow genotyping data associated with estimating the heterozygosity of the in-bred line.This dataset is associated with the following publication:Burns, F., L. Cogburn, G. Ankley , D. Villeneuve , E. Waits , Y. Chang, V. Llaca, S. Deschamps, R. Jackson, and R. Hoke. Sequencing and De novo Draft Assemblies of the Fathead Minnow (Pimphales promelas)Reference Genome. ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY. Society of Environmental Toxicology and Chemistry, Pensacola, FL, USA, 35(1): 212-217, (2016).

Deep analysis of wild Vitis flower transcriptome reveals unexplored genome regions associated with sex specification.

PubMed

Ramos, Miguel Jesus Nunes; Coito, João Lucas; Fino, Joana; Cunha, Jorge; Silva, Helena; de Almeida, Patrícia Gomes; Costa, Maria Manuela Ribeiro; Amâncio, Sara; Paulo, Octávio S; Rocheta, Margarida

2017-01-01

RNA-seq of Vitis during early stages of bud development, in male, female and hermaphrodite flowers, identified new loci outside of annotated gene models, suggesting their involvement in sex establishment. The molecular mechanisms responsible for flower sex specification remain unclear for most plant species. In the case of V. vinifera ssp. vinifera, it is not fully understood what determines hermaphroditism in the domesticated subspecies and male or female flowers in wild dioecious relatives (Vitis vinifera ssp. sylvestris). Here, we describe a de novo assembly of the transcriptome of three flower developmental stages from the three Vitis vinifera flower types. The validation of de novo assembly showed a correlation of 0.825. The main goals of this work were the identification of V. v. sylvestris exclusive transcripts and the characterization of differential gene expression during flower development. RNA from several flower developmental stages was used previously to generate Illumina sequence reads. Through a sequential de novo assembly strategy one comprehensive transcriptome comprising 95,516 non-redundant transcripts was assembled. From this dataset 81,064 transcripts were annotated to V. v. vinifera reference transcriptome and 11,084 were annotated against V. v. vinifera reference genome. Moreover, we found 3368 transcripts that could not be mapped to Vitis reference genome. From all the non-redundant transcripts that were assembled, bioinformatics analysis identified 133 specific of V. v. sylvestris and 516 transcripts differentially expressed among the three flower types. The detection of transcription from areas of the genome not currently annotated suggests active transcription of previously unannotated genomic loci during early stages of bud development.

Draft genome of a Xanthomonas perforans strain associated with pith necrosis.

PubMed

Torelli, Emanuela; Aiello, Dalia; Polizzi, Giancarlo; Firrao, Giuseppe; Cirvilleri, Gabriella

2015-02-01

Xanthomonas perforans causes bacterial spot of tomato and pepper. A genome draft of an unusual isolate (strain 4P1S2), differing in that it was associated with stem pith necrosis, was assembled from Illumina MiSeq sequencing data using the draft of X. perforans strain 91-118 as a reference. The resulting draft (accession number JRWW00000000) largely overlapped with the reference draft. In addition, the reads not mapping on the reference assembly were selected and used for a further assembly, that revealed a large putative plasmid. The analysis of the predicted proteins showed only few gene features that could be potentially implicated in the switch of a phytopathological behavior. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

Use of a draft genome of coffee (Coffea arabica) to identify SNPs associated with caffeine content.

PubMed

Tran, Hue T M; Ramaraj, Thiruvarangan; Furtado, Agnelo; Lee, Leonard Slade; Henry, Robert J

2018-03-07

Arabica coffee (Coffea arabica) has a small gene pool limiting genetic improvement. Selection for caffeine content within this gene pool would be assisted by identification of the genes controlling this important trait. Sequencing of DNA bulks from 18 genotypes with extreme high- or low-caffeine content from a population of 232 genotypes was used to identify linked polymorphisms. To obtain a reference genome, a whole genome assembly of arabica coffee (variety K7) was achieved by sequencing using short read (Illumina) and long-read (PacBio) technology. Assembly was performed using a range of assembly tools resulting in 76 409 scaffolds with a scaffold N50 of 54 544 bp and a total scaffold length of 1448 Mb. Validation of the genome assembly using different tools showed high completeness of the genome. More than 99% of transcriptome sequences mapped to the C. arabica draft genome, and 89% of BUSCOs were present. The assembled genome annotated using AUGUSTUS yielded 99 829 gene models. Using the draft arabica genome as reference in mapping and variant calling allowed the detection of 1444 nonsynonymous single nucleotide polymorphisms (SNPs) associated with caffeine content. Based on Kyoto Encyclopaedia of Genes and Genomes pathway-based analysis, 65 caffeine-associated SNPs were discovered, among which 11 SNPs were associated with genes encoding enzymes involved in the conversion of substrates, which participate in the caffeine biosynthesis pathways. This analysis demonstrated the complex genetic control of this key trait in coffee. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

A complete high-quality MinION nanopore assembly of an extensively drug-resistant Mycobacterium tuberculosis Beijing lineage strain identifies novel variation in repetitive PE/PPE gene regions.

PubMed

Bainomugisa, Arnold; Duarte, Tania; Lavu, Evelyn; Pandey, Sushil; Coulter, Chris; Marais, Ben J; Coin, Lachlan M

2018-06-15

A better understanding of the genomic changes that facilitate the emergence and spread of drug-resistant Mycobacterium tuberculosis strains is currently required. Here, we report the use of the MinION nanopore sequencer (Oxford Nanopore Technologies) to sequence and assemble an extensively drug-resistant (XDR) isolate, which is part of a modern Beijing sub-lineage strain, prevalent in Western Province, Papua New Guinea. Using 238-fold coverage obtained from a single flow-cell, de novo assembly of nanopore reads resulted into one contiguous assembly with 99.92 % assembly accuracy. Incorporation of complementary short read sequences (Illumina) as part of consensus error correction resulted in a 4 404 064 bp genome with 99.98 % assembly accuracy. This assembly had an average nucleotide identity of 99.7 % relative to the reference genome, H37Rv. We assembled nearly all GC-rich repetitive PE/PPE family genes (166/168) and identified variants within these genes. With an estimated genotypic error rate of 5.3 % from MinION data, we demonstrated identification of variants to include the conventional drug resistance mutations, and those that contribute to the resistance phenotype (efflux pumps/transporter) and virulence. Reference-based alignment of the assembly allowed detection of deletions and insertions. MinION sequencing provided a fully annotated assembly of a transmissible XDR strain from an endemic setting and showed its utility to provide further understanding of genomic processes within Mycobacterium tuberculosis.

Assembling the Setaria italica L. Beauv. genome into nine chromosomes and insights into regions affecting growth and drought tolerance

PubMed Central

Tsai, Kevin J.; Lu, Mei-Yeh Jade; Yang, Kai-Jung; Li, Mengyun; Teng, Yuchuan; Chen, Shihmay; Ku, Maurice S. B.; Li, Wen-Hsiung

2016-01-01

The diploid C4 plant foxtail millet (Setaria italica L. Beauv.) is an important crop in many parts of Africa and Asia for the vast consumption of its grain and ability to grow in harsh environments, but remains understudied in terms of complete genomic architecture. To date, there have been only two genome assembly and annotation efforts with neither assembly reaching over 86% of the estimated genome size. We have combined de novo assembly with custom reference-guided improvements on a popular cultivar of foxtail millet and have achieved a genome assembly of 477 Mbp in length, which represents over 97% of the estimated 490 Mbp. The assembly anchors over 98% of the predicted genes to the nine assembled nuclear chromosomes and contains more functional annotation gene models than previous assemblies. Our annotation has identified a large number of unique gene ontology terms related to metabolic activities, a region of chromosome 9 with several growth factor proteins, and regions syntenic with pearl millet or maize genomic regions that have been previously shown to affect growth. The new assembly and annotation for this important species can be used for detailed investigation and future innovations in growth for millet and other grains. PMID:27734962

Assembling the Setaria italica L. Beauv. genome into nine chromosomes and insights into regions affecting growth and drought tolerance.

PubMed

Tsai, Kevin J; Lu, Mei-Yeh Jade; Yang, Kai-Jung; Li, Mengyun; Teng, Yuchuan; Chen, Shihmay; Ku, Maurice S B; Li, Wen-Hsiung

2016-10-13

The diploid C 4 plant foxtail millet (Setaria italica L. Beauv.) is an important crop in many parts of Africa and Asia for the vast consumption of its grain and ability to grow in harsh environments, but remains understudied in terms of complete genomic architecture. To date, there have been only two genome assembly and annotation efforts with neither assembly reaching over 86% of the estimated genome size. We have combined de novo assembly with custom reference-guided improvements on a popular cultivar of foxtail millet and have achieved a genome assembly of 477 Mbp in length, which represents over 97% of the estimated 490 Mbp. The assembly anchors over 98% of the predicted genes to the nine assembled nuclear chromosomes and contains more functional annotation gene models than previous assemblies. Our annotation has identified a large number of unique gene ontology terms related to metabolic activities, a region of chromosome 9 with several growth factor proteins, and regions syntenic with pearl millet or maize genomic regions that have been previously shown to affect growth. The new assembly and annotation for this important species can be used for detailed investigation and future innovations in growth for millet and other grains.

Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics.

PubMed

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

2012-02-01

Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics. Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae). Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40× and 30×, respectively. Divergence from the reference significantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC. Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be efficiently obtained for molecular phylogenetics studies.

A clone-free, single molecule map of the domestic cow (Bos taurus) genome.

PubMed

Zhou, Shiguo; Goldstein, Steve; Place, Michael; Bechner, Michael; Patino, Diego; Potamousis, Konstantinos; Ravindran, Prabu; Pape, Louise; Rincon, Gonzalo; Hernandez-Ortiz, Juan; Medrano, Juan F; Schwartz, David C

2015-08-28

The cattle (Bos taurus) genome was originally selected for sequencing due to its economic importance and unique biology as a model organism for understanding other ruminants, or mammals. Currently, there are two cattle genome sequence assemblies (UMD3.1 and Btau4.6) from groups using dissimilar assembly algorithms, which were complemented by genetic and physical map resources. However, past comparisons between these assemblies revealed substantial differences. Consequently, such discordances have engendered ambiguities when using reference sequence data, impacting genomic studies in cattle and motivating construction of a new optical map resource--BtOM1.0--to guide comparisons and improvements to the current sequence builds. Accordingly, our comprehensive comparisons of BtOM1.0 against the UMD3.1 and Btau4.6 sequence builds tabulate large-to-immediate scale discordances requiring mediation. The optical map, BtOM1.0, spanning the B. taurus genome (Hereford breed, L1 Dominette 01449) was assembled from an optical map dataset consisting of 2,973,315 (439 X; raw dataset size before assembly) single molecule optical maps (Rmaps; 1 Rmap = 1 restriction mapped DNA molecule) generated by the Optical Mapping System. The BamHI map spans 2,575.30 Mb and comprises 78 optical contigs assembled by a combination of iterative (using the reference sequence: UMD3.1) and de novo assembly techniques. BtOM1.0 is a high-resolution physical map featuring an average restriction fragment size of 8.91 Kb. Comparisons of BtOM1.0 vs. UMD3.1, or Btau4.6, revealed that Btau4.6 presented far more discordances (7,463) vs. UMD3.1 (4,754). Overall, we found that Btau4.6 presented almost double the number of discordances than UMD3.1 across most of the 6 categories of sequence vs. map discrepancies, which are: COMPLEX (misassembly), DELs (extraneous sequences), INSs (missing sequences), ITs (Inverted/Translocated sequences), ECs (extra restriction cuts) and MCs (missing restriction cuts). Alignments of UMD3.1 and Btau4.6 to BtOM1.0 reveal discordances commensurate with previous reports, and affirm the NCBI's current designation of UMD3.1 sequence assembly as the "reference assembly" and the Btau4.6 as the "alternate assembly." The cattle genome optical map, BtOM1.0, when used as a comprehensive and largely independent guide, will greatly assist improvements to existing sequence builds, and later serve as an accurate physical scaffold for studies concerning the comparative genomics of cattle breeds.

The diploid genome sequence of an Asian individual

PubMed Central

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

2009-01-01

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

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum.

PubMed

VanBuren, Robert; Bryant, Doug; Edger, Patrick P; Tang, Haibao; Burgess, Diane; Challabathula, Dinakar; Spittle, Kristi; Hall, Richard; Gu, Jenny; Lyons, Eric; Freeling, Michael; Bartels, Dorothea; Ten Hallers, Boudewijn; Hastie, Alex; Michael, Todd P; Mockler, Todd C

2015-11-26

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.

The Past, Present, and Future of Human Centromere Genomics

PubMed Central

Aldrup-MacDonald, Megan E.; Sullivan, Beth A.

2014-01-01

The centromere is the chromosomal locus essential for chromosome inheritance and genome stability. Human centromeres are located at repetitive alpha satellite DNA arrays that compose approximately 5% of the genome. Contiguous alpha satellite DNA sequence is absent from the assembled reference genome, limiting current understanding of centromere organization and function. Here, we review the progress in centromere genomics spanning the discovery of the sequence to its molecular characterization and the work done during the Human Genome Project era to elucidate alpha satellite structure and sequence variation. We discuss exciting recent advances in alpha satellite sequence assembly that have provided important insight into the abundance and complex organization of this sequence on human chromosomes. In light of these new findings, we offer perspectives for future studies of human centromere assembly and function. PMID:24683489

Genome assembly from synthetic long read clouds

PubMed Central

Kuleshov, Volodymyr; Snyder, Michael P.; Batzoglou, Serafim

2016-01-01

Motivation: Despite rapid progress in sequencing technology, assembling de novo the genomes of new species as well as reconstructing complex metagenomes remains major technological challenges. New synthetic long read (SLR) technologies promise significant advances towards these goals; however, their applicability is limited by high sequencing requirements and the inability of current assembly paradigms to cope with combinations of short and long reads. Results: Here, we introduce Architect, a new de novo scaffolder aimed at SLR technologies. Unlike previous assembly strategies, Architect does not require a costly subassembly step; instead it assembles genomes directly from the SLR’s underlying short reads, which we refer to as read clouds. This enables a 4- to 20-fold reduction in sequencing requirements and a 5-fold increase in assembly contiguity on both genomic and metagenomic datasets relative to state-of-the-art assembly strategies aimed directly at fully subassembled long reads. Availability and Implementation: Our source code is freely available at https://github.com/kuleshov/architect. Contact: kuleshov@stanford.edu PMID:27307620

Trinity: Transcriptome Assembly for Genetic and Functional Analysis of Cancer | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

The cancer transcriptome is shaped by genetic changes, variation in gene transcription, mRNA processing, editing and stability, and the cancer microbiome. Deciphering this variation and understanding its implications on tumorigenesis requires sophisticated computational analyses. Most RNA-Seq analyses rely on methods that first map short reads to a reference genome, and then compare them to annotated transcripts or assemble them. However, this strategy can be limited when the cancer genome is substantially different than the reference or for detecting sequences from the cancer microbiome.

Software for pre-processing Illumina next-generation sequencing short read sequences

PubMed Central

2014-01-01

Background When compared to Sanger sequencing technology, next-generation sequencing (NGS) technologies are hindered by shorter sequence read length, higher base-call error rate, non-uniform coverage, and platform-specific sequencing artifacts. These characteristics lower the quality of their downstream analyses, e.g. de novo and reference-based assembly, by introducing sequencing artifacts and errors that may contribute to incorrect interpretation of data. Although many tools have been developed for quality control and pre-processing of NGS data, none of them provide flexible and comprehensive trimming options in conjunction with parallel processing to expedite pre-processing of large NGS datasets. Methods We developed ngsShoRT (next-generation sequencing Short Reads Trimmer), a flexible and comprehensive open-source software package written in Perl that provides a set of algorithms commonly used for pre-processing NGS short read sequences. We compared the features and performance of ngsShoRT with existing tools: CutAdapt, NGS QC Toolkit and Trimmomatic. We also compared the effects of using pre-processed short read sequences generated by different algorithms on de novo and reference-based assembly for three different genomes: Caenorhabditis elegans, Saccharomyces cerevisiae S288c, and Escherichia coli O157 H7. Results Several combinations of ngsShoRT algorithms were tested on publicly available Illumina GA II, HiSeq 2000, and MiSeq eukaryotic and bacteria genomic short read sequences with the focus on removing sequencing artifacts and low-quality reads and/or bases. Our results show that across three organisms and three sequencing platforms, trimming improved the mean quality scores of trimmed sequences. Using trimmed sequences for de novo and reference-based assembly improved assembly quality as well as assembler performance. In general, ngsShoRT outperformed comparable trimming tools in terms of trimming speed and improvement of de novo and reference-based assembly as measured by assembly contiguity and correctness. Conclusions Trimming of short read sequences can improve the quality of de novo and reference-based assembly and assembler performance. The parallel processing capability of ngsShoRT reduces trimming time and improves the memory efficiency when dealing with large datasets. We recommend combining sequencing artifacts removal, and quality score based read filtering and base trimming as the most consistent method for improving sequence quality and downstream assemblies. ngsShoRT source code, user guide and tutorial are available at http://research.bioinformatics.udel.edu/genomics/ngsShoRT/. ngsShoRT can be incorporated as a pre-processing step in genome and transcriptome assembly projects. PMID:24955109

A post-assembly genome-improvement toolkit (PAGIT) to obtain annotated genomes from contigs.

PubMed

Swain, Martin T; Tsai, Isheng J; Assefa, Samual A; Newbold, Chris; Berriman, Matthew; Otto, Thomas D

2012-06-07

Genome projects now produce draft assemblies within weeks owing to advanced high-throughput sequencing technologies. For milestone projects such as Escherichia coli or Homo sapiens, teams of scientists were employed to manually curate and finish these genomes to a high standard. Nowadays, this is not feasible for most projects, and the quality of genomes is generally of a much lower standard. This protocol describes software (PAGIT) that is used to improve the quality of draft genomes. It offers flexible functionality to close gaps in scaffolds, correct base errors in the consensus sequence and exploit reference genomes (if available) in order to improve scaffolding and generating annotations. The protocol is most accessible for bacterial and small eukaryotic genomes (up to 300 Mb), such as pathogenic bacteria, malaria and parasitic worms. Applying PAGIT to an E. coli assembly takes ∼24 h: it doubles the average contig size and annotates over 4,300 gene models.

Draft genome and reference transcriptomic resources for the urticating pine defoliator Thaumetopoea pityocampa (Lepidoptera: Notodontidae).

PubMed

Gschloessl, B; Dorkeld, F; Berges, H; Beydon, G; Bouchez, O; Branco, M; Bretaudeau, A; Burban, C; Dubois, E; Gauthier, P; Lhuillier, E; Nichols, J; Nidelet, S; Rocha, S; Sauné, L; Streiff, R; Gautier, M; Kerdelhué, C

2018-05-01

The pine processionary moth Thaumetopoea pityocampa (Lepidoptera: Notodontidae) is the main pine defoliator in the Mediterranean region. Its urticating larvae cause severe human and animal health concerns in the invaded areas. This species shows a high phenotypic variability for various traits, such as phenology, fecundity and tolerance to extreme temperatures. This study presents the construction and analysis of extensive genomic and transcriptomic resources, which are an obligate prerequisite to understand their underlying genetic architecture. Using a well-studied population from Portugal with peculiar phenological characteristics, the karyotype was first determined and a first draft genome of 537 Mb total length was assembled into 68,292 scaffolds (N50 = 164 kb). From this genome assembly, 29,415 coding genes were predicted. To circumvent some limitations for fine-scale physical mapping of genomic regions of interest, a 3X coverage BAC library was also developed. In particular, 11 BACs from this library were individually sequenced to assess the assembly quality. Additionally, de novo transcriptomic resources were generated from various developmental stages sequenced with HiSeq and MiSeq Illumina technologies. The reads were de novo assembled into 62,376 and 63,175 transcripts, respectively. Then, a robust subset of the genome-predicted coding genes, the de novo transcriptome assemblies and previously published 454/Sanger data were clustered to obtain a high-quality and comprehensive reference transcriptome consisting of 29,701 bona fide unigenes. These sequences covered 99% of the cegma and 88% of the busco highly conserved eukaryotic genes and 84% of the busco arthropod gene set. Moreover, 90% of these transcripts could be localized on the draft genome. The described information is available via a genome annotation portal (http://bipaa.genouest.org/sp/thaumetopoea_pityocampa/). © 2018 John Wiley & Sons Ltd.

Assembly and diploid architecture of an individual human genome via single-molecule technologies

PubMed Central

Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

2015-01-01

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality. PMID:26121404

Assembly and diploid architecture of an individual human genome via single-molecule technologies.

PubMed

Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

2015-08-01

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

Single sample resolution of rare microbial dark matter in a marine invertebrate metagenome

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

Miller, Ian J.; Weyna, Theodore R.; Fong, Stephen S.

Direct, untargeted sequencing of environmental samples (metagenomics) and de novo genome assembly enable the study of uncultured and phylogenetically divergent organisms. However, separating individual genomes from a mixed community has often relied on the differential-coverage analysis of multiple, deeply sequenced samples. In the metagenomic investigation of the marine bryozoan Bugula neritina, we uncovered seven bacterial genomes associated with a single B. neritina individual that appeared to be transient associates, two of which were unique to one individual and undetectable using certain “universal” 16S rRNA primers and probes. We recovered high quality genome assemblies for several rare instances of “microbial darkmore » matter,” or phylogenetically divergent bacteria lacking genomes in reference databases, from a single tissue sample that was not subjected to any physical or chemical pre-treatment. One of these rare, divergent organisms has a small (593 kbp), poorly annotated genome with low GC content (20.9%) and a 16S rRNA gene with just 65% sequence similarity to the closest reference sequence. Lastly, our findings illustrate the importance of sampling strategy and de novo assembly of metagenomic reads to understand the extent and function of bacterial biodiversity.« less

Single sample resolution of rare microbial dark matter in a marine invertebrate metagenome

DOE PAGES

Miller, Ian J.; Weyna, Theodore R.; Fong, Stephen S.; ...

2016-09-29

Direct, untargeted sequencing of environmental samples (metagenomics) and de novo genome assembly enable the study of uncultured and phylogenetically divergent organisms. However, separating individual genomes from a mixed community has often relied on the differential-coverage analysis of multiple, deeply sequenced samples. In the metagenomic investigation of the marine bryozoan Bugula neritina, we uncovered seven bacterial genomes associated with a single B. neritina individual that appeared to be transient associates, two of which were unique to one individual and undetectable using certain “universal” 16S rRNA primers and probes. We recovered high quality genome assemblies for several rare instances of “microbial darkmore » matter,” or phylogenetically divergent bacteria lacking genomes in reference databases, from a single tissue sample that was not subjected to any physical or chemical pre-treatment. One of these rare, divergent organisms has a small (593 kbp), poorly annotated genome with low GC content (20.9%) and a 16S rRNA gene with just 65% sequence similarity to the closest reference sequence. Lastly, our findings illustrate the importance of sampling strategy and de novo assembly of metagenomic reads to understand the extent and function of bacterial biodiversity.« less

Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly of Malassezia sympodialis

PubMed Central

Tellgren-Roth, Christian; Baudo, Charles D.; Kennell, John C.; Sun, Sheng; Billmyre, R. Blake; Schröder, Markus S.; Andersson, Anna; Holm, Tina; Sigurgeirsson, Benjamin; Wu, Guangxi; Sankaranarayanan, Sundar Ram; Siddharthan, Rahul; Sanyal, Kaustuv; Lundeberg, Joakim; Nystedt, Björn; Boekhout, Teun; Dawson, Thomas L.; Heitman, Joseph

2017-01-01

Abstract Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies. PMID:28100699

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

A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly

PubMed Central

2013-01-01

Background The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes. Results We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters. Conclusions These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies. PMID:23496952

A comparison across non-model animals suggests an optimal sequencing depth for de novo transcriptome assembly.

PubMed

Francis, Warren R; Christianson, Lynne M; Kiko, Rainer; Powers, Meghan L; Shaner, Nathan C; Haddock, Steven H D

2013-03-12

The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes. We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters. These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies.

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

USDA-ARS?s Scientific Manuscript database

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

Whole transcriptome analysis using next-generation sequencing of model species Setaria viridis to support C4 photosynthesis research.

PubMed

Xu, Jiajia; Li, Yuanyuan; Ma, Xiuling; Ding, Jianfeng; Wang, Kai; Wang, Sisi; Tian, Ye; Zhang, Hui; Zhu, Xin-Guang

2013-09-01

Setaria viridis is an emerging model species for genetic studies of C4 photosynthesis. Many basic molecular resources need to be developed to support for this species. In this paper, we performed a comprehensive transcriptome analysis from multiple developmental stages and tissues of S. viridis using next-generation sequencing technologies. Sequencing of the transcriptome from multiple tissues across three developmental stages (seed germination, vegetative growth, and reproduction) yielded a total of 71 million single end 100 bp long reads. Reference-based assembly using Setaria italica genome as a reference generated 42,754 transcripts. De novo assembly generated 60,751 transcripts. In addition, 9,576 and 7,056 potential simple sequence repeats (SSRs) covering S. viridis genome were identified when using the reference based assembled transcripts and the de novo assembled transcripts, respectively. This identified transcripts and SSR provided by this study can be used for both reverse and forward genetic studies based on S. viridis.

The Drosophila genome nexus: a population genomic resource of 623 Drosophila melanogaster genomes, including 197 from a single ancestral range population.

PubMed

Lack, Justin B; Cardeno, Charis M; Crepeau, Marc W; Taylor, William; Corbett-Detig, Russell B; Stevens, Kristian A; Langley, Charles H; Pool, John E

2015-04-01

Hundreds of wild-derived Drosophila melanogaster genomes have been published, but rigorous comparisons across data sets are precluded by differences in alignment methodology. The most common approach to reference-based genome assembly is a single round of alignment followed by quality filtering and variant detection. We evaluated variations and extensions of this approach and settled on an assembly strategy that utilizes two alignment programs and incorporates both substitutions and short indels to construct an updated reference for a second round of mapping prior to final variant detection. Utilizing this approach, we reassembled published D. melanogaster population genomic data sets and added unpublished genomes from several sub-Saharan populations. Most notably, we present aligned data from phase 3 of the Drosophila Population Genomics Project (DPGP3), which provides 197 genomes from a single ancestral range population of D. melanogaster (from Zambia). The large sample size, high genetic diversity, and potentially simpler demographic history of the DPGP3 sample will make this a highly valuable resource for fundamental population genetic research. The complete set of assemblies described here, termed the Drosophila Genome Nexus, presently comprises 623 consistently aligned genomes and is publicly available in multiple formats with supporting documentation and bioinformatic tools. This resource will greatly facilitate population genomic analysis in this model species by reducing the methodological differences between data sets. Copyright © 2015 by the Genetics Society of America.

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

PubMed Central

2011-01-01

Background BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. Results This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Conclusions Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed. PMID:21794110

Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes.

PubMed

Feltus, Frank A; Saski, Christopher A; Mockaitis, Keithanne; Haiminen, Niina; Parida, Laxmi; Smith, Zachary; Ford, James; Staton, Margaret E; Ficklin, Stephen P; Blackmon, Barbara P; Cheng, Chun-Huai; Schnell, Raymond J; Kuhn, David N; Motamayor, Juan-Carlos

2011-07-27

BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

Wild emmer genome architecture and diversity elucidate wheat evolution and domestication.

PubMed

Avni, Raz; Nave, Moran; Barad, Omer; Baruch, Kobi; Twardziok, Sven O; Gundlach, Heidrun; Hale, Iago; Mascher, Martin; Spannagl, Manuel; Wiebe, Krystalee; Jordan, Katherine W; Golan, Guy; Deek, Jasline; Ben-Zvi, Batsheva; Ben-Zvi, Gil; Himmelbach, Axel; MacLachlan, Ron P; Sharpe, Andrew G; Fritz, Allan; Ben-David, Roi; Budak, Hikmet; Fahima, Tzion; Korol, Abraham; Faris, Justin D; Hernandez, Alvaro; Mikel, Mark A; Levy, Avraham A; Steffenson, Brian; Maccaferri, Marco; Tuberosa, Roberto; Cattivelli, Luigi; Faccioli, Primetta; Ceriotti, Aldo; Kashkush, Khalil; Pourkheirandish, Mohammad; Komatsuda, Takao; Eilam, Tamar; Sela, Hanan; Sharon, Amir; Ohad, Nir; Chamovitz, Daniel A; Mayer, Klaus F X; Stein, Nils; Ronen, Gil; Peleg, Zvi; Pozniak, Curtis J; Akhunov, Eduard D; Distelfeld, Assaf

2017-07-07

Wheat ( Triticum spp.) is one of the founder crops that likely drove the Neolithic transition to sedentary agrarian societies in the Fertile Crescent more than 10,000 years ago. Identifying genetic modifications underlying wheat's domestication requires knowledge about the genome of its allo-tetraploid progenitor, wild emmer ( T. turgidum ssp. dicoccoides ). We report a 10.1-gigabase assembly of the 14 chromosomes of wild tetraploid wheat, as well as analyses of gene content, genome architecture, and genetic diversity. With this fully assembled polyploid wheat genome, we identified the causal mutations in Brittle Rachis 1 ( TtBtr1 ) genes controlling shattering, a key domestication trait. A study of genomic diversity among wild and domesticated accessions revealed genomic regions bearing the signature of selection under domestication. This reference assembly will serve as a resource for accelerating the genome-assisted improvement of modern wheat varieties. Copyright © 2017, American Association for the Advancement of Science.

BAUM: improving genome assembly by adaptive unique mapping and local overlap-layout-consensus approach.

PubMed

Wang, Anqi; Wang, Zhanyu; Li, Zheng; Li, Lei M

2018-06-15

It is highly desirable to assemble genomes of high continuity and consistency at low cost. The current bottleneck of draft genome continuity using the second generation sequencing (SGS) reads is primarily caused by uncertainty among repetitive sequences. Even though the single-molecule real-time sequencing technology is very promising to overcome the uncertainty issue, its relatively high cost and error rate add burden on budget or computation. Many long-read assemblers take the overlap-layout-consensus (OLC) paradigm, which is less sensitive to sequencing errors, heterozygosity and variability of coverage. However, current assemblers of SGS data do not sufficiently take advantage of the OLC approach. Aiming at minimizing uncertainty, the proposed method BAUM, breaks the whole genome into regions by adaptive unique mapping; then the local OLC is used to assemble each region in parallel. BAUM can (i) perform reference-assisted assembly based on the genome of a close species (ii) or improve the results of existing assemblies that are obtained based on short or long sequencing reads. The tests on two eukaryote genomes, a wild rice Oryza longistaminata and a parrot Melopsittacus undulatus, show that BAUM achieved substantial improvement on genome size and continuity. Besides, BAUM reconstructed a considerable amount of repetitive regions that failed to be assembled by existing short read assemblers. We also propose statistical approaches to control the uncertainty in different steps of BAUM. http://www.zhanyuwang.xin/wordpress/index.php/2017/07/21/baum. Supplementary data are available at Bioinformatics online.

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

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

VanBuren, Robert; Bryant, Doug; Edger, Patrick P.

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

DOE PAGES

VanBuren, Robert; Bryant, Doug; Edger, Patrick P.; ...

2015-11-11

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less

iDoComp: a compression scheme for assembled genomes

PubMed Central

Ochoa, Idoia; Hernaez, Mikel; Weissman, Tsachy

2015-01-01

Motivation: With the release of the latest next-generation sequencing (NGS) machine, the HiSeq X by Illumina, the cost of sequencing a Human has dropped to a mere $4000. Thus we are approaching a milestone in the sequencing history, known as the $1000 genome era, where the sequencing of individuals is affordable, opening the doors to effective personalized medicine. Massive generation of genomic data, including assembled genomes, is expected in the following years. There is crucial need for compression of genomes guaranteed of performing well simultaneously on different species, from simple bacteria to humans, which will ease their transmission, dissemination and analysis. Further, most of the new genomes to be compressed will correspond to individuals of a species from which a reference already exists on the database. Thus, it is natural to propose compression schemes that assume and exploit the availability of such references. Results: We propose iDoComp, a compressor of assembled genomes presented in FASTA format that compresses an individual genome using a reference genome for both the compression and the decompression. In terms of compression efficiency, iDoComp outperforms previously proposed algorithms in most of the studied cases, with comparable or better running time. For example, we observe compression gains of up to 60% in several cases, including H.sapiens data, when comparing with the best compression performance among the previously proposed algorithms. Availability: iDoComp is written in C and can be downloaded from: http://www.stanford.edu/~iochoa/iDoComp.html (We also provide a full explanation on how to run the program and an example with all the necessary files to run it.). Contact: iochoa@stanford.edu Supplementary information: Supplementary Data are available at Bioinformatics online. PMID:25344501

Re-annotation, improved large-scale assembly and establishment of a catalogue of noncoding loci for the genome of the model brown alga Ectocarpus.

PubMed

Cormier, Alexandre; Avia, Komlan; Sterck, Lieven; Derrien, Thomas; Wucher, Valentin; Andres, Gwendoline; Monsoor, Misharl; Godfroy, Olivier; Lipinska, Agnieszka; Perrineau, Marie-Mathilde; Van De Peer, Yves; Hitte, Christophe; Corre, Erwan; Coelho, Susana M; Cock, J Mark

2017-04-01

The genome of the filamentous brown alga Ectocarpus was the first to be completely sequenced from within the brown algal group and has served as a key reference genome both for this lineage and for the stramenopiles. We present a complete structural and functional reannotation of the Ectocarpus genome. The large-scale assembly of the Ectocarpus genome was significantly improved and genome-wide gene re-annotation using extensive RNA-seq data improved the structure of 11 108 existing protein-coding genes and added 2030 new loci. A genome-wide analysis of splicing isoforms identified an average of 1.6 transcripts per locus. A large number of previously undescribed noncoding genes were identified and annotated, including 717 loci that produce long noncoding RNAs. Conservation of lncRNAs between Ectocarpus and another brown alga, the kelp Saccharina japonica, suggests that at least a proportion of these loci serve a function. Finally, a large collection of single nucleotide polymorphism-based markers was developed for genetic analyses. These resources are available through an updated and improved genome database. This study significantly improves the utility of the Ectocarpus genome as a high-quality reference for the study of many important aspects of brown algal biology and as a reference for genomic analyses across the stramenopiles. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Finishing bacterial genome assemblies with Mix.

PubMed

Soueidan, Hayssam; Maurier, Florence; Groppi, Alexis; Sirand-Pugnet, Pascal; Tardy, Florence; Citti, Christine; Dupuy, Virginie; Nikolski, Macha

2013-01-01

Among challenges that hamper reaping the benefits of genome assembly are both unfinished assemblies and the ensuing experimental costs. First, numerous software solutions for genome de novo assembly are available, each having its advantages and drawbacks, without clear guidelines as to how to choose among them. Second, these solutions produce draft assemblies that often require a resource intensive finishing phase. In this paper we address these two aspects by developing Mix , a tool that mixes two or more draft assemblies, without relying on a reference genome and having the goal to reduce contig fragmentation and thus speed-up genome finishing. The proposed algorithm builds an extension graph where vertices represent extremities of contigs and edges represent existing alignments between these extremities. These alignment edges are used for contig extension. The resulting output assembly corresponds to a set of paths in the extension graph that maximizes the cumulative contig length. We evaluate the performance of Mix on bacterial NGS data from the GAGE-B study and apply it to newly sequenced Mycoplasma genomes. Resulting final assemblies demonstrate a significant improvement in the overall assembly quality. In particular, Mix is consistent by providing better overall quality results even when the choice is guided solely by standard assembly statistics, as is the case for de novo projects. Mix is implemented in Python and is available at https://github.com/cbib/MIX, novel data for our Mycoplasma study is available at http://services.cbib.u-bordeaux2.fr/mix/.

Genomic structural differences between cattle and river buffalo identified through a combination and genomic and transcriptomic analysis

USDA-ARS?s Scientific Manuscript database

Water buffalo (Bubalus bubalis L.) is an important livestock species worldwide. Like many other livestock species, water buffalo lacks high quality and continuous reference genome assembly required for fine-scale comparative genomics studies. In this work, we present a dataset, which characterizes g...

The W22 genome: a foundation for maize functional genomics and transposon biology

USDA-ARS?s Scientific Manuscript database

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using small-read sequencing technologies. We show that significant structural heterogeneity exists in ...

Fine mapping of copy number variations on two cattle genome assemblies using high density SNP array

USDA-ARS?s Scientific Manuscript database

Btau_4.0 and UMD3.1 are two distinct cattle reference genome assemblies. In our previous study using the low density BovineSNP50 array, we reported a copy number variation (CNV) analysis on Btau_4.0 with 521 animals of 21 cattle breeds, yielding 682 CNV regions with a total length of 139.8 megabases...

Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly of Malassezia sympodialis.

PubMed

Zhu, Yafeng; Engström, Pär G; Tellgren-Roth, Christian; Baudo, Charles D; Kennell, John C; Sun, Sheng; Billmyre, R Blake; Schröder, Markus S; Andersson, Anna; Holm, Tina; Sigurgeirsson, Benjamin; Wu, Guangxi; Sankaranarayanan, Sundar Ram; Siddharthan, Rahul; Sanyal, Kaustuv; Lundeberg, Joakim; Nystedt, Björn; Boekhout, Teun; Dawson, Thomas L; Heitman, Joseph; Scheynius, Annika; Lehtiö, Janne

2017-03-17

Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene annotation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodialis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Endogenous avian leukosis viral loci in the Red Jungle Fowl genome assembly.

PubMed

Benkel, Bernhard; Rutherford, Katherine

2014-12-01

The current build (galGal4) of the genome of the ancestor of the modern chicken, the Red Jungle Fowl, contains a single endogenous avian leukosis viral element (ALVE) on chromosome 1 (designated RSV-LTR; family ERVK). The assembly shows the ALVE provirus juxtaposed with a member of a second family of avian endogenous retroviruses (designated GGERV20; family ERVL); however, the status of the 3' end of the ALVE element as well as its flanking region remain unclear due to a gap in the reference genome sequence. In this study, we filled the gap in the assembly using a combination of long-range PCR (LR-PCR) and a short contig present in the unassembled portion of the reference genome database. Our results demonstrate that the ALVE element (ALVE-JFevB) is inserted into the putative envelope region of a GGERV20 element, roughly 1 kbp from its 3' end, and that ALVE-JFevB is complete, and depending on its expression status, potentially capable of directing the production of virus. Moreover, the unassembled portion of the genome database contains junction fragments for a second, previously characterized endogenous proviral element, ALVE-6. ©2014 Poultry Science Association Inc.

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

Statistical Significance of Optical Map Alignments

PubMed Central

Sarkar, Deepayan; Goldstein, Steve; Schwartz, David C.

2012-01-01

Abstract The Optical Mapping System constructs ordered restriction maps spanning entire genomes through the assembly and analysis of large datasets comprising individually analyzed genomic DNA molecules. Such restriction maps uniquely reveal mammalian genome structure and variation, but also raise computational and statistical questions beyond those that have been solved in the analysis of smaller, microbial genomes. We address the problem of how to filter maps that align poorly to a reference genome. We obtain map-specific thresholds that control errors and improve iterative assembly. We also show how an optimal self-alignment score provides an accurate approximation to the probability of alignment, which is useful in applications seeking to identify structural genomic abnormalities. PMID:22506568

Improvement of genome assembly completeness and identification of novel full-length protein-coding genes by RNA-seq in the giant panda genome.

PubMed

Chen, Meili; Hu, Yibo; Liu, Jingxing; Wu, Qi; Zhang, Chenglin; Yu, Jun; Xiao, Jingfa; Wei, Fuwen; Wu, Jiayan

2015-12-11

High-quality and complete gene models are the basis of whole genome analyses. The giant panda (Ailuropoda melanoleuca) genome was the first genome sequenced on the basis of solely short reads, but the genome annotation had lacked the support of transcriptomic evidence. In this study, we applied RNA-seq to globally improve the genome assembly completeness and to detect novel expressed transcripts in 12 tissues from giant pandas, by using a transcriptome reconstruction strategy that combined reference-based and de novo methods. Several aspects of genome assembly completeness in the transcribed regions were effectively improved by the de novo assembled transcripts, including genome scaffolding, the detection of small-size assembly errors, the extension of scaffold/contig boundaries, and gap closure. Through expression and homology validation, we detected three groups of novel full-length protein-coding genes. A total of 12.62% of the novel protein-coding genes were validated by proteomic data. GO annotation analysis showed that some of the novel protein-coding genes were involved in pigmentation, anatomical structure formation and reproduction, which might be related to the development and evolution of the black-white pelage, pseudo-thumb and delayed embryonic implantation of giant pandas. The updated genome annotation will help further giant panda studies from both structural and functional perspectives.

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes).

PubMed

Dessimoz, Christophe; Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-09-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references.

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes)

PubMed Central

Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro

2011-01-01

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references. PMID:21712341

GenomePeek—an online tool for prokaryotic genome and metagenome analysis

DOE PAGES

McNair, Katelyn; Edwards, Robert A.

2015-06-16

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

Subgenome-anchored physical frameworks of the allotetraploid Upland cotton (Gossypium hirsutum L.) genome, and an approach toward reference-grade assemblies of polyploids

USDA-ARS?s Scientific Manuscript database

Like many agricultural crops, the cultivated cotton genome is large and polyploid (~2.5Gb), consisting of two very similar repeat-rich subgenomes, whose size and complexity pose significant challenges for accurate genome reconstruction using whole-genome shotgun approaches. A strategy for accurately...

Gee Fu: a sequence version and web-services database tool for genomic assembly, genome feature and NGS data.

PubMed

Ramirez-Gonzalez, Ricardo; Caccamo, Mario; MacLean, Daniel

2011-10-01

Scientists now use high-throughput sequencing technologies and short-read assembly methods to create draft genome assemblies in just days. Tools and pipelines like the assembler, and the workflow management environments make it easy for a non-specialist to implement complicated pipelines to produce genome assemblies and annotations very quickly. Such accessibility results in a proliferation of assemblies and associated files, often for many organisms. These assemblies get used as a working reference by lots of different workers, from a bioinformatician doing gene prediction or a bench scientist designing primers for PCR. Here we describe Gee Fu, a database tool for genomic assembly and feature data, including next-generation sequence alignments. Gee Fu is an instance of a Ruby-On-Rails web application on a feature database that provides web and console interfaces for input, visualization of feature data via AnnoJ, access to data through a web-service interface, an API for direct data access by Ruby scripts and access to feature data stored in BAM files. Gee Fu provides a platform for storing and sharing different versions of an assembly and associated features that can be accessed and updated by bench biologists and bioinformaticians in ways that are easy and useful for each. http://tinyurl.com/geefu dan.maclean@tsl.ac.uk.

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

PubMed

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

2016-07-01

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

Three invariant Hi-C interaction patterns: Applications to genome assembly.

PubMed

Oddes, Sivan; Zelig, Aviv; Kaplan, Noam

2018-06-01

Assembly of reference-quality genomes from next-generation sequencing data is a key challenge in genomics. Recently, we and others have shown that Hi-C data can be used to address several outstanding challenges in the field of genome assembly. This principle has since been developed in academia and industry, and has been used in the assembly of several major genomes. In this paper, we explore the central principles underlying Hi-C-based assembly approaches, by quantitatively defining and characterizing three invariant Hi-C interaction patterns on which these approaches can build: Intrachromosomal interaction enrichment, distance-dependent interaction decay and local interaction smoothness. Specifically, we evaluate to what degree each invariant pattern holds on a single locus level in different species, cell types and Hi-C map resolutions. We find that these patterns are generally consistent across species and cell types but are affected by sequencing depth, and that matrix balancing improves consistency of loci with all three invariant patterns. Finally, we overview current Hi-C-based assembly approaches in light of these invariant patterns and demonstrate how local interaction smoothness can be used to easily detect scaffolding errors in extremely sparse Hi-C maps. We suggest that simultaneously considering all three invariant patterns may lead to better Hi-C-based genome assembly methods. Copyright © 2018 Elsevier Inc. All rights reserved.

Tips and tricks for the assembly of a Corynebacterium pseudotuberculosis genome using a semiconductor sequencer.

PubMed

Ramos, Rommel Thiago Jucá; Carneiro, Adriana Ribeiro; Soares, Siomar de Castro; dos Santos, Anderson Rodrigues; Almeida, Sintia; Guimarães, Luis; Figueira, Flávia; Barbosa, Eudes; Tauch, Andreas; Azevedo, Vasco; Silva, Artur

2013-03-01

New sequencing platforms have enabled rapid decoding of complete prokaryotic genomes at relatively low cost. The Ion Torrent platform is an example of these technologies, characterized by lower coverage, generating challenges for the genome assembly. One particular problem is the lack of genomes that enable reference-based assembly, such as the one used in the present study, Corynebacterium pseudotuberculosis biovar equi, which causes high economic losses in the US equine industry. The quality treatment strategy incorporated into the assembly pipeline enabled a 16-fold greater use of the sequencing data obtained compared with traditional quality filter approaches. Data preprocessing prior to the de novo assembly enabled the use of known methodologies in the next-generation sequencing data assembly. Moreover, manual curation was proved to be essential for ensuring a quality assembly, which was validated by comparative genomics with other species of the genus Corynebacterium. The present study presents a modus operandi that enables a greater and better use of data obtained from semiconductor sequencing for obtaining the complete genome from a prokaryotic microorganism, C. pseudotuberculosis, which is not a traditional biological model such as Escherichia coli. © 2012 The Authors. Published by Society for Applied Microbiology and Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Harnessing Whole Genome Sequencing in Medical Mycology.

PubMed

Cuomo, Christina A

2017-01-01

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

Genome Sequence of Torulaspora delbrueckii NRRL Y-50541, Isolated from Mezcal Fermentation

PubMed Central

Gomez-Angulo, Jorge; Vega-Alvarado, Leticia; Escalante-García, Zazil; Grande, Ricardo; Gschaedler-Mathis, Anne; Amaya-Delgado, Lorena

2015-01-01

Torulaspora delbrueckii presents metabolic features interesting for biotechnological applications (in the dairy and wine industries). Recently, the T. delbrueckii CBS 1146 genome, which has been maintained under laboratory conditions since 1970, was published. Thus, a genome of a new mezcal yeast was sequenced and characterized and showed genetic differences and a higher genome assembly quality, offering a better reference genome. PMID:26205871

Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

PubMed

Seoane-Zonjic, Pedro; Cañas, Rafael A; Bautista, Rocío; Gómez-Maldonado, Josefa; Arrillaga, Isabel; Fernández-Pozo, Noé; Claros, M Gonzalo; Cánovas, Francisco M; Ávila, Concepción

2016-02-27

In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members. The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a Ruby gem for this class of analyses.

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

Bennetzen, Jeffrey L; Yang, Xiaohan; Ye, Chuyu

We generated a high-quality reference genome sequence for foxtail millet (Setaria italica). The {approx}400-Mb assembly covers {approx}80% of the genome and >95% of the gene space. The assembly was anchored to a 992-locus genetic map and was annotated by comparison with >1.3 million expressed sequence tag reads. We produced more than 580 million RNA-Seq reads to facilitate expression analyses. We also sequenced Setaria viridis, the ancestral wild relative of S. italica, and identified regions of differential single-nucleotide polymorphism density, distribution of transposable elements, small RNA content, chromosomal rearrangement and segregation distortion. The genus Setaria includes natural and cultivated species thatmore » demonstrate a wide capacity for adaptation. The genetic basis of this adaptation was investigated by comparing five sequenced grass genomes. We also used the diploid Setaria genome to evaluate the ongoing genome assembly of a related polyploid, switchgrass (Panicum virgatum).« less

Icarus: visualizer for de novo assembly evaluation.

PubMed

Mikheenko, Alla; Valin, Gleb; Prjibelski, Andrey; Saveliev, Vladislav; Gurevich, Alexey

2016-11-01

: Data visualization plays an increasingly important role in NGS data analysis. With advances in both sequencing and computational technologies, it has become a new bottleneck in genomics studies. Indeed, evaluation of de novo genome assemblies is one of the areas that can benefit from the visualization. However, even though multiple quality assessment methods are now available, existing visualization tools are hardly suitable for this purpose. Here, we present Icarus-a novel genome visualizer for accurate assessment and analysis of genomic draft assemblies, which is based on the tool QUAST. Icarus can be used in studies where a related reference genome is available, as well as for non-model organisms. The tool is available online and as a standalone application. http://cab.spbu.ru/software/icarus CONTACT: aleksey.gurevich@spbu.ruSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Construction of Reference Chromosome-Scale Pseudomolecules for Potato: Integrating the Potato Genome with Genetic and Physical Maps

PubMed Central

Sharma, Sanjeev Kumar; Bolser, Daniel; de Boer, Jan; Sønderkær, Mads; Amoros, Walter; Carboni, Martin Federico; D’Ambrosio, Juan Martín; de la Cruz, German; Di Genova, Alex; Douches, David S.; Eguiluz, Maria; Guo, Xiao; Guzman, Frank; Hackett, Christine A.; Hamilton, John P.; Li, Guangcun; Li, Ying; Lozano, Roberto; Maass, Alejandro; Marshall, David; Martinez, Diana; McLean, Karen; Mejía, Nilo; Milne, Linda; Munive, Susan; Nagy, Istvan; Ponce, Olga; Ramirez, Manuel; Simon, Reinhard; Thomson, Susan J.; Torres, Yerisf; Waugh, Robbie; Zhang, Zhonghua; Huang, Sanwen; Visser, Richard G. F.; Bachem, Christian W. B.; Sagredo, Boris; Feingold, Sergio E.; Orjeda, Gisella; Veilleux, Richard E.; Bonierbale, Merideth; Jacobs, Jeanne M. E.; Milbourne, Dan; Martin, David Michael Alan; Bryan, Glenn J.

2013-01-01

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker−based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal “pseudomolecules”. PMID:24062527

Dissection of the Octoploid Strawberry Genome by Deep Sequencing of the Genomes of Fragaria Species

PubMed Central

Hirakawa, Hideki; Shirasawa, Kenta; Kosugi, Shunichi; Tashiro, Kosuke; Nakayama, Shinobu; Yamada, Manabu; Kohara, Mistuyo; Watanabe, Akiko; Kishida, Yoshie; Fujishiro, Tsunakazu; Tsuruoka, Hisano; Minami, Chiharu; Sasamoto, Shigemi; Kato, Midori; Nanri, Keiko; Komaki, Akiko; Yanagi, Tomohiro; Guoxin, Qin; Maeda, Fumi; Ishikawa, Masami; Kuhara, Satoru; Sato, Shusei; Tabata, Satoshi; Isobe, Sachiko N.

2014-01-01

Cultivated strawberry (Fragaria x ananassa) is octoploid and shows allogamous behaviour. The present study aims at dissecting this octoploid genome through comparison with its wild relatives, F. iinumae, F. nipponica, F. nubicola, and F. orientalis by de novo whole-genome sequencing on an Illumina and Roche 454 platforms. The total length of the assembled Illumina genome sequences obtained was 698 Mb for F. x ananassa, and ∼200 Mb each for the four wild species. Subsequently, a virtual reference genome termed FANhybrid_r1.2 was constructed by integrating the sequences of the four homoeologous subgenomes of F. x ananassa, from which heterozygous regions in the Roche 454 and Illumina genome sequences were eliminated. The total length of FANhybrid_r1.2 thus created was 173.2 Mb with the N50 length of 5137 bp. The Illumina-assembled genome sequences of F. x ananassa and the four wild species were then mapped onto the reference genome, along with the previously published F. vesca genome sequence to establish the subgenomic structure of F. x ananassa. The strategy adopted in this study has turned out to be successful in dissecting the genome of octoploid F. x ananassa and appears promising when applied to the analysis of other polyploid plant species. PMID:24282021

RNASeq-based genome annotation and identification of long-noncoding RNAs in the grapevine cultivar 'Riesling'

USDA-ARS?s Scientific Manuscript database

The technological advances of RNA-seq and de novo transcriptome assembly have enabled genome annotation and transcriptome profiling in heterozygous species. This is a promising approach to improving the annotation of the reference genome sequence of grapevine (Vitis vinifera L.), a species of high-l...

Genome sequences of three strains of Aspergillus flavus for the biological control of Aflatoxin

USDA-ARS?s Scientific Manuscript database

The genomes of three strains of Aspergillus flavus with demonstrated utility for the biological control of aflatoxin were sequenced. These sequences were assembled with MIRA and annotated with Augustus using A. flavus strain 3357 (NCBI EQ963472) as a reference. Each strain had a genome of 36.3 to ...

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

Short read Illumina data for the de novo assembly of a non-model snail species transcriptome (Radix balthica, Basommatophora, Pulmonata), and a comparison of assembler performance

PubMed Central

2011-01-01

Background Until recently, read lengths on the Solexa/Illumina system were too short to reliably assemble transcriptomes without a reference sequence, especially for non-model organisms. However, with read lengths up to 100 nucleotides available in the current version, an assembly without reference genome should be possible. For this study we created an EST data set for the common pond snail Radix balthica by Illumina sequencing of a normalized transcriptome. Performance of three different short read assemblers was compared with respect to: the number of contigs, their length, depth of coverage, their quality in various BLAST searches and the alignment to mitochondrial genes. Results A single sequencing run of a normalized RNA pool resulted in 16,923,850 paired end reads with median read length of 61 bases. The assemblies generated by VELVET, OASES, and SeqMan NGEN differed in the total number of contigs, contig length, the number and quality of gene hits obtained by BLAST searches against various databases, and contig performance in the mt genome comparison. While VELVET produced the highest overall number of contigs, a large fraction of these were of small size (< 200bp), and gave redundant hits in BLAST searches and the mt genome alignment. The best overall contig performance resulted from the NGEN assembly. It produced the second largest number of contigs, which on average were comparable to the OASES contigs but gave the highest number of gene hits in two out of four BLAST searches against different reference databases. A subsequent meta-assembly of the four contig sets resulted in larger contigs, less redundancy and a higher number of BLAST hits. Conclusion Our results document the first de novo transcriptome assembly of a non-model species using Illumina sequencing data. We show that de novo transcriptome assembly using this approach yields results useful for downstream applications, in particular if a meta-assembly of contig sets is used to increase contig quality. These results highlight the ongoing need for improvements in assembly methodology. PMID:21679424

Haemonchus contortus: Genome Structure, Organization and Comparative Genomics.

PubMed

Laing, R; Martinelli, A; Tracey, A; Holroyd, N; Gilleard, J S; Cotton, J A

2016-01-01

One of the first genome sequencing projects for a parasitic nematode was that for Haemonchus contortus. The open access data from the Wellcome Trust Sanger Institute provided a valuable early resource for the research community, particularly for the identification of specific genes and genetic markers. Later, a second sequencing project was initiated by the University of Melbourne, and the two draft genome sequences for H. contortus were published back-to-back in 2013. There is a pressing need for long-range genomic information for genetic mapping, population genetics and functional genomic studies, so we are continuing to improve the Wellcome Trust Sanger Institute assembly to provide a finished reference genome for H. contortus. This review describes this process, compares the H. contortus genome assemblies with draft genomes from other members of the strongylid group and discusses future directions for parasite genomics using the H. contortus model. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome Sequence of Torulaspora delbrueckii NRRL Y-50541, Isolated from Mezcal Fermentation.

PubMed

Gomez-Angulo, Jorge; Vega-Alvarado, Leticia; Escalante-García, Zazil; Grande, Ricardo; Gschaedler-Mathis, Anne; Amaya-Delgado, Lorena; Arrizon, Javier; Sanchez-Flores, Alejandro

2015-07-23

Torulaspora delbrueckii presents metabolic features interesting for biotechnological applications (in the dairy and wine industries). Recently, the T. delbrueckii CBS 1146 genome, which has been maintained under laboratory conditions since 1970, was published. Thus, a genome of a new mezcal yeast was sequenced and characterized and showed genetic differences and a higher genome assembly quality, offering a better reference genome. Copyright © 2015 Gomez-Angulo et al.

Rice SNP-seek database update: new SNPs, indels, and queries.

PubMed

Mansueto, Locedie; Fuentes, Roven Rommel; Borja, Frances Nikki; Detras, Jeffery; Abriol-Santos, Juan Miguel; Chebotarov, Dmytro; Sanciangco, Millicent; Palis, Kevin; Copetti, Dario; Poliakov, Alexandre; Dubchak, Inna; Solovyev, Victor; Wing, Rod A; Hamilton, Ruaraidh Sackville; Mauleon, Ramil; McNally, Kenneth L; Alexandrov, Nickolai

2017-01-04

We describe updates to the Rice SNP-Seek Database since its first release. We ran a new SNP-calling pipeline followed by filtering that resulted in complete, base, filtered and core SNP datasets. Besides the Nipponbare reference genome, the pipeline was run on genome assemblies of IR 64, 93-11, DJ 123 and Kasalath. New genotype query and display features are added for reference assemblies, SNP datasets and indels. JBrowse now displays BAM, VCF and other annotation tracks, the additional genome assemblies and an embedded VISTA genome comparison viewer. Middleware is redesigned for improved performance by using a hybrid of HDF5 and RDMS for genotype storage. Query modules for genotypes, varieties and genes are improved to handle various constraints. An integrated list manager allows the user to pass query parameters for further analysis. The SNP Annotator adds traits, ontology terms, effects and interactions to markers in a list. Web-service calls were implemented to access most data. These features enable seamless querying of SNP-Seek across various biological entities, a step toward semi-automated gene-trait association discovery. URL: http://snp-seek.irri.org. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome assembly and annotation of Arabidopsis halleri, a model for heavy metal hyperaccumulation and evolutionary ecology.

PubMed

Briskine, Roman V; Paape, Timothy; Shimizu-Inatsugi, Rie; Nishiyama, Tomoaki; Akama, Satoru; Sese, Jun; Shimizu, Kentaro K

2017-09-01

The self-incompatible species Arabidopsis halleri is a close relative of the self-compatible model plant Arabidopsis thaliana. The broad European and Asian distribution and heavy metal hyperaccumulation ability make A. halleri a useful model for ecological genomics studies. We used long-insert mate-pair libraries to improve the genome assembly of the A. halleri ssp. gemmifera Tada mine genotype (W302) collected from a site with high contamination by heavy metals in Japan. After five rounds of forced selfing, heterozygosity was reduced to 0.04%, which facilitated subsequent genome assembly. Our assembly now covers 196 Mb or 78% of the estimated genome size and achieved scaffold N50 length of 712 kb. To validate assembly and annotation, we used synteny of A. halleri Tada mine with a previously published high-quality reference assembly of a closely related species, Arabidopsis lyrata. Further validation of the assembly quality comes from synteny and phylogenetic analysis of the HEAVY METAL ATPASE4 (HMA4) and METAL TOLERANCE PROTEIN1 (MTP1) regions using published sequences from European A. halleri for comparison. Three tandemly duplicated copies of HMA4, key gene involved in cadmium and zinc hyperaccumulation, were assembled on a single scaffold. The assembly will enhance the genomewide studies of A. halleri as well as the allopolyploid Arabidopsis kamchatica derived from A. lyrata and A. halleri. © 2016 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

The ecoresponsive genome of Daphnia pulex

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

Colbourne, John K.; Pfrender, Michael E.; Gilbert, Donald

2011-02-04

This document provides supporting material related to the sequencing of the ecoresponsive genome of Daphnia pulex. This material includes information on materials and methods and supporting text, as well as supplemental figures, tables, and references. The coverage of materials and methods addresses genome sequence, assembly, and mapping to chromosomes, gene inventory, attributes of a compact genome, the origin and preservation of Daphnia pulex genes, implications of Daphnia's genome structure, evolutionary diversification of duplicated genes, functional significance of expanded gene families, and ecoresponsive genes. Supporting text covers chromosome studies, gene homology among Daphnia genomes, micro-RNA and transposable elements and the 46more » Daphnia pulex opsins. 36 figures, 50 tables, 183 references.« less

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

Fast-SG: an alignment-free algorithm for hybrid assembly.

PubMed

Di Genova, Alex; Ruz, Gonzalo A; Sagot, Marie-France; Maass, Alejandro

2018-05-01

Long-read sequencing technologies are the ultimate solution for genome repeats, allowing near reference-level reconstructions of large genomes. However, long-read de novo assembly pipelines are computationally intense and require a considerable amount of coverage, thereby hindering their broad application to the assembly of large genomes. Alternatively, hybrid assembly methods that combine short- and long-read sequencing technologies can reduce the time and cost required to produce de novo assemblies of large genomes. Here, we propose a new method, called Fast-SG, that uses a new ultrafast alignment-free algorithm specifically designed for constructing a scaffolding graph using light-weight data structures. Fast-SG can construct the graph from either short or long reads. This allows the reuse of efficient algorithms designed for short-read data and permits the definition of novel modular hybrid assembly pipelines. Using comprehensive standard datasets and benchmarks, we show how Fast-SG outperforms the state-of-the-art short-read aligners when building the scaffoldinggraph and can be used to extract linking information from either raw or error-corrected long reads. We also show how a hybrid assembly approach using Fast-SG with shallow long-read coverage (5X) and moderate computational resources can produce long-range and accurate reconstructions of the genomes of Arabidopsis thaliana (Ler-0) and human (NA12878). Fast-SG opens a door to achieve accurate hybrid long-range reconstructions of large genomes with low effort, high portability, and low cost.

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

A Hybrid Approach for the Automated Finishing of Bacterial Genomes

PubMed Central

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

2013-01-01

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

ATLAS (Automatic Tool for Local Assembly Structures) - A Comprehensive Infrastructure for Assembly, Annotation, and Genomic Binning of Metagenomic and Metaranscripomic Data

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

White, Richard A.; Brown, Joseph M.; Colby, Sean M.

ATLAS (Automatic Tool for Local Assembly Structures) is a comprehensive multiomics data analysis pipeline that is massively parallel and scalable. ATLAS contains a modular analysis pipeline for assembly, annotation, quantification and genome binning of metagenomics and metatranscriptomics data and a framework for reference metaproteomic database construction. ATLAS transforms raw sequence data into functional and taxonomic data at the microbial population level and provides genome-centric resolution through genome binning. ATLAS provides robust taxonomy based on majority voting of protein coding open reading frames rolled-up at the contig level using modified lowest common ancestor (LCA) analysis. ATLAS provides robust taxonomy based onmore » majority voting of protein coding open reading frames rolled-up at the contig level using modified lowest common ancestor (LCA) analysis. ATLAS is user-friendly, easy install through bioconda maintained as open-source on GitHub, and is implemented in Snakemake for modular customizable workflows.« less

X. couchianus and X. hellerii genome models provide genomic variation insight among Xiphophorus species.

PubMed

Shen, Yingjia; Chalopin, Domitille; Garcia, Tzintzuni; Boswell, Mikki; Boswell, William; Shiryev, Sergey A; Agarwala, Richa; Volff, Jean-Nicolas; Postlethwait, John H; Schartl, Manfred; Minx, Patrick; Warren, Wesley C; Walter, Ronald B

2016-01-07

Xiphophorus fishes are represented by 26 live-bearing species of tropical fish that express many attributes (e.g., viviparity, genetic and phenotypic variation, ecological adaptation, varied sexual developmental mechanisms, ability to produce fertile interspecies hybrids) that have made attractive research models for over 85 years. Use of various interspecies hybrids to investigate the genetics underlying spontaneous and induced tumorigenesis has resulted in the development and maintenance of pedigreed Xiphophorus lines specifically bred for research. The recent availability of the X. maculatus reference genome assembly now provides unprecedented opportunities for novel and exciting comparative research studies among Xiphophorus species. We present sequencing, assembly and annotation of two new genomes representing Xiphophorus couchianus and Xiphophorus hellerii. The final X. couchianus and X. hellerii assemblies have total sizes of 708 Mb and 734 Mb and correspond to 98 % and 102 % of the X. maculatus Jp 163 A genome size, respectively. The rates of single nucleotide change range from 1 per 52 bp to 1 per 69 bp among the three genomes and the impact of putatively damaging variants are presented. In addition, a survey of transposable elements allowed us to deduce an ancestral TE landscape, uncovered potential active TEs and document a recent burst of TEs during evolution of this genus. Two new Xiphophorus genomes and their corresponding transcriptomes were efficiently assembled, the former using a novel guided assembly approach. Three assembled genome sequences within this single vertebrate order of new world live-bearing fishes will accelerate our understanding of relationship between environmental adaptation and genome evolution. In addition, these genome resources provide capability to determine allele specific gene regulation among interspecies hybrids produced by crossing any of the three species that are known to produce progeny predisposed to tumor development.

A Near-Complete Haplotype-Phased Genome of the Dikaryotic Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici Reveals High Interhaplotype Diversity

PubMed Central

Sperschneider, Jana; Garnica, Diana P.; Miller, Marisa E.; Taylor, Jennifer M.; Dodds, Peter N.; Park, Robert F.

2018-01-01

ABSTRACT A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales. In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies. PMID:29463659

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.

Whole genome assembly of a natto production strain Bacillus subtilis natto from very short read data.

PubMed

Nishito, Yukari; Osana, Yasunori; Hachiya, Tsuyoshi; Popendorf, Kris; Toyoda, Atsushi; Fujiyama, Asao; Itaya, Mitsuhiro; Sakakibara, Yasubumi

2010-04-16

Bacillus subtilis natto is closely related to the laboratory standard strain B. subtilis Marburg 168, and functions as a starter for the production of the traditional Japanese food "natto" made from soybeans. Although re-sequencing whole genomes of several laboratory domesticated B. subtilis 168 derivatives has already been attempted using short read sequencing data, the assembly of the whole genome sequence of a closely related strain, B. subtilis natto, from very short read data is more challenging, particularly with our aim to assemble one fully connected scaffold from short reads around 35 bp in length. We applied a comparative genome assembly method, which combines de novo assembly and reference guided assembly, to one of the B. subtilis natto strains. We successfully assembled 28 scaffolds and managed to avoid substantial fragmentation. Completion of the assembly through long PCR experiments resulted in one connected scaffold for B. subtilis natto. Based on the assembled genome sequence, our orthologous gene analysis between natto BEST195 and Marburg 168 revealed that 82.4% of 4375 predicted genes in BEST195 are one-to-one orthologous to genes in 168, with two genes in-paralog, 3.2% are deleted in 168, 14.3% are inserted in BEST195, and 5.9% of genes present in 168 are deleted in BEST195. The natto genome contains the same alleles in the promoter region of degQ and the coding region of swrAA as the wild strain, RO-FF-1. These are specific for gamma-PGA production ability, which is related to natto production. Further, the B. subtilis natto strain completely lacked a polyketide synthesis operon, disrupted the plipastatin production operon, and possesses previously unidentified transposases. The determination of the whole genome sequence of Bacillus subtilis natto provided detailed analyses of a set of genes related to natto production, demonstrating the number and locations of insertion sequences that B. subtilis natto harbors but B. subtilis 168 lacks. Multiple genome-level comparisons among five closely related Bacillus species were also carried out. The determined genome sequence of B. subtilis natto and gene annotations are available from the Natto genome browser http://natto-genome.org/.

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.

Whole genome sequences of the USMARC sheep diversity panel v 2.4 aligned to the ovine reference genome assembly

USDA-ARS?s Scientific Manuscript database

A searchable and publicly viewable set of mapped genomes from 96 rams from 9 US sheep breeds was created. The nine pure breeds were selected to represent genetic diversity for traits such as fertility, prolificacy, maternal ability, growth rate, carcass leanness, wool quality, mature weight, and lo...

Comparative sequence alignment reveals River Buffalo genomic structural differences compared with cattle

USDA-ARS?s Scientific Manuscript database

Water buffalo (Bubalus bubalis L.) represent a significant livestock species with high economic importance and promising characteristics for production; however, like many other livestock species, they lack a highly polished and contiguous reference genome assembly for use in high-resolution compara...

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-02-01

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

A reference genetic map of C. clementina hort. ex Tan.; citrus evolution inferences from comparative mapping

PubMed Central

2012-01-01

Background Most modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a ‘Mediterranean’ mandarin × sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine. Results Five parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between ‘Mediterranean’ mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Conclusions A reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the ‘Mediterranean’ mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents. PMID:23126659

RADseq dataset with 90% missing data fully resolves recent radiation of Petalidium (Acanthaceae) in the ultra-arid deserts of Namibia.

PubMed

Tripp, Erin A; Tsai, Yi-Hsin Erica; Zhuang, Yongbin; Dexter, Kyle G

2017-10-01

Deserts, even those at tropical latitudes, often have strikingly low levels of plant diversity, particularly within genera. One remarkable exception to this pattern is the genus Petalidium (Acanthaceae), in which 37 of 40 named species occupy one of the driest environments on Earth, the Namib Desert of Namibia and neighboring Angola. To contribute to understanding this enigmatic diversity, we generated RADseq data for 47 accessions of Petalidium representing 22 species. We explored the impacts of 18 different combinations of assembly parameters in de novo assembly of the data across nine levels of missing data plus a best practice assembly using a reference Acanthaceae genome for a total of 171 sequence datasets assembled. RADseq data assembled at several thresholds of missing data, including 90% missing data, yielded phylogenetic hypotheses of Petalidium that were confidently and nearly fully resolved, which is notable given that divergence time analyses suggest a crown age for African species of 3.6-1.4 Ma. De novo assembly of our data yielded the most strongly supported and well-resolved topologies; in contrast, reference-based assembly performed poorly, perhaps due in part to moderate phylogenetic divergence between the reference genome, Ruellia speciosa , and the ingroup. Overall, we found that Petalidium , despite the harshness of the environment in which species occur, shows a net diversification rate (0.8-2.1 species per my) on par with those of diverse genera in tropical, Mediterranean, and alpine environments.

Genomic resources and draft assemblies of the human and porcine varieties of scabies mites, Sarcoptes scabiei var. hominis and var. suis.

PubMed

Mofiz, Ehtesham; Holt, Deborah C; Seemann, Torsten; Currie, Bart J; Fischer, Katja; Papenfuss, Anthony T

2016-06-02

The scabies mite, Sarcoptes scabiei, is a parasitic arachnid and cause of the infectious skin disease scabies in humans and mange in other animal species. Scabies infections are a major health problem, particularly in remote Indigenous communities in Australia, where secondary group A streptococcal and Staphylococcus aureus infections of scabies sores are thought to drive the high rate of rheumatic heart disease and chronic kidney disease. We sequenced the genome of two samples of Sarcoptes scabiei var. hominis obtained from unrelated patients with crusted scabies located in different parts of northern Australia using the Illumina HiSeq. We also sequenced samples of Sarcoptes scabiei var. suis from a pig model. Because of the small size of the scabies mite, these data are derived from pools of thousands of mites and are metagenomic, including host and microbiome DNA. We performed cleaning and de novo assembly and present Sarcoptes scabiei var. hominis and var. suis draft reference genomes. We have constructed a preliminary annotation of this reference comprising 13,226 putative coding sequences based on sequence similarity to known proteins. We have developed extensive genomic resources for the scabies mite, including reference genomes and a preliminary annotation.

Draft Genome Assemblies of Enterobacter aerogenes CDC 6003-71, Enterobacter cloacae CDC 442-68, and Pantoea agglomerans UA 0804-01.

PubMed

Minogue, T D; Daligault, H E; Davenport, K W; Bishop-Lilly, K A; Bruce, D C; Chain, P S; Coyne, S R; Chertkov, O; Freitas, T; Frey, K G; Jaissle, J; Koroleva, G I; Ladner, J T; Palacios, G F; Redden, C L; Xu, Y; Johnson, S L

2014-10-23

The Enterobacteriaceae are environmental and enteric microbes. We sequenced the genomes of two Enterobacter reference strains, E. aerogenes CDC 6003-71 and E. cloacae CDC 442-68, as well as one near neighbor used as an exclusionary reference for diagnostics, Pantoea agglomerans CDC UA0804-01. The genome sizes range from 4.72 to 5.55 Mbp and have G+C contents from 54.6 to 55.1%. Copyright © 2014 Minogue et al.

VirSorter: mining viral signal from microbial genomic data.

PubMed

Roux, Simon; Enault, Francois; Hurwitz, Bonnie L; Sullivan, Matthew B

2015-01-01

Viruses of microbes impact all ecosystems where microbes drive key energy and substrate transformations including the oceans, humans and industrial fermenters. However, despite this recognized importance, our understanding of viral diversity and impacts remains limited by too few model systems and reference genomes. One way to fill these gaps in our knowledge of viral diversity is through the detection of viral signal in microbial genomic data. While multiple approaches have been developed and applied for the detection of prophages (viral genomes integrated in a microbial genome), new types of microbial genomic data are emerging that are more fragmented and larger scale, such as Single-cell Amplified Genomes (SAGs) of uncultivated organisms or genomic fragments assembled from metagenomic sequencing. Here, we present VirSorter, a tool designed to detect viral signal in these different types of microbial sequence data in both a reference-dependent and reference-independent manner, leveraging probabilistic models and extensive virome data to maximize detection of novel viruses. Performance testing shows that VirSorter's prophage prediction capability compares to that of available prophage predictors for complete genomes, but is superior in predicting viral sequences outside of a host genome (i.e., from extrachromosomal prophages, lytic infections, or partially assembled prophages). Furthermore, VirSorter outperforms existing tools for fragmented genomic and metagenomic datasets, and can identify viral signal in assembled sequence (contigs) as short as 3kb, while providing near-perfect identification (>95% Recall and 100% Precision) on contigs of at least 10kb. Because VirSorter scales to large datasets, it can also be used in "reverse" to more confidently identify viral sequence in viral metagenomes by sorting away cellular DNA whether derived from gene transfer agents, generalized transduction or contamination. Finally, VirSorter is made available through the iPlant Cyberinfrastructure that provides a web-based user interface interconnected with the required computing resources. VirSorter thus complements existing prophage prediction softwares to better leverage fragmented, SAG and metagenomic datasets in a way that will scale to modern sequencing. Given these features, VirSorter should enable the discovery of new viruses in microbial datasets, and further our understanding of uncultivated viral communities across diverse ecosystems.

VirSorter: mining viral signal from microbial genomic data

PubMed Central

Roux, Simon; Enault, Francois; Hurwitz, Bonnie L.

2015-01-01

Viruses of microbes impact all ecosystems where microbes drive key energy and substrate transformations including the oceans, humans and industrial fermenters. However, despite this recognized importance, our understanding of viral diversity and impacts remains limited by too few model systems and reference genomes. One way to fill these gaps in our knowledge of viral diversity is through the detection of viral signal in microbial genomic data. While multiple approaches have been developed and applied for the detection of prophages (viral genomes integrated in a microbial genome), new types of microbial genomic data are emerging that are more fragmented and larger scale, such as Single-cell Amplified Genomes (SAGs) of uncultivated organisms or genomic fragments assembled from metagenomic sequencing. Here, we present VirSorter, a tool designed to detect viral signal in these different types of microbial sequence data in both a reference-dependent and reference-independent manner, leveraging probabilistic models and extensive virome data to maximize detection of novel viruses. Performance testing shows that VirSorter’s prophage prediction capability compares to that of available prophage predictors for complete genomes, but is superior in predicting viral sequences outside of a host genome (i.e., from extrachromosomal prophages, lytic infections, or partially assembled prophages). Furthermore, VirSorter outperforms existing tools for fragmented genomic and metagenomic datasets, and can identify viral signal in assembled sequence (contigs) as short as 3kb, while providing near-perfect identification (>95% Recall and 100% Precision) on contigs of at least 10kb. Because VirSorter scales to large datasets, it can also be used in “reverse” to more confidently identify viral sequence in viral metagenomes by sorting away cellular DNA whether derived from gene transfer agents, generalized transduction or contamination. Finally, VirSorter is made available through the iPlant Cyberinfrastructure that provides a web-based user interface interconnected with the required computing resources. VirSorter thus complements existing prophage prediction softwares to better leverage fragmented, SAG and metagenomic datasets in a way that will scale to modern sequencing. Given these features, VirSorter should enable the discovery of new viruses in microbial datasets, and further our understanding of uncultivated viral communities across diverse ecosystems. PMID:26038737

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.

Improving the goat long-read assembly with optical mapping

USDA-ARS?s Scientific Manuscript database

Reference genome assemblies provide important context in genetics by standardizing the order of genes and providing a universal set of coordinates for individual nucleotides. Often due to the high complexity of genic regions and higher copy number of genes involved in immune function, immunity-relat...

Upgrading short-read animal genome assemblies to chromosome level using comparative genomics and a universal probe set.

PubMed

Damas, Joana; O'Connor, Rebecca; Farré, Marta; Lenis, Vasileios Panagiotis E; Martell, Henry J; Mandawala, Anjali; Fowler, Katie; Joseph, Sunitha; Swain, Martin T; Griffin, Darren K; Larkin, Denis M

2017-05-01

Most recent initiatives to sequence and assemble new species' genomes de novo fail to achieve the ultimate endpoint to produce contigs, each representing one whole chromosome. Even the best-assembled genomes (using contemporary technologies) consist of subchromosomal-sized scaffolds. To circumvent this problem, we developed a novel approach that combines computational algorithms to merge scaffolds into chromosomal fragments, PCR-based scaffold verification, and physical mapping to chromosomes. Multigenome-alignment-guided probe selection led to the development of a set of universal avian BAC clones that permit rapid anchoring of multiple scaffolds to chromosomes on all avian genomes. As proof of principle, we assembled genomes of the pigeon ( Columbia livia ) and peregrine falcon ( Falco peregrinus ) to chromosome levels comparable, in continuity, to avian reference genomes. Both species are of interest for breeding, cultural, food, and/or environmental reasons. Pigeon has a typical avian karyotype (2n = 80), while falcon (2n = 50) is highly rearranged compared to the avian ancestor. By using chromosome breakpoint data, we established that avian interchromosomal breakpoints appear in the regions of low density of conserved noncoding elements (CNEs) and that the chromosomal fission sites are further limited to long CNE "deserts." This corresponds with fission being the rarest type of rearrangement in avian genome evolution. High-throughput multiple hybridization and rapid capture strategies using the current BAC set provide the basis for assembling numerous avian (and possibly other reptilian) species, while the overall strategy for scaffold assembly and mapping provides the basis for an approach that (provided metaphases can be generated) could be applied to any animal genome. © 2017 Damas et al.; Published by Cold Spring Harbor Laboratory Press.

Upgrading short-read animal genome assemblies to chromosome level using comparative genomics and a universal probe set

PubMed Central

O'Connor, Rebecca; Lenis, Vasileios Panagiotis E.; Martell, Henry J.; Mandawala, Anjali; Fowler, Katie; Joseph, Sunitha; Swain, Martin T.; Griffin, Darren K.; Larkin, Denis M.

2017-01-01

Most recent initiatives to sequence and assemble new species’ genomes de novo fail to achieve the ultimate endpoint to produce contigs, each representing one whole chromosome. Even the best-assembled genomes (using contemporary technologies) consist of subchromosomal-sized scaffolds. To circumvent this problem, we developed a novel approach that combines computational algorithms to merge scaffolds into chromosomal fragments, PCR-based scaffold verification, and physical mapping to chromosomes. Multigenome-alignment-guided probe selection led to the development of a set of universal avian BAC clones that permit rapid anchoring of multiple scaffolds to chromosomes on all avian genomes. As proof of principle, we assembled genomes of the pigeon (Columbia livia) and peregrine falcon (Falco peregrinus) to chromosome levels comparable, in continuity, to avian reference genomes. Both species are of interest for breeding, cultural, food, and/or environmental reasons. Pigeon has a typical avian karyotype (2n = 80), while falcon (2n = 50) is highly rearranged compared to the avian ancestor. By using chromosome breakpoint data, we established that avian interchromosomal breakpoints appear in the regions of low density of conserved noncoding elements (CNEs) and that the chromosomal fission sites are further limited to long CNE “deserts.” This corresponds with fission being the rarest type of rearrangement in avian genome evolution. High-throughput multiple hybridization and rapid capture strategies using the current BAC set provide the basis for assembling numerous avian (and possibly other reptilian) species, while the overall strategy for scaffold assembly and mapping provides the basis for an approach that (provided metaphases can be generated) could be applied to any animal genome. PMID:27903645

Assembled genomic and tissue-specific transcriptomic data resources for two genetically distinct lines of Cowpea ( Vigna unguiculata (L.) Walp)

PubMed Central

Spriggs, Andrew; Henderson, Steven T.; Hand, Melanie L.; Johnson, Susan D.; Taylor, Jennifer M.; Koltunow, Anna

2018-01-01

Cowpea ( Vigna unguiculata (L.) Walp) is an important legume crop for food security in areas of low-input and smallholder farming throughout Africa and Asia. Genetic improvements are required to increase yield and resilience to biotic and abiotic stress and to enhance cowpea crop performance. An integrated cowpea genomic and gene expression data resource has the potential to greatly accelerate breeding and the delivery of novel genetic traits for cowpea. Extensive genomic resources for cowpea have been absent from the public domain; however, a recent early release reference genome for IT97K-499-35 ( Vigna unguiculata v1.0, NSF, UCR, USAID, DOE-JGI, http://phytozome.jgi.doe.gov/) has now been established in a collaboration between the Joint Genome Institute (JGI) and University California (UC) Riverside. Here we release supporting genomic and transcriptomic data for IT97K-499-35 and a second transformable cowpea variety, IT86D-1010. The transcriptome resource includes six tissue-specific datasets for each variety, with particular emphasis on reproductive tissues that extend and support the V. unguiculata v1.0 reference. Annotations have been included in our resource to allow direct mapping to the v1.0 cowpea reference. Access to this resource provided here is supported by raw and assembled data downloads. PMID:29528046

Assembled genomic and tissue-specific transcriptomic data resources for two genetically distinct lines of Cowpea ( Vigna unguiculata (L.) Walp).

PubMed

Spriggs, Andrew; Henderson, Steven T; Hand, Melanie L; Johnson, Susan D; Taylor, Jennifer M; Koltunow, Anna

2018-02-09

Cowpea ( Vigna unguiculata (L.) Walp) is an important legume crop for food security in areas of low-input and smallholder farming throughout Africa and Asia. Genetic improvements are required to increase yield and resilience to biotic and abiotic stress and to enhance cowpea crop performance. An integrated cowpea genomic and gene expression data resource has the potential to greatly accelerate breeding and the delivery of novel genetic traits for cowpea. Extensive genomic resources for cowpea have been absent from the public domain; however, a recent early release reference genome for IT97K-499-35 ( Vigna unguiculata  v1.0, NSF, UCR, USAID, DOE-JGI, http://phytozome.jgi.doe.gov/) has now been established in a collaboration between the Joint Genome Institute (JGI) and University California (UC) Riverside. Here we release supporting genomic and transcriptomic data for IT97K-499-35 and a second transformable cowpea variety, IT86D-1010. The transcriptome resource includes six tissue-specific datasets for each variety, with particular emphasis on reproductive tissues that extend and support the V. unguiculata v1.0 reference. Annotations have been included in our resource to allow direct mapping to the v1.0 cowpea reference. Access to this resource provided here is supported by raw and assembled data downloads.

De Novo Transcriptome Assembly and Analyses of Gene Expression during Photomorphogenesis in Diploid Wheat Triticum monococcum

PubMed Central

Naithani, Sushma; Sullivan, Chris; Preece, Justin; Tiwari, Vijay K.; Elser, Justin; Leonard, Jeffrey M.; Sage, Abigail; Gresham, Cathy; Kerhornou, Arnaud; Bolser, Dan; McCarthy, Fiona; Kersey, Paul; Lazo, Gerard R.; Jaiswal, Pankaj

2014-01-01

Background Triticum monococcum (2n) is a close ancestor of T. urartu, the A-genome progenitor of cultivated hexaploid wheat, and is therefore a useful model for the study of components regulating photomorphogenesis in diploid wheat. In order to develop genetic and genomic resources for such a study, we constructed genome-wide transcriptomes of two Triticum monococcum subspecies, the wild winter wheat T. monococcum ssp. aegilopoides (accession G3116) and the domesticated spring wheat T. monococcum ssp. monococcum (accession DV92) by generating de novo assemblies of RNA-Seq data derived from both etiolated and green seedlings. Principal Findings The de novo transcriptome assemblies of DV92 and G3116 represent 120,911 and 117,969 transcripts, respectively. We successfully mapped ∼90% of these transcripts from each accession to barley and ∼95% of the transcripts to T. urartu genomes. However, only ∼77% transcripts mapped to the annotated barley genes and ∼85% transcripts mapped to the annotated T. urartu genes. Differential gene expression analyses revealed 22% more light up-regulated and 35% more light down-regulated transcripts in the G3116 transcriptome compared to DV92. The DV92 and G3116 mRNA sequence reads aligned against the reference barley genome led to the identification of ∼500,000 single nucleotide polymorphism (SNP) and ∼22,000 simple sequence repeat (SSR) sites. Conclusions De novo transcriptome assemblies of two accessions of the diploid wheat T. monococcum provide new empirical transcriptome references for improving Triticeae genome annotations, and insights into transcriptional programming during photomorphogenesis. The SNP and SSR sites identified in our analysis provide additional resources for the development of molecular markers. PMID:24821410

It’s More Than Stamp Collecting: How Genome Sequencing Can Unify Biological Research

PubMed Central

Richards, Stephen

2015-01-01

The availability of reference genome sequences, especially the human reference, has revolutionized the study of biology. However, whilst the genomes of some species have been fully sequenced, a wide range of biological problems still cannot be effectively studied for lack of genome sequence information. Here, I identify neglected areas of biology and describe how both targeted species sequencing and more broad taxonomic surveys of the tree of life can address important biological questions. I enumerate the significant benefits that would accrue from sequencing a broader range of taxa, as well as discuss the technical advances in sequencing and assembly methods that would allow for wide-ranging application of whole-genome analysis. Finally, I suggest that in addition to “Big Science” survey initiatives to sequence the tree of life, a modified infrastructure-funding paradigm would better support reference genome sequence generation for research communities most in need. PMID:26003218

It's more than stamp collecting: how genome sequencing can unify biological research.

PubMed

Richards, Stephen

2015-07-01

The availability of reference genome sequences, especially the human reference, has revolutionized the study of biology. However, while the genomes of some species have been fully sequenced, a wide range of biological problems still cannot be effectively studied for lack of genome sequence information. Here, I identify neglected areas of biology and describe how both targeted species sequencing and more broad taxonomic surveys of the tree of life can address important biological questions. I enumerate the significant benefits that would accrue from sequencing a broader range of taxa, as well as discuss the technical advances in sequencing and assembly methods that would allow for wide-ranging application of whole-genome analysis. Finally, I suggest that in addition to 'big science' survey initiatives to sequence the tree of life, a modified infrastructure-funding paradigm would better support reference genome sequence generation for research communities most in need. Copyright © 2015 Elsevier Ltd. All rights reserved.

RNA-Seq analysis and transcriptome assembly for blackberry (Rubus sp. Var. Lochness) fruit.

PubMed

Garcia-Seco, Daniel; Zhang, Yang; Gutierrez-Mañero, Francisco J; Martin, Cathie; Ramos-Solano, Beatriz

2015-01-22

There is an increasing interest in berries, especially blackberries in the diet, because of recent reports of their health benefits due to their high content of flavonoids. A broad range of genomic tools are available for other Rosaceae species but these tools are still lacking in the Rubus genus, thus limiting gene discovery and the breeding of improved varieties. De novo RNA-seq of ripe blackberries grown under field conditions was performed using Illumina Hiseq 2000. Almost 9 billion nucleotide bases were sequenced in total. Following assembly, 42,062 consensus sequences were detected. For functional annotation, 33,040 (NR), 32,762 (NT), 21,932 (Swiss-Prot), 20,134 (KEGG), 13,676 (COG), 24,168 (GO) consensus sequences were annotated using different databases; in total 34,552 annotated sequences were identified. For protein prediction analysis, the number of coding DNA sequences (CDS) that mapped to the protein database was 32,540. Non redundant (NR), annotation showed that 25,418 genes (73.5%) has the highest similarity with Fragaria vesca subspecies vesca. Reanalysis was undertaken by aligning the reads with this reference genome for a deeper analysis of the transcriptome. We demonstrated that de novo assembly, using Trinity and later annotation with Blast using different databases, were complementary to alignment to the reference sequence using SOAPaligner/SOAP2. The Fragaria reference genome belongs to a species in the same family as blackberry (Rosaceae) but to a different genus. Since blackberries are tetraploids, the possibility of artefactual gene chimeras resulting from mis-assembly was tested with one of the genes sequenced by RNAseq, Chalcone Synthase (CHS). cDNAs encoding this protein were cloned and sequenced. Primers designed to the assembled sequences accurately distinguished different contigs, at least for chalcone synthase genes. We prepared and analysed transcriptome data from ripe blackberries, for which prior genomic information was limited. This new sequence information will improve the knowledge of this important and healthy fruit, providing an invaluable new tool for biological research.

Genome Sequencing and Analysis of Yersina pestis KIM D27, an Avirulent Strain Exempt from Select Agent Regulation

PubMed Central

Losada, Liliana; Varga, John J.; Hostetler, Jessica; Radune, Diana; Kim, Maria; Durkin, Scott; Schneewind, Olaf; Nierman, William C.

2011-01-01

Yersinia pestis is the causative agent of the plague. Y. pestis KIM 10+ strain was passaged and selected for loss of the 102 kb pgm locus, resulting in an attenuated strain, KIM D27. In this study, whole genome sequencing was performed on KIM D27 in order to identify any additional differences. Initial assemblies of 454 data were highly fragmented, and various bioinformatic tools detected between 15 and 465 SNPs and INDELs when comparing both strains, the vast majority associated with A or T homopolymer sequences. Consequently, Illumina sequencing was performed to improve the quality of the assembly. Hybrid sequence assemblies were performed and a total of 56 validated SNP/INDELs and 5 repeat differences were identified in the D27 strain relative to published KIM 10+ sequence. However, further analysis showed that 55 of these SNP/INDELs and 3 repeats were errors in the KIM 10+ reference sequence. We conclude that both 454 and Illumina sequencing were required to obtain the most accurate and rapid sequence results for Y. pestis KIMD27. SNP and INDELS calls were most accurate when both Newbler and CLC Genomics Workbench were employed. For purposes of obtaining high quality genome sequence differences between strains, any identified differences should be verified in both the new and reference genomes. PMID:21559501

Genome sequencing and analysis of Yersina pestis KIM D27, an avirulent strain exempt from select agent regulation.

PubMed

Losada, Liliana; Varga, John J; Hostetler, Jessica; Radune, Diana; Kim, Maria; Durkin, Scott; Schneewind, Olaf; Nierman, William C

2011-04-29

Yersinia pestis is the causative agent of the plague. Y. pestis KIM 10+ strain was passaged and selected for loss of the 102 kb pgm locus, resulting in an attenuated strain, KIM D27. In this study, whole genome sequencing was performed on KIM D27 in order to identify any additional differences. Initial assemblies of 454 data were highly fragmented, and various bioinformatic tools detected between 15 and 465 SNPs and INDELs when comparing both strains, the vast majority associated with A or T homopolymer sequences. Consequently, Illumina sequencing was performed to improve the quality of the assembly. Hybrid sequence assemblies were performed and a total of 56 validated SNP/INDELs and 5 repeat differences were identified in the D27 strain relative to published KIM 10+ sequence. However, further analysis showed that 55 of these SNP/INDELs and 3 repeats were errors in the KIM 10+ reference sequence. We conclude that both 454 and Illumina sequencing were required to obtain the most accurate and rapid sequence results for Y. pestis KIMD27. SNP and INDELS calls were most accurate when both Newbler and CLC Genomics Workbench were employed. For purposes of obtaining high quality genome sequence differences between strains, any identified differences should be verified in both the new and reference genomes.

Draft Genome Sequence of Microbacterium sp. Strain UCD-TDU (Phylum Actinobacteria)

PubMed Central

Bendiks, Zachary A.; Lang, Jenna M.; Darling, Aaron E.; Coil, David A.

2013-01-01

Here, we present the draft genome sequence of Microbacterium sp. strain UCD-TDU, a member of the phylum Actinobacteria. The assembly contains 3,746,321 bp (in 8 scaffolds). This strain was isolated from a residential toilet as part of an undergraduate student research project to sequence reference genomes of microbes from the built environment. PMID:23516225

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

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome.

PubMed

Bickhart, Derek M; Rosen, Benjamin D; Koren, Sergey; Sayre, Brian L; Hastie, Alex R; Chan, Saki; Lee, Joyce; Lam, Ernest T; Liachko, Ivan; Sullivan, Shawn T; Burton, Joshua N; Huson, Heather J; Nystrom, John C; Kelley, Christy M; Hutchison, Jana L; Zhou, Yang; Sun, Jiajie; Crisà, Alessandra; Ponce de León, F Abel; Schwartz, John C; Hammond, John A; Waldbieser, Geoffrey C; Schroeder, Steven G; Liu, George E; Dunham, Maitreya J; Shendure, Jay; Sonstegard, Tad S; Phillippy, Adam M; Van Tassell, Curtis P; Smith, Timothy P L

2017-04-01

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ∼400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

PubMed Central

Bickhart, Derek M.; Rosen, Benjamin D.; Koren, Sergey; Sayre, Brian L.; Hastie, Alex R.; Chan, Saki; Lee, Joyce; Lam, Ernest T.; Liachko, Ivan; Sullivan, Shawn T.; Burton, Joshua N.; Huson, Heather J.; Nystrom, John C.; Kelley, Christy M.; Hutchison, Jana L.; Zhou, Yang; Sun, Jiajie; Crisà, Alessandra; de León, F. Abel Ponce; Schwartz, John C.; Hammond, John A.; Waldbieser, Geoffrey C.; Schroeder, Steven G.; Liu, George E.; Dunham, Maitreya J.; Shendure, Jay; Sonstegard, Tad S.; Phillippy, Adam M.; Van Tassell, Curtis P.; Smith, Timothy P.L.

2018-01-01

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus), based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ~400-fold improvement in continuity due to properly assembled gaps compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex ever produced for an individual of a ruminant species. PMID:28263316

Resolving the problem of multiple accessions of the same transcript deposited across various public databases.

PubMed

Weirick, Tyler; John, David; Uchida, Shizuka

2017-03-01

Maintaining the consistency of genomic annotations is an increasingly complex task because of the iterative and dynamic nature of assembly and annotation, growing numbers of biological databases and insufficient integration of annotations across databases. As information exchange among databases is poor, a 'novel' sequence from one reference annotation could be annotated in another. Furthermore, relationships to nearby or overlapping annotated transcripts are even more complicated when using different genome assemblies. To better understand these problems, we surveyed current and previous versions of genomic assemblies and annotations across a number of public databases containing long noncoding RNA. We identified numerous discrepancies of transcripts regarding their genomic locations, transcript lengths and identifiers. Further investigation showed that the positional differences between reference annotations of essentially the same transcript could lead to differences in its measured expression at the RNA level. To aid in resolving these problems, we present the algorithm 'Universal Genomic Accession Hash (UGAHash)' and created an open source web tool to encourage the usage of the UGAHash algorithm. The UGAHash web tool (http://ugahash.uni-frankfurt.de) can be accessed freely without registration. The web tool allows researchers to generate Universal Genomic Accessions for genomic features or to explore annotations deposited in the public databases of the past and present versions. We anticipate that the UGAHash web tool will be a valuable tool to check for the existence of transcripts before judging the newly discovered transcripts as novel. © The Author 2016. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Revealing misassembled segments in the bovine reference genome by high resolution linkage disequilibrium scan

USDA-ARS?s Scientific Manuscript database

Misassembly signatures, created by shuffling the order of sequences while assembling a genome, can be easily seen by analyzing the unexpected behaviour of the linkage disequilibrium (LD) decay. A heuristic process was proposed to identify those misassembly signatures and presented the ones found in ...

rnaQUAST: a quality assessment tool for de novo transcriptome assemblies.

PubMed

Bushmanova, Elena; Antipov, Dmitry; Lapidus, Alla; Suvorov, Vladimir; Prjibelski, Andrey D

2016-07-15

Ability to generate large RNA-Seq datasets created a demand for both de novo and reference-based transcriptome assemblers. However, while many transcriptome assemblers are now available, there is still no unified quality assessment tool for RNA-Seq assemblies. We present rnaQUAST-a tool for evaluating RNA-Seq assembly quality and benchmarking transcriptome assemblers using reference genome and gene database. rnaQUAST calculates various metrics that demonstrate completeness and correctness levels of the assembled transcripts, and outputs them in a user-friendly report. rnaQUAST is implemented in Python and is freely available at http://bioinf.spbau.ru/en/rnaquast ap@bioinf.spbau.ru Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

PARRoT- a homology-based strategy to quantify and compare RNA-sequencing from non-model organisms.

PubMed

Gan, Ruei-Chi; Chen, Ting-Wen; Wu, Timothy H; Huang, Po-Jung; Lee, Chi-Ching; Yeh, Yuan-Ming; Chiu, Cheng-Hsun; Huang, Hsien-Da; Tang, Petrus

2016-12-22

Next-generation sequencing promises the de novo genomic and transcriptomic analysis of samples of interests. However, there are only a few organisms having reference genomic sequences and even fewer having well-defined or curated annotations. For transcriptome studies focusing on organisms lacking proper reference genomes, the common strategy is de novo assembly followed by functional annotation. However, things become even more complicated when multiple transcriptomes are compared. Here, we propose a new analysis strategy and quantification methods for quantifying expression level which not only generate a virtual reference from sequencing data, but also provide comparisons between transcriptomes. First, all reads from the transcriptome datasets are pooled together for de novo assembly. The assembled contigs are searched against NCBI NR databases to find potential homolog sequences. Based on the searched result, a set of virtual transcripts are generated and served as a reference transcriptome. By using the same reference, normalized quantification values including RC (read counts), eRPKM (estimated RPKM) and eTPM (estimated TPM) can be obtained that are comparable across transcriptome datasets. In order to demonstrate the feasibility of our strategy, we implement it in the web service PARRoT. PARRoT stands for Pipeline for Analyzing RNA Reads of Transcriptomes. It analyzes gene expression profiles for two transcriptome sequencing datasets. For better understanding of the biological meaning from the comparison among transcriptomes, PARRoT further provides linkage between these virtual transcripts and their potential function through showing best hits in SwissProt, NR database, assigning GO terms. Our demo datasets showed that PARRoT can analyze two paired-end transcriptomic datasets of approximately 100 million reads within just three hours. In this study, we proposed and implemented a strategy to analyze transcriptomes from non-reference organisms which offers the opportunity to quantify and compare transcriptome profiles through a homolog based virtual transcriptome reference. By using the homolog based reference, our strategy effectively avoids the problems that may cause from inconsistencies among transcriptomes. This strategy will shed lights on the field of comparative genomics for non-model organism. We have implemented PARRoT as a web service which is freely available at http://parrot.cgu.edu.tw .

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano.

PubMed

Wasik, Kaja; Gurtowski, James; Zhou, Xin; Ramos, Olivia Mendivil; Delás, M Joaquina; Battistoni, Giorgia; El Demerdash, Osama; Falciatori, Ilaria; Vizoso, Dita B; Smith, Andrew D; Ladurner, Peter; Schärer, Lukas; McCombie, W Richard; Hannon, Gregory J; Schatz, Michael

2015-10-06

The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell-fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ∼75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.

Revealing the missing expressed genes beyond the human reference genome by RNA-Seq.

PubMed

Chen, Geng; Li, Ruiyuan; Shi, Leming; Qi, Junyi; Hu, Pengzhan; Luo, Jian; Liu, Mingyao; Shi, Tieliu

2011-12-02

The complete and accurate human reference genome is important for functional genomics researches. Therefore, the incomplete reference genome and individual specific sequences have significant effects on various studies. we used two RNA-Seq datasets from human brain tissues and 10 mixed cell lines to investigate the completeness of human reference genome. First, we demonstrated that in previously identified ~5 Mb Asian and ~5 Mb African novel sequences that are absent from the human reference genome of NCBI build 36, ~211 kb and ~201 kb of them could be transcribed, respectively. Our results suggest that many of those transcribed regions are not specific to Asian and African, but also present in Caucasian. Then, we found that the expressions of 104 RefSeq genes that are unalignable to NCBI build 37 in brain and cell lines are higher than 0.1 RPKM. 55 of them are conserved across human, chimpanzee and macaque, suggesting that there are still a significant number of functional human genes absent from the human reference genome. Moreover, we identified hundreds of novel transcript contigs that cannot be aligned to NCBI build 37, RefSeq genes and EST sequences. Some of those novel transcript contigs are also conserved among human, chimpanzee and macaque. By positioning those contigs onto the human genome, we identified several large deletions in the reference genome. Several conserved novel transcript contigs were further validated by RT-PCR. Our findings demonstrate that a significant number of genes are still absent from the incomplete human reference genome, highlighting the importance of further refining the human reference genome and curating those missing genes. Our study also shows the importance of de novo transcriptome assembly. The comparative approach between reference genome and other related human genomes based on the transcriptome provides an alternative way to refine the human reference genome.

Reference-free comparative genomics of 174 chloroplasts.

PubMed

Kua, Chai-Shian; Ruan, Jue; Harting, John; Ye, Cheng-Xi; Helmus, Matthew R; Yu, Jun; Cannon, Charles H

2012-01-01

Direct analysis of unassembled genomic data could greatly increase the power of short read DNA sequencing technologies and allow comparative genomics of organisms without a completed reference available. Here, we compare 174 chloroplasts by analyzing the taxanomic distribution of short kmers across genomes [1]. We then assemble de novo contigs centered on informative variation. The localized de novo contigs can be separated into two major classes: tip = unique to a single genome and group = shared by a subset of genomes. Prior to assembly, we found that ~18% of the chloroplast was duplicated in the inverted repeat (IR) region across a four-fold difference in genome sizes, from a highly reduced parasitic orchid [2] to a massive algal chloroplast [3], including gnetophytes [4] and cycads [5]. The conservation of this ratio between single copy and duplicated sequence was basal among green plants, independent of photosynthesis and mechanism of genome size change, and different in gymnosperms and lower plants. Major lineages in the angiosperm clade differed in the pattern of shared kmers and de novo contigs. For example, parasitic plants demonstrated an expected accelerated overall rate of evolution, while the hemi-parasitic genomes contained a great deal more novel sequence than holo-parasitic plants, suggesting different mechanisms at different stages of genomic contraction. Additionally, the legumes are diverging more quickly and in different ways than other major families. Small duplicated fragments of the rrn23 genes were deeply conserved among seed plants, including among several species without the IR regions, indicating a crucial functional role of this duplication. Localized de novo assembly of informative kmers greatly reduces the complexity of large comparative analyses by confining the analysis to a small partition of data and genomes relevant to the specific question, allowing direct analysis of next-gen sequence data from previously unstudied genomes and rapid discovery of informative candidate regions.

Reference-Free Comparative Genomics of 174 Chloroplasts

PubMed Central

Kua, Chai-Shian; Ruan, Jue; Harting, John; Ye, Cheng-Xi; Helmus, Matthew R.; Yu, Jun; Cannon, Charles H.

2012-01-01

Direct analysis of unassembled genomic data could greatly increase the power of short read DNA sequencing technologies and allow comparative genomics of organisms without a completed reference available. Here, we compare 174 chloroplasts by analyzing the taxanomic distribution of short kmers across genomes [1]. We then assemble de novo contigs centered on informative variation. The localized de novo contigs can be separated into two major classes: tip = unique to a single genome and group = shared by a subset of genomes. Prior to assembly, we found that ∼18% of the chloroplast was duplicated in the inverted repeat (IR) region across a four-fold difference in genome sizes, from a highly reduced parasitic orchid [2] to a massive algal chloroplast [3], including gnetophytes [4] and cycads [5]. The conservation of this ratio between single copy and duplicated sequence was basal among green plants, independent of photosynthesis and mechanism of genome size change, and different in gymnosperms and lower plants. Major lineages in the angiosperm clade differed in the pattern of shared kmers and de novo contigs. For example, parasitic plants demonstrated an expected accelerated overall rate of evolution, while the hemi-parasitic genomes contained a great deal more novel sequence than holo-parasitic plants, suggesting different mechanisms at different stages of genomic contraction. Additionally, the legumes are diverging more quickly and in different ways than other major families. Small duplicated fragments of the rrn23 genes were deeply conserved among seed plants, including among several species without the IR regions, indicating a crucial functional role of this duplication. Localized de novo assembly of informative kmers greatly reduces the complexity of large comparative analyses by confining the analysis to a small partition of data and genomes relevant to the specific question, allowing direct analysis of next-gen sequence data from previously unstudied genomes and rapid discovery of informative candidate regions. PMID:23185288

Separating homeologs by phasing in the tetraploid wheat transcriptome.

PubMed

Krasileva, Ksenia V; Buffalo, Vince; Bailey, Paul; Pearce, Stephen; Ayling, Sarah; Tabbita, Facundo; Soria, Marcelo; Wang, Shichen; Akhunov, Eduard; Uauy, Cristobal; Dubcovsky, Jorge

2013-06-25

The high level of identity among duplicated homoeologous genomes in tetraploid pasta wheat presents substantial challenges for de novo transcriptome assembly. To solve this problem, we develop a specialized bioinformatics workflow that optimizes transcriptome assembly and separation of merged homoeologs. To evaluate our strategy, we sequence and assemble the transcriptome of one of the diploid ancestors of pasta wheat, and compare both assemblies with a benchmark set of 13,472 full-length, non-redundant bread wheat cDNAs. A total of 489 million 100 bp paired-end reads from tetraploid wheat assemble in 140,118 contigs, including 96% of the benchmark cDNAs. We used a comparative genomics approach to annotate 66,633 open reading frames. The multiple k-mer assembly strategy increases the proportion of cDNAs assembled full-length in a single contig by 22% relative to the best single k-mer size. Homoeologs are separated using a post-assembly pipeline that includes polymorphism identification, phasing of SNPs, read sorting, and re-assembly of phased reads. Using a reference set of genes, we determine that 98.7% of SNPs analyzed are correctly separated by phasing. Our study shows that de novo transcriptome assembly of tetraploid wheat benefit from multiple k-mer assembly strategies more than diploid wheat. Our results also demonstrate that phasing approaches originally designed for heterozygous diploid organisms can be used to separate the close homoeologous genomes of tetraploid wheat. The predicted tetraploid wheat proteome and gene models provide a valuable tool for the wheat research community and for those interested in comparative genomic studies.

Separating homeologs by phasing in the tetraploid wheat transcriptome

PubMed Central

2013-01-01

Background The high level of identity among duplicated homoeologous genomes in tetraploid pasta wheat presents substantial challenges for de novo transcriptome assembly. To solve this problem, we develop a specialized bioinformatics workflow that optimizes transcriptome assembly and separation of merged homoeologs. To evaluate our strategy, we sequence and assemble the transcriptome of one of the diploid ancestors of pasta wheat, and compare both assemblies with a benchmark set of 13,472 full-length, non-redundant bread wheat cDNAs. Results A total of 489 million 100 bp paired-end reads from tetraploid wheat assemble in 140,118 contigs, including 96% of the benchmark cDNAs. We used a comparative genomics approach to annotate 66,633 open reading frames. The multiple k-mer assembly strategy increases the proportion of cDNAs assembled full-length in a single contig by 22% relative to the best single k-mer size. Homoeologs are separated using a post-assembly pipeline that includes polymorphism identification, phasing of SNPs, read sorting, and re-assembly of phased reads. Using a reference set of genes, we determine that 98.7% of SNPs analyzed are correctly separated by phasing. Conclusions Our study shows that de novo transcriptome assembly of tetraploid wheat benefit from multiple k-mer assembly strategies more than diploid wheat. Our results also demonstrate that phasing approaches originally designed for heterozygous diploid organisms can be used to separate the close homoeologous genomes of tetraploid wheat. The predicted tetraploid wheat proteome and gene models provide a valuable tool for the wheat research community and for those interested in comparative genomic studies. PMID:23800085

Whole genome sequences of the USMARC beef cattle diversity panel v2.9 aligned to the bovine reference genome assembly

USDA-ARS?s Scientific Manuscript database

A searchable and publicly viewable set of mapped genomes from 96 beef sires from 19 popular breeds of U.S. cattle was created. These sires with minimal pedigree relationships, represent >99% of the germplasm used in the US beef industry circa 2000. The group is estimated to contain more than 187 u...

BusyBee Web: metagenomic data analysis by bootstrapped supervised binning and annotation

PubMed Central

Kiefer, Christina; Fehlmann, Tobias; Backes, Christina

2017-01-01

Abstract Metagenomics-based studies of mixed microbial communities are impacting biotechnology, life sciences and medicine. Computational binning of metagenomic data is a powerful approach for the culture-independent recovery of population-resolved genomic sequences, i.e. from individual or closely related, constituent microorganisms. Existing binning solutions often require a priori characterized reference genomes and/or dedicated compute resources. Extending currently available reference-independent binning tools, we developed the BusyBee Web server for the automated deconvolution of metagenomic data into population-level genomic bins using assembled contigs (Illumina) or long reads (Pacific Biosciences, Oxford Nanopore Technologies). A reversible compression step as well as bootstrapped supervised binning enable quick turnaround times. The binning results are represented in interactive 2D scatterplots. Moreover, bin quality estimates, taxonomic annotations and annotations of antibiotic resistance genes are computed and visualized. Ground truth-based benchmarks of BusyBee Web demonstrate comparably high performance to state-of-the-art binning solutions for assembled contigs and markedly improved performance for long reads (median F1 scores: 70.02–95.21%). Furthermore, the applicability to real-world metagenomic datasets is shown. In conclusion, our reference-independent approach automatically bins assembled contigs or long reads, exhibits high sensitivity and precision, enables intuitive inspection of the results, and only requires FASTA-formatted input. The web-based application is freely accessible at: https://ccb-microbe.cs.uni-saarland.de/busybee. PMID:28472498

Optimizing and benchmarking de novo transcriptome sequencing: from library preparation to assembly evaluation.

PubMed

Hara, Yuichiro; Tatsumi, Kaori; Yoshida, Michio; Kajikawa, Eriko; Kiyonari, Hiroshi; Kuraku, Shigehiro

2015-11-18

RNA-seq enables gene expression profiling in selected spatiotemporal windows and yields massive sequence information with relatively low cost and time investment, even for non-model species. However, there remains a large room for optimizing its workflow, in order to take full advantage of continuously developing sequencing capacity. Transcriptome sequencing for three embryonic stages of Madagascar ground gecko (Paroedura picta) was performed with the Illumina platform. The output reads were assembled de novo for reconstructing transcript sequences. In order to evaluate the completeness of transcriptome assemblies, we prepared a reference gene set consisting of vertebrate one-to-one orthologs. To take advantage of increased read length of >150 nt, we demonstrated shortened RNA fragmentation time, which resulted in a dramatic shift of insert size distribution. To evaluate products of multiple de novo assembly runs incorporating reads with different RNA sources, read lengths, and insert sizes, we introduce a new reference gene set, core vertebrate genes (CVG), consisting of 233 genes that are shared as one-to-one orthologs by all vertebrate genomes examined (29 species)., The completeness assessment performed by the computational pipelines CEGMA and BUSCO referring to CVG, demonstrated higher accuracy and resolution than with the gene set previously established for this purpose. As a result of the assessment with CVG, we have derived the most comprehensive transcript sequence set of the Madagascar ground gecko by means of assembling individual libraries followed by clustering the assembled sequences based on their overall similarities. Our results provide several insights into optimizing de novo RNA-seq workflow, including the coordination between library insert size and read length, which manifested in improved connectivity of assemblies. The approach and assembly assessment with CVG demonstrated here would be applicable to transcriptome analysis of other species as well as whole genome analyses.

Innovative assembly strategy contributes to understanding the evolution and conservation genetics of the endangered Solenodon paradoxus from the island of Hispaniola.

PubMed

Grigorev, Kirill; Kliver, Sergey; Dobrynin, Pavel; Komissarov, Aleksey; Wolfsberger, Walter; Krasheninnikova, Ksenia; Afanador-Herna Ndez, Yashira M; Brandt, Adam L; Paulino, Liz A; Carreras, Rosanna; Rodríguez, Luis E; Nu N Ez, Adrell; Brandt, Jessica R; Silva, Filipe; Herna Ndez-Martich, J David; Majeske, Audrey J; Antunes, Agostinho; Roca, Alfred L; O'Brien, Stephen J; Martínez-Cruzado, Juan Carlos; Oleksyk, Taras K

2018-03-16

Solenodons are insectivores living in Hispaniola and Cuba that form an isolated branch in the tree of placental mammals highly divergent from other eulipothyplan insectivores The history, unique biology and adaptations of these enigmatic venomous species could be illuminated by the availability of genome data, but a whole genome assembly for solenodons has not been previously performed, partially due to the difficulty in obtaining samples from the field. Island isolation and reduced numbers have likely resulted in high homozygosity within the Hispaniolan solenodon (Solenodon paradoxus), thus we tested the performance of several assembly strategies on the genome of this genetically impoverished species. The string-graph based assembly strategy seemed a better choice compared to the conventional de Bruijn graph approach, due to the high levels of homozygosity, which is often a hallmark of endemic or endangered species. A consensus reference genome was assembled from sequences of five individuals from the southern subspecies (S. p. woodi). In addition, we obtained additional sequence from one sample of the northern subspecies (S. p. paradoxus). The resulting genome assemblies were compared to each other, and annotated for genes, with a specific emphasis on venom genes, repeats, variable microsatellite loci and other genomic variants. Phylogenetic positioning and selection signatures were inferred based on 4,416 single copy orthologs from 10 other mammals. We estimated that solenodons diverged from other extant mammals 73.6 Mya. Patterns of SNP variation allowed us to infer population demography, which supported a subspecies split within the Hispaniolan solenodon at least 300 Kya.

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

PubMed

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

2013-04-25

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

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

PubMed Central

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

2013-01-01

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

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

PGSB/MIPS Plant Genome Information Resources and Concepts for the Analysis of Complex Grass Genomes.

PubMed

Spannagl, Manuel; Bader, Kai; Pfeifer, Matthias; Nussbaumer, Thomas; Mayer, Klaus F X

2016-01-01

PGSB (Plant Genome and Systems Biology; formerly MIPS-Munich Institute for Protein Sequences) has been involved in developing, implementing and maintaining plant genome databases for more than a decade. Genome databases and analysis resources have focused on individual genomes and aim to provide flexible and maintainable datasets for model plant genomes as a backbone against which experimental data, e.g., from high-throughput functional genomics, can be organized and analyzed. In addition, genomes from both model and crop plants form a scaffold for comparative genomics, assisted by specialized tools such as the CrowsNest viewer to explore conserved gene order (synteny) between related species on macro- and micro-levels.The genomes of many economically important Triticeae plants such as wheat, barley, and rye present a great challenge for sequence assembly and bioinformatic analysis due to their enormous complexity and large genome size. Novel concepts and strategies have been developed to deal with these difficulties and have been applied to the genomes of wheat, barley, rye, and other cereals. This includes the GenomeZipper concept, reference-guided exome assembly, and "chromosome genomics" based on flow cytometry sorted chromosomes.

When Genomics Is Not Enough: Experimental Evidence for a Decrease in LINE-1 Activity During the Evolution of Australian Marsupials

PubMed Central

Gallus, Susanne; Lammers, Fritjof

2016-01-01

The autonomous transposable element LINE-1 is a highly abundant element that makes up between 15% and 20% of therian mammal genomes. Since their origin before the divergence of marsupials and placental mammals, LINE-1 elements have contributed actively to the genome landscape. A previous in silico screen of the Tasmanian devil genome revealed a lack of functional coding LINE-1 sequences. In this study we present the results of an in vitro analysis from a partial LINE-1 reverse transcriptase coding sequence in five marsupial species. Our experimental screen supports the in silico findings of the genome-wide degradation of LINE-1 sequences in the Tasmanian devil, and identifies a high frequency of degraded LINE-1 sequences in other Australian marsupials. The comparison between the experimentally obtained LINE-1 sequences and reference genome assemblies suggests that conclusions from in silico analyses of retrotransposition activity can be influenced by incomplete genome assemblies from short reads. PMID:27389686

Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce

PubMed Central

Reyes-Chin-Wo, Sebastian; Wang, Zhiwen; Yang, Xinhua; Kozik, Alexander; Arikit, Siwaret; Song, Chi; Xia, Liangfeng; Froenicke, Lutz; Lavelle, Dean O.; Truco, María-José; Xia, Rui; Zhu, Shilin; Xu, Chunyan; Xu, Huaqin; Xu, Xun; Cox, Kyle; Korf, Ian; Meyers, Blake C.; Michelmore, Richard W.

2017-01-01

Lettuce (Lactuca sativa) is a major crop and a member of the large, highly successful Compositae family of flowering plants. Here we present a reference assembly for the species and family. This was generated using whole-genome shotgun Illumina reads plus in vitro proximity ligation data to create large superscaffolds; it was validated genetically and superscaffolds were oriented in genetic bins ordered along nine chromosomal pseudomolecules. We identify several genomic features that may have contributed to the success of the family, including genes encoding Cycloidea-like transcription factors, kinases, enzymes involved in rubber biosynthesis and disease resistance proteins that are expanded in the genome. We characterize 21 novel microRNAs, one of which may trigger phasiRNAs from numerous kinase transcripts. We provide evidence for a whole-genome triplication event specific but basal to the Compositae. We detect 26% of the genome in triplicated regions containing 30% of all genes that are enriched for regulatory sequences and depleted for genes involved in defence. PMID:28401891

The Agassiz’s desert tortoise genome provides a resource for the conservation of a threatened species

PubMed Central

Tollis, Marc; DeNardo, Dale F.; Cornelius, John A.; Dolby, Greer A.; Edwards, Taylor; Henen, Brian T.; Karl, Alice E.; Murphy, Robert W.

2017-01-01

Agassiz’s desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. This G. agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex. PMID:28562605

The Agassiz's desert tortoise genome provides a resource for the conservation of a threatened species.

PubMed

Tollis, Marc; DeNardo, Dale F; Cornelius, John A; Dolby, Greer A; Edwards, Taylor; Henen, Brian T; Karl, Alice E; Murphy, Robert W; Kusumi, Kenro

2017-01-01

Agassiz's desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. This G. agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex.

De novo assembly of highly polymorphic metagenomic data using in situ generated reference sequences and a novel BLAST-based assembly pipeline.

PubMed

Lin, You-Yu; Hsieh, Chia-Hung; Chen, Jiun-Hong; Lu, Xuemei; Kao, Jia-Horng; Chen, Pei-Jer; Chen, Ding-Shinn; Wang, Hurng-Yi

2017-04-26

The accuracy of metagenomic assembly is usually compromised by high levels of polymorphism due to divergent reads from the same genomic region recognized as different loci when sequenced and assembled together. A viral quasispecies is a group of abundant and diversified genetically related viruses found in a single carrier. Current mainstream assembly methods, such as Velvet and SOAPdenovo, were not originally intended for the assembly of such metagenomics data, and therefore demands for new methods to provide accurate and informative assembly results for metagenomic data. In this study, we present a hybrid method for assembling highly polymorphic data combining the partial de novo-reference assembly (PDR) strategy and the BLAST-based assembly pipeline (BBAP). The PDR strategy generates in situ reference sequences through de novo assembly of a randomly extracted partial data set which is subsequently used for the reference assembly for the full data set. BBAP employs a greedy algorithm to assemble polymorphic reads. We used 12 hepatitis B virus quasispecies NGS data sets from a previous study to assess and compare the performance of both PDR and BBAP. Analyses suggest the high polymorphism of a full metagenomic data set leads to fragmentized de novo assembly results, whereas the biased or limited representation of external reference sequences included fewer reads into the assembly with lower assembly accuracy and variation sensitivity. In comparison, the PDR generated in situ reference sequence incorporated more reads into the final PDR assembly of the full metagenomics data set along with greater accuracy and higher variation sensitivity. BBAP assembly results also suggest higher assembly efficiency and accuracy compared to other assembly methods. Additionally, BBAP assembly recovered HBV structural variants that were not observed amongst assembly results of other methods. Together, PDR/BBAP assembly results were significantly better than other compared methods. Both PDR and BBAP independently increased the assembly efficiency and accuracy of highly polymorphic data, and assembly performances were further improved when used together. BBAP also provides nucleotide frequency information. Together, PDR and BBAP provide powerful tools for metagenomic data studies.

The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis-Acting Element That Is Essential for Nucleocapsid Assembly.

PubMed

Huang, Zhihong; Pan, Mengjia; Zhu, Silei; Zhang, Hao; Wu, Wenbi; Yuan, Meijin; Yang, Kai

2017-03-01

Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83 -encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis -acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis -acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis -acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process. IMPORTANCE Virus nucleocapsid assembly usually requires specific cis -acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome replication intermediates, and the encapsidation of the viral genome into procapsids. In linear DNA viruses, such elements generally locate at the ends of the viral genome; however, most of these elements remain unidentified in circular DNA viruses (including baculovirus) due to their circular genomic conformation. Here, we identified a nucleocapsid assembly-essential element in the AcMNPV (the archetype of baculovirus) genome. This finding provides an important reference for studies of nucleocapsid assembly-related elements in baculoviruses and other circular DNA viruses. Moreover, as most of the previous studies of baculovirus nucleocapsid assembly have been focused on viral proteins, our study provides a novel entry point to investigate this mechanism via cis -acting elements in the viral genome. Copyright © 2017 American Society for Microbiology.

The UCSC Genome Browser database: extensions and updates 2013.

PubMed

Meyer, Laurence R; Zweig, Ann S; Hinrichs, Angie S; Karolchik, Donna; Kuhn, Robert M; Wong, Matthew; Sloan, Cricket A; Rosenbloom, Kate R; Roe, Greg; Rhead, Brooke; Raney, Brian J; Pohl, Andy; Malladi, Venkat S; Li, Chin H; Lee, Brian T; Learned, Katrina; Kirkup, Vanessa; Hsu, Fan; Heitner, Steve; Harte, Rachel A; Haeussler, Maximilian; Guruvadoo, Luvina; Goldman, Mary; Giardine, Belinda M; Fujita, Pauline A; Dreszer, Timothy R; Diekhans, Mark; Cline, Melissa S; Clawson, Hiram; Barber, Galt P; Haussler, David; Kent, W James

2013-01-01

The University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analysing and sharing both publicly available and user-generated genomic datasets. As of September 2012, genomic sequence and a basic set of annotation 'tracks' are provided for 63 organisms, including 26 mammals, 13 non-mammal vertebrates, 3 invertebrate deuterostomes, 13 insects, 6 worms, yeast and sea hare. In the past year 19 new genome assemblies have been added, and we anticipate releasing another 28 in early 2013. Further, a large number of annotation tracks have been either added, updated by contributors or remapped to the latest human reference genome. Among these are an updated UCSC Genes track for human and mouse assemblies. We have also introduced several features to improve usability, including new navigation menus. This article provides an update to the UCSC Genome Browser database, which has been previously featured in the Database issue of this journal.

Comparative Transcriptomic Approaches Exploring Contamination Stress Tolerance in Salix sp. Reveal the Importance for a Metaorganismal de Novo Assembly Approach for Nonmodel Plants1[OPEN

PubMed Central

Brereton, Nicholas J. B.; Marleau, Julie; Nissim, Werther Guidi; Labrecque, Michel; Joly, Simon; Pitre, Frederic E.

2016-01-01

Metatranscriptomic study of nonmodel organisms requires strategies that retain the highly resolved genetic information generated from model organisms while allowing for identification of the unexpected. A real-world biological application of phytoremediation, the field growth of 10 Salix cultivars on polluted soils, was used as an exemplar nonmodel and multifaceted crop response well-disposed to the study of gene expression. Sequence reads were assembled de novo to create 10 independent transcriptomes, a global transcriptome, and were mapped against the Salix purpurea 94006 reference genome. Annotation of assembled contigs was performed without a priori assumption of the originating organism. Global transcriptome construction from 3.03 billion paired-end reads revealed 606,880 unique contigs annotated from 1588 species, often common in all 10 cultivars. Comparisons between transcriptomic and metatranscriptomic methodologies provide clear evidence that nonnative RNA can mistakenly map to reference genomes, especially to conserved regions of common housekeeping genes, such as actin, α/β-tubulin, and elongation factor 1-α. In Salix, Rubisco activase transcripts were down-regulated in contaminated trees across all 10 cultivars, whereas thiamine thizole synthase and CP12, a Calvin Cycle master regulator, were uniformly up-regulated. De novo assembly approaches, with unconstrained annotation, can improve data quality; care should be taken when exploring such plant genetics to reduce de facto data exclusion by mapping to a single reference genome alone. Salix gene expression patterns strongly suggest cultivar-wide alteration of specific photosynthetic apparatus and protection of the antenna complexes from oxidation damage in contaminated trees, providing an insight into common stress tolerance strategies in a real-world phytoremediation system. PMID:27002060

What's in your next-generation sequence data? An exploration of unmapped DNA and RNA sequence reads from the bovine reference individual

USDA-ARS?s Scientific Manuscript database

BACKGROUND: Next-generation sequencing projects commonly commence by aligning reads to a reference genome assembly. While improvements in alignment algorithms and computational hardware have greatly enhanced the efficiency and accuracy of alignments, a significant percentage of reads often remain u...

A New Chicken Genome Assembly Provides Insight into Avian Genome Structure.

PubMed

Warren, Wesley C; Hillier, LaDeana W; Tomlinson, Chad; Minx, Patrick; Kremitzki, Milinn; Graves, Tina; Markovic, Chris; Bouk, Nathan; Pruitt, Kim D; Thibaud-Nissen, Francoise; Schneider, Valerie; Mansour, Tamer A; Brown, C Titus; Zimin, Aleksey; Hawken, Rachel; Abrahamsen, Mitch; Pyrkosz, Alexis B; Morisson, Mireille; Fillon, Valerie; Vignal, Alain; Chow, William; Howe, Kerstin; Fulton, Janet E; Miller, Marcia M; Lovell, Peter; Mello, Claudio V; Wirthlin, Morgan; Mason, Andrew S; Kuo, Richard; Burt, David W; Dodgson, Jerry B; Cheng, Hans H

2017-01-05

The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3), built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb, including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31, and 33, and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 protein-coding) over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B, encompassing two segments: one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts. Copyright © 2017 Warren et al.

Draft genomes of two blister beetles Hycleus cichorii and Hycleus phaleratus

PubMed Central

Wu, Yuan-Ming; Li, Jiang

2018-01-01

Abstract Background Commonly known as blister beetles or Spanish fly, there are more than 1500 species in the Meloidae family (Hexapoda: Coleoptera: Tenebrionoidea) that produce the potent defensive blistering agent cantharidin. Cantharidin and its derivatives have been used to treat cancers such as liver, stomach, lung, and esophageal cancers. Hycleus cichorii and Hycleus phaleratus are the most commercially important blister beetles in China due to their ability to biosynthesize this potent vesicant. However, there is a lack of genome reference, which has hindered development of studies on the biosynthesis of cantharidin and a better understanding of its biology and pharmacology. Results We report 2 draft genomes and quantified gene sets for the blister beetles H. cichorii and H. phaleratus, 2 complex genomes with >72% repeats and approximately 1% heterozygosity, using Illumina sequencing data. An integrated assembly pipeline was performed for assembly, and most of the coding regions were obtained. Benchmarking universal single-copy orthologs (BUSCO) assessment showed that our assembly obtained more than 98% of the Endopterygota universal single-copy orthologs. Comparison analysis showed that the completeness of coding genes in our assembly was comparable to other beetle genomes such as Dendroctonus ponderosae and Agrilus planipennis. Gene annotation yielded 13 813 and 13 725 protein-coding genes in H. cichorii and H. phaleratus, of which approximately 89% were functionally annotated. BUSCO assessment showed that approximately 86% and 84% of the Endopterygota universal single-copy orthologs were annotated completely in these 2 gene sets, whose completeness is comparable to that of D. ponderosae and A. planipennis. Conclusions Assembly of both blister beetle genomes provides a valuable resource for future biosynthesis of cantharidin and comparative genomic studies of blister beetles and other beetles. PMID:29444297

Draft genomes of two blister beetles Hycleus cichorii and Hycleus phaleratus.

PubMed

Wu, Yuan-Ming; Li, Jiang; Chen, Xiang-Sheng

2018-03-01

Commonly known as blister beetles or Spanish fly, there are more than 1500 species in the Meloidae family (Hexapoda: Coleoptera: Tenebrionoidea) that produce the potent defensive blistering agent cantharidin. Cantharidin and its derivatives have been used to treat cancers such as liver, stomach, lung, and esophageal cancers. Hycleus cichorii and Hycleus phaleratus are the most commercially important blister beetles in China due to their ability to biosynthesize this potent vesicant. However, there is a lack of genome reference, which has hindered development of studies on the biosynthesis of cantharidin and a better understanding of its biology and pharmacology. We report 2 draft genomes and quantified gene sets for the blister beetles H. cichorii and H. phaleratus, 2 complex genomes with >72% repeats and approximately 1% heterozygosity, using Illumina sequencing data. An integrated assembly pipeline was performed for assembly, and most of the coding regions were obtained. Benchmarking universal single-copy orthologs (BUSCO) assessment showed that our assembly obtained more than 98% of the Endopterygota universal single-copy orthologs. Comparison analysis showed that the completeness of coding genes in our assembly was comparable to other beetle genomes such as Dendroctonus ponderosae and Agrilus planipennis. Gene annotation yielded 13 813 and 13 725 protein-coding genes in H. cichorii and H. phaleratus, of which approximately 89% were functionally annotated. BUSCO assessment showed that approximately 86% and 84% of the Endopterygota universal single-copy orthologs were annotated completely in these 2 gene sets, whose completeness is comparable to that of D. ponderosae and A. planipennis. Assembly of both blister beetle genomes provides a valuable resource for future biosynthesis of cantharidin and comparative genomic studies of blister beetles and other beetles.

The Asian arowana (Scleropages formosus) genome provides new insights into the evolution of an early lineage of teleosts

PubMed Central

Bian, Chao; Hu, Yinchang; Ravi, Vydianathan; Kuznetsova, Inna S.; Shen, Xueyan; Mu, Xidong; Sun, Ying; You, Xinxin; Li, Jia; Li, Xiaofeng; Qiu, Ying; Tay, Boon-Hui; Thevasagayam, Natascha May; Komissarov, Aleksey S.; Trifonov, Vladimir; Kabilov, Marsel; Tupikin, Alexey; Luo, Jianren; Liu, Yi; Song, Hongmei; Liu, Chao; Wang, Xuejie; Gu, Dangen; Yang, Yexin; Li, Wujiao; Polgar, Gianluca; Fan, Guangyi; Zeng, Peng; Zhang, He; Xiong, Zijun; Tang, Zhujing; Peng, Chao; Ruan, Zhiqiang; Yu, Hui; Chen, Jieming; Fan, Mingjun; Huang, Yu; Wang, Min; Zhao, Xiaomeng; Hu, Guojun; Yang, Huanming; Wang, Jian; Wang, Jun; Xu, Xun; Song, Linsheng; Xu, Gangchun; Xu, Pao; Xu, Junmin; O’Brien, Stephen J.; Orbán, László; Venkatesh, Byrappa; Shi, Qiong

2016-01-01

The Asian arowana (Scleropages formosus), one of the world’s most expensive cultivated ornamental fishes, is an endangered species. It represents an ancient lineage of teleosts: the Osteoglossomorpha. Here, we provide a high-quality chromosome-level reference genome of a female golden-variety arowana using a combination of deep shotgun sequencing and high-resolution linkage mapping. In addition, we have also generated two draft genome assemblies for the red and green varieties. Phylogenomic analysis supports a sister group relationship between Osteoglossomorpha (bonytongues) and Elopomorpha (eels and relatives), with the two clades together forming a sister group of Clupeocephala which includes all the remaining teleosts. The arowana genome retains the full complement of eight Hox clusters unlike the African butterfly fish (Pantodon buchholzi), another bonytongue fish, which possess only five Hox clusters. Differential gene expression among three varieties provides insights into the genetic basis of colour variation. A potential heterogametic sex chromosome is identified in the female arowana karyotype, suggesting that the sex is determined by a ZW/ZZ sex chromosomal system. The high-quality reference genome of the golden arowana and the draft assemblies of the red and green varieties are valuable resources for understanding the biology, adaptation and behaviour of Asian arowanas. PMID:27089831

Single-molecule sequencing and Hi-C-based proximity-guided assembly of amaranth (Amaranthus hypochondriacus) chromosomes provide insights into genome evolution.

PubMed

Lightfoot, D J; Jarvis, D E; Ramaraj, T; Lee, R; Jellen, E N; Maughan, P J

2017-08-31

Amaranth (Amaranthus hypochondriacus) was a food staple among the ancient civilizations of Central and South America that has recently received increased attention due to the high nutritional value of the seeds, with the potential to help alleviate malnutrition and food security concerns, particularly in arid and semiarid regions of the developing world. Here, we present a reference-quality assembly of the amaranth genome which will assist the agronomic development of the species. Utilizing single-molecule, real-time sequencing (Pacific Biosciences) and chromatin interaction mapping (Hi-C) to close assembly gaps and scaffold contigs, respectively, we improved our previously reported Illumina-based assembly to produce a chromosome-scale assembly with a scaffold N50 of 24.4 Mb. The 16 largest scaffolds contain 98% of the assembly and likely represent the haploid chromosomes (n = 16). To demonstrate the accuracy and utility of this approach, we produced physical and genetic maps and identified candidate genes for the betalain pigmentation pathway. The chromosome-scale assembly facilitated a genome-wide syntenic comparison of amaranth with other Amaranthaceae species, revealing chromosome loss and fusion events in amaranth that explain the reduction from the ancestral haploid chromosome number (n = 18) for a tetraploid member of the Amaranthaceae. The assembly method reported here minimizes cost by relying primarily on short-read technology and is one of the first reported uses of in vivo Hi-C for assembly of a plant genome. Our analyses implicate chromosome loss and fusion as major evolutionary events in the 2n = 32 amaranths and clearly establish the homoeologous relationship among most of the subgenome chromosomes, which will facilitate future investigations of intragenomic changes that occurred post polyploidization.

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

PubMed

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

2013-01-01

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

Improving amphibian genomic resources: a multitissue reference transcriptome of an iconic invader.

PubMed

Richardson, Mark F; Sequeira, Fernando; Selechnik, Daniel; Carneiro, Miguel; Vallinoto, Marcelo; Reid, Jack G; West, Andrea J; Crossland, Michael R; Shine, Richard; Rollins, Lee A

2018-01-01

Cane toads (Rhinella marina) are an iconic invasive species introduced to 4 continents and well utilized for studies of rapid evolution in introduced environments. Despite the long introduction history of this species, its profound ecological impacts, and its utility for demonstrating evolutionary principles, genetic information is sparse. Here we produce a de novo transcriptome spanning multiple tissues and life stages to enable investigation of the genetic basis of previously identified rapid phenotypic change over the introduced range. Using approximately 1.9 billion reads from developing tadpoles and 6 adult tissue-specific cDNA libraries, as well as a transcriptome assembly pipeline encompassing 100 separate de novo assemblies, we constructed 62 202 transcripts, of which we functionally annotated ∼50%. Our transcriptome assembly exhibits 90% full-length completeness of the Benchmarking Universal Single-Copy Orthologs data set. Robust assembly metrics and comparisons with several available anuran transcriptomes and genomes indicate that our cane toad assembly is one of the most complete anuran genomic resources available. This comprehensive anuran transcriptome will provide a valuable resource for investigation of genes under selection during invasion in cane toads, but will also greatly expand our general knowledge of anuran genomes, which are underrepresented in the literature. The data set is publically available in NCBI and GigaDB to serve as a resource for other researchers. © The Authors 2017. Published by Oxford University Press.

Improving amphibian genomic resources: a multitissue reference transcriptome of an iconic invader

PubMed Central

Reid, Jack G; Crossland, Michael R

2018-01-01

Abstract Background Cane toads (Rhinella marina) are an iconic invasive species introduced to 4 continents and well utilized for studies of rapid evolution in introduced environments. Despite the long introduction history of this species, its profound ecological impacts, and its utility for demonstrating evolutionary principles, genetic information is sparse. Here we produce a de novo transcriptome spanning multiple tissues and life stages to enable investigation of the genetic basis of previously identified rapid phenotypic change over the introduced range. Findings Using approximately 1.9 billion reads from developing tadpoles and 6 adult tissue-specific cDNA libraries, as well as a transcriptome assembly pipeline encompassing 100 separate de novo assemblies, we constructed 62 202 transcripts, of which we functionally annotated ∼50%. Our transcriptome assembly exhibits 90% full-length completeness of the Benchmarking Universal Single-Copy Orthologs data set. Robust assembly metrics and comparisons with several available anuran transcriptomes and genomes indicate that our cane toad assembly is one of the most complete anuran genomic resources available. Conclusions This comprehensive anuran transcriptome will provide a valuable resource for investigation of genes under selection during invasion in cane toads, but will also greatly expand our general knowledge of anuran genomes, which are underrepresented in the literature. The data set is publically available in NCBI and GigaDB to serve as a resource for other researchers. PMID:29186423

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize

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

Hirsch, Candice N.; Hirsch, Cory D.; Brohammer, Alex B.

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison ofmore » these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.« less

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize

DOE PAGES

Hirsch, Candice N.; Hirsch, Cory D.; Brohammer, Alex B.; ...

2016-11-01

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison ofmore » these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.« less

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize[OPEN

PubMed Central

Soifer, Ilya; Barad, Omer; Shem-Tov, Doron; Baruch, Kobi; Lu, Fei; Hernandez, Alvaro G.; Wright, Chris L.; Koehler, Klaus; Buell, C. Robin; de Leon, Natalia

2016-01-01

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison of these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools. PMID:27803309

High-resolution genetic maps of Eucalyptus improve Eucalyptus grandis genome assembly.

PubMed

Bartholomé, Jérôme; Mandrou, Eric; Mabiala, André; Jenkins, Jerry; Nabihoudine, Ibouniyamine; Klopp, Christophe; Schmutz, Jeremy; Plomion, Christophe; Gion, Jean-Marc

2015-06-01

Genetic maps are key tools in genetic research as they constitute the framework for many applications, such as quantitative trait locus analysis, and support the assembly of genome sequences. The resequencing of the two parents of a cross between Eucalyptus urophylla and Eucalyptus grandis was used to design a single nucleotide polymorphism (SNP) array of 6000 markers evenly distributed along the E. grandis genome. The genotyping of 1025 offspring enabled the construction of two high-resolution genetic maps containing 1832 and 1773 markers with an average marker interval of 0.45 and 0.5 cM for E. grandis and E. urophylla, respectively. The comparison between genetic maps and the reference genome highlighted 85% of collinear regions. A total of 43 noncollinear regions and 13 nonsynthetic regions were detected and corrected in the new genome assembly. This improved version contains 4943 scaffolds totalling 691.3 Mb of which 88.6% were captured by the 11 chromosomes. The mapping data were also used to investigate the effect of population size and number of markers on linkage mapping accuracy. This study provides the most reliable linkage maps for Eucalyptus and version 2.0 of the E. grandis genome. © 2014 CIRAD. New Phytologist © 2014 New Phytologist Trust.

Challenges imposed by minor reference alleles on the identification and reporting of clinical variants from exome data.

PubMed

Koko, Mahmoud; Abdallah, Mohammed O E; Amin, Mutaz; Ibrahim, Muntaser

2018-01-15

The conventional variant calling of pathogenic alleles in exome and genome sequencing requires the presence of the non-pathogenic alleles as genome references. This hinders the correct identification of variants with minor and/or pathogenic reference alleles warranting additional approaches for variant calling. More than 26,000 Exome Aggregation Consortium (ExAC) variants have a minor reference allele including variants with known ClinVar disease alleles. For instance, in a number of variants related to clotting disorders, the phenotype-associated allele is a human genome reference allele (rs6025, rs6003, rs1799983, and rs2227564 using the assembly hg19). We highlighted how the current variant calling standards miss homozygous reference disease variants in these sites and provided a bioinformatic panel that can be used to screen these variants using commonly available variant callers. We present exome sequencing results from an individual with venous thrombosis to emphasize how pathogenic alleles in clinically relevant variants escape variant calling while non-pathogenic alleles are detected. This article highlights the importance of specialized variant calling strategies in clinical variants with minor reference alleles especially in the context of personal genomes and exomes. We provide here a simple strategy to screen potential disease-causing variants when present in homozygous reference state.

NGSPanPipe: A Pipeline for Pan-genome Identification in Microbial Strains from Experimental Reads.

PubMed

Kulsum, Umay; Kapil, Arti; Singh, Harpreet; Kaur, Punit

2018-01-01

Recent advancements in sequencing technologies have decreased both time span and cost for sequencing the whole bacterial genome. High-throughput Next-Generation Sequencing (NGS) technology has led to the generation of enormous data concerning microbial populations publically available across various repositories. As a consequence, it has become possible to study and compare the genomes of different bacterial strains within a species or genus in terms of evolution, ecology and diversity. Studying the pan-genome provides insights into deciphering microevolution, global composition and diversity in virulence and pathogenesis of a species. It can also assist in identifying drug targets and proposing vaccine candidates. The effective analysis of these large genome datasets necessitates the development of robust tools. Current methods to develop pan-genome do not support direct input of raw reads from the sequencer machine but require preprocessing of reads as an assembled protein/gene sequence file or the binary matrix of orthologous genes/proteins. We have designed an easy-to-use integrated pipeline, NGSPanPipe, which can directly identify the pan-genome from short reads. The output from the pipeline is compatible with other pan-genome analysis tools. We evaluated our pipeline with other methods for developing pan-genome, i.e. reference-based assembly and de novo assembly using simulated reads of Mycobacterium tuberculosis. The single script pipeline (pipeline.pl) is applicable for all bacterial strains. It integrates multiple in-house Perl scripts and is freely accessible from https://github.com/Biomedinformatics/NGSPanPipe .

A computational genomics pipeline for prokaryotic sequencing projects.

PubMed

Kislyuk, Andrey O; Katz, Lee S; Agrawal, Sonia; Hagen, Matthew S; Conley, Andrew B; Jayaraman, Pushkala; Nelakuditi, Viswateja; Humphrey, Jay C; Sammons, Scott A; Govil, Dhwani; Mair, Raydel D; Tatti, Kathleen M; Tondella, Maria L; Harcourt, Brian H; Mayer, Leonard W; Jordan, I King

2010-08-01

New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems.

The genome of the Gulf pipefish enables understanding of evolutionary innovations.

PubMed

Small, C M; Bassham, S; Catchen, J; Amores, A; Fuiten, A M; Brown, R S; Jones, A G; Cresko, W A

2016-12-20

Evolutionary origins of derived morphologies ultimately stem from changes in protein structure, gene regulation, and gene content. A well-assembled, annotated reference genome is a central resource for pursuing these molecular phenomena underlying phenotypic evolution. We explored the genome of the Gulf pipefish (Syngnathus scovelli), which belongs to family Syngnathidae (pipefishes, seahorses, and seadragons). These fishes have dramatically derived bodies and a remarkable novelty among vertebrates, the male brood pouch. We produce a reference genome, condensed into chromosomes, for the Gulf pipefish. Gene losses and other changes have occurred in pipefish hox and dlx clusters and in the tbx and pitx gene families, candidate mechanisms for the evolution of syngnathid traits, including an elongated axis and the loss of ribs, pelvic fins, and teeth. We measure gene expression changes in pregnant versus non-pregnant brood pouch tissue and characterize the genomic organization of duplicated metalloprotease genes (patristacins) recruited into the function of this novel structure. Phylogenetic inference using ultraconserved sequences provides an alternative hypothesis for the relationship between orders Syngnathiformes and Scombriformes. Comparisons of chromosome structure among percomorphs show that chromosome number in a pipefish ancestor became reduced via chromosomal fusions. The collected findings from this first syngnathid reference genome open a window into the genomic underpinnings of highly derived morphologies, demonstrating that de novo production of high quality and useful reference genomes is within reach of even small research groups.

The Whole-Genome and Transcriptome of the Manila Clam (Ruditapes philippinarum).

PubMed

Mun, Seyoung; Kim, Yun-Ji; Markkandan, Kesavan; Shin, Wonseok; Oh, Sumin; Woo, Jiyoung; Yoo, Jongsu; An, Hyesuck; Han, Kyudong

2017-06-01

The manila clam, Ruditapes philippinarum, is an important bivalve species in worldwide aquaculture including Korea. The aquaculture production of R. philippinarum is under threat from diverse environmental factors including viruses, microorganisms, parasites, and water conditions with subsequently declining production. In spite of its importance as a marine resource, the reference genome of R. philippinarum for comprehensive genetic studies is largely unexplored. Here, we report the de novo whole-genome and transcriptome assembly of R. philippinarum across three different tissues (foot, gill, and adductor muscle), and provide the basic data for advanced studies in selective breeding and disease control in order to obtain successful aquaculture systems. An approximately 2.56 Gb high quality whole-genome was assembled with various library construction methods. A total of 108,034 protein coding gene models were predicted and repetitive elements including simple sequence repeats and noncoding RNAs were identified to further understanding of the genetic background of R. philippinarum for genomics-assisted breeding. Comparative analysis with the bivalve marine invertebrates uncover that the gene family related to complement C1q was enriched. Furthermore, we performed transcriptome analysis with three different tissues in order to support genome annotation and then identified 41,275 transcripts which were annotated. The R. philippinarum genome resource will markedly advance a wide range of potential genetic studies, a reference genome for comparative analysis of bivalve species and unraveling mechanisms of biological processes in molluscs. We believe that the R. philippinarum genome will serve as an initial platform for breeding better-quality clams using a genomic approach. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Single-molecule optical genome mapping of a human HapMap and a colorectal cancer cell line.

PubMed

Teo, Audrey S M; Verzotto, Davide; Yao, Fei; Nagarajan, Niranjan; Hillmer, Axel M

2015-01-01

Next-generation sequencing (NGS) technologies have changed our understanding of the variability of the human genome. However, the identification of genome structural variations based on NGS approaches with read lengths of 35-300 bases remains a challenge. Single-molecule optical mapping technologies allow the analysis of DNA molecules of up to 2 Mb and as such are suitable for the identification of large-scale genome structural variations, and for de novo genome assemblies when combined with short-read NGS data. Here we present optical mapping data for two human genomes: the HapMap cell line GM12878 and the colorectal cancer cell line HCT116. High molecular weight DNA was obtained by embedding GM12878 and HCT116 cells, respectively, in agarose plugs, followed by DNA extraction under mild conditions. Genomic DNA was digested with KpnI and 310,000 and 296,000 DNA molecules (≥ 150 kb and 10 restriction fragments), respectively, were analyzed per cell line using the Argus optical mapping system. Maps were aligned to the human reference by OPTIMA, a new glocal alignment method. Genome coverage of 6.8× and 5.7× was obtained, respectively; 2.9× and 1.7× more than the coverage obtained with previously available software. Optical mapping allows the resolution of large-scale structural variations of the genome, and the scaffold extension of NGS-based de novo assemblies. OPTIMA is an efficient new alignment method; our optical mapping data provide a resource for genome structure analyses of the human HapMap reference cell line GM12878, and the colorectal cancer cell line HCT116.

Construction of a map-based reference genome sequence for barley, Hordeum vulgare L.

PubMed Central

Beier, Sebastian; Himmelbach, Axel; Colmsee, Christian; Zhang, Xiao-Qi; Barrero, Roberto A.; Zhang, Qisen; Li, Lin; Bayer, Micha; Bolser, Daniel; Taudien, Stefan; Groth, Marco; Felder, Marius; Hastie, Alex; Šimková, Hana; Staňková, Helena; Vrána, Jan; Chan, Saki; Muñoz-Amatriaín, María; Ounit, Rachid; Wanamaker, Steve; Schmutzer, Thomas; Aliyeva-Schnorr, Lala; Grasso, Stefano; Tanskanen, Jaakko; Sampath, Dharanya; Heavens, Darren; Cao, Sujie; Chapman, Brett; Dai, Fei; Han, Yong; Li, Hua; Li, Xuan; Lin, Chongyun; McCooke, John K.; Tan, Cong; Wang, Songbo; Yin, Shuya; Zhou, Gaofeng; Poland, Jesse A.; Bellgard, Matthew I.; Houben, Andreas; Doležel, Jaroslav; Ayling, Sarah; Lonardi, Stefano; Langridge, Peter; Muehlbauer, Gary J.; Kersey, Paul; Clark, Matthew D.; Caccamo, Mario; Schulman, Alan H.; Platzer, Matthias; Close, Timothy J.; Hansson, Mats; Zhang, Guoping; Braumann, Ilka; Li, Chengdao; Waugh, Robbie; Scholz, Uwe; Stein, Nils; Mascher, Martin

2017-01-01

Barley (Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. ‘Morex’ was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX). PMID:28448065

Draft genome sequence of the silver pomfret fish, Pampus argenteus.

PubMed

AlMomin, Sabah; Kumar, Vinod; Al-Amad, Sami; Al-Hussaini, Mohsen; Dashti, Talal; Al-Enezi, Khaznah; Akbar, Abrar

2016-01-01

Silver pomfret, Pampus argenteus, is a fish species from coastal waters. Despite its high commercial value, this edible fish has not been sequenced. Hence, its genetic and genomic studies have been limited. We report the first draft genome sequence of the silver pomfret obtained using a Next Generation Sequencing (NGS) technology. We assembled 38.7 Gb of nucleotides into scaffolds of 350 Mb with N50 of about 1.5 kb, using high quality paired end reads. These scaffolds represent 63.7% of the estimated silver pomfret genome length. The newly sequenced and assembled genome has 11.06% repetitive DNA regions, and this percentage is comparable to that of the tilapia genome. The genome analysis predicted 16 322 genes. About 91% of these genes showed homology with known proteins. Many gene clusters were annotated to protein and fatty-acid metabolism pathways that may be important in the context of the meat texture and immune system developmental processes. The reference genome can pave the way for the identification of many other genomic features that could improve breeding and population-management strategies, and it can also help characterize the genetic diversity of P. argenteus.

Centromere reference models for human chromosomes X and Y satellite arrays

PubMed Central

Miga, Karen H.; Newton, Yulia; Jain, Miten; Altemose, Nicolas; Willard, Huntington F.; Kent, W. James

2014-01-01

The human genome sequence remains incomplete, with multimegabase-sized gaps representing the endogenous centromeres and other heterochromatic regions. Available sequence-based studies within these sites in the genome have demonstrated a role in centromere function and chromosome pairing, necessary to ensure proper chromosome segregation during cell division. A common genomic feature of these regions is the enrichment of long arrays of near-identical tandem repeats, known as satellite DNAs, which offer a limited number of variant sites to differentiate individual repeat copies across millions of bases. This substantial sequence homogeneity challenges available assembly strategies and, as a result, centromeric regions are omitted from ongoing genomic studies. To address this problem, we utilize monomer sequence and ordering information obtained from whole-genome shotgun reads to model two haploid human satellite arrays on chromosomes X and Y, resulting in an initial characterization of 3.83 Mb of centromeric DNA within an individual genome. To further expand the utility of each centromeric reference sequence model, we evaluate sites within the arrays for short-read mappability and chromosome specificity. Because satellite DNAs evolve in a concerted manner, we use these centromeric assemblies to assess the extent of sequence variation among 366 individuals from distinct human populations. We thus identify two satellite array variants in both X and Y centromeres, as determined by array length and sequence composition. This study provides an initial sequence characterization of a regional centromere and establishes a foundation to extend genomic characterization to these sites as well as to other repeat-rich regions within complex genomes. PMID:24501022

Multi-scale structural community organisation of the human genome.

PubMed

Boulos, Rasha E; Tremblay, Nicolas; Arneodo, Alain; Borgnat, Pierre; Audit, Benjamin

2017-04-11

Structural interaction frequency matrices between all genome loci are now experimentally achievable thanks to high-throughput chromosome conformation capture technologies. This ensues a new methodological challenge for computational biology which consists in objectively extracting from these data the structural motifs characteristic of genome organisation. We deployed the fast multi-scale community mining algorithm based on spectral graph wavelets to characterise the networks of intra-chromosomal interactions in human cell lines. We observed that there exist structural domains of all sizes up to chromosome length and demonstrated that the set of structural communities forms a hierarchy of chromosome segments. Hence, at all scales, chromosome folding predominantly involves interactions between neighbouring sites rather than the formation of links between distant loci. Multi-scale structural decomposition of human chromosomes provides an original framework to question structural organisation and its relationship to functional regulation across the scales. By construction the proposed methodology is independent of the precise assembly of the reference genome and is thus directly applicable to genomes whose assembly is not fully determined.

Evolutionary signals of selection on cognition from the great tit genome and methylome

PubMed Central

Laine, Veronika N.; Gossmann, Toni I.; Schachtschneider, Kyle M.; Garroway, Colin J.; Madsen, Ole; Verhoeven, Koen J. F.; de Jager, Victor; Megens, Hendrik-Jan; Warren, Wesley C.; Minx, Patrick; Crooijmans, Richard P. M. A.; Corcoran, Pádraic; Adriaensen, Frank; Belda, Eduardo; Bushuev, Andrey; Cichon, Mariusz; Charmantier, Anne; Dingemanse, Niels; Doligez, Blandine; Eeva, Tapio; Erikstad, Kjell Einar; Fedorov, Slava; Hau, Michaela; Hille, Sabine; Hinde, Camilla; Kempenaers, Bart; Kerimov, Anvar; Krist, Milos; Mand, Raivo; Matthysen, Erik; Nager, Reudi; Norte, Claudia; Orell, Markku; Richner, Heinz; Slagsvold, Tore; Tilgar, Vallo; Tinbergen, Joost; Torok, Janos; Tschirren, Barbara; Yuta, Tera; Sheldon, Ben C.; Slate, Jon; Zeng, Kai; van Oers, Kees; Visser, Marcel E.; Groenen, Martien A. M.

2016-01-01

For over 50 years, the great tit (Parus major) has been a model species for research in evolutionary, ecological and behavioural research; in particular, learning and cognition have been intensively studied. Here, to provide further insight into the molecular mechanisms behind these important traits, we de novo assemble a great tit reference genome and whole-genome re-sequence another 29 individuals from across Europe. We show an overrepresentation of genes related to neuronal functions, learning and cognition in regions under positive selection, as well as increased CpG methylation in these regions. In addition, great tit neuronal non-CpG methylation patterns are very similar to those observed in mammals, suggesting a universal role in neuronal epigenetic regulation which can affect learning-, memory- and experience-induced plasticity. The high-quality great tit genome assembly will play an instrumental role in furthering the integration of ecological, evolutionary, behavioural and genomic approaches in this model species. PMID:26805030

A comprehensive transcriptome assembly of Pigeonpea (Cajanus cajan L.) using sanger and second-generation sequencing platforms.

PubMed

Kudapa, Himabindu; Bharti, Arvind K; Cannon, Steven B; Farmer, Andrew D; Mulaosmanovic, Benjamin; Kramer, Robin; Bohra, Abhishek; Weeks, Nathan T; Crow, John A; Tuteja, Reetu; Shah, Trushar; Dutta, Sutapa; Gupta, Deepak K; Singh, Archana; Gaikwad, Kishor; Sharma, Tilak R; May, Gregory D; Singh, Nagendra K; Varshney, Rajeev K

2012-09-01

A comprehensive transcriptome assembly for pigeonpea has been developed by analyzing 128.9 million short Illumina GA IIx single end reads, 2.19 million single end FLX/454 reads, and 18 353 Sanger expressed sequenced tags from more than 16 genotypes. The resultant transcriptome assembly, referred to as CcTA v2, comprised 21 434 transcript assembly contigs (TACs) with an N50 of 1510 bp, the largest one being ~8 kb. Of the 21 434 TACs, 16 622 (77.5%) could be mapped on to the soybean genome build 1.0.9 under fairly stringent alignment parameters. Based on knowledge of intron junctions, 10 009 primer pairs were designed from 5033 TACs for amplifying intron spanning regions (ISRs). By using in silico mapping of BAC-end-derived SSR loci of pigeonpea on the soybean genome as a reference, putative mapping positions at the chromosome level were predicted for 6284 ISR markers, covering all 11 pigeonpea chromosomes. A subset of 128 ISR markers were analyzed on a set of eight genotypes. While 116 markers were validated, 70 markers showed one to three alleles, with an average of 0.16 polymorphism information content (PIC) value. In summary, the CcTA v2 transcript assembly and ISR markers will serve as a useful resource to accelerate genetic research and breeding applications in pigeonpea.

Genomics of Compositae crops: reference transcriptome assemblies and evidence of hybridization with wild relatives.

PubMed

Hodgins, Kathryn A; Lai, Zhao; Oliveira, Luiz O; Still, David W; Scascitelli, Moira; Barker, Michael S; Kane, Nolan C; Dempewolf, Hannes; Kozik, Alex; Kesseli, Richard V; Burke, John M; Michelmore, Richard W; Rieseberg, Loren H

2014-01-01

Although the Compositae harbours only two major food crops, sunflower and lettuce, many other species in this family are utilized by humans and have experienced various levels of domestication. Here, we have used next-generation sequencing technology to develop 15 reference transcriptome assemblies for Compositae crops or their wild relatives. These data allow us to gain insight into the evolutionary and genomic consequences of plant domestication. Specifically, we performed Illumina sequencing of Cichorium endivia, Cichorium intybus, Echinacea angustifolia, Iva annua, Helianthus tuberosus, Dahlia hybrida, Leontodon taraxacoides and Glebionis segetum, as well 454 sequencing of Guizotia scabra, Stevia rebaudiana, Parthenium argentatum and Smallanthus sonchifolius. Illumina reads were assembled using Trinity, and 454 reads were assembled using MIRA and CAP3. We evaluated the coverage of the transcriptomes using BLASTX analysis of a set of ultra-conserved orthologs (UCOs) and recovered most of these genes (88-98%). We found a correlation between contig length and read length for the 454 assemblies, and greater contig lengths for the 454 compared with the Illumina assemblies. This suggests that longer reads can aid in the assembly of more complete transcripts. Finally, we compared the divergence of orthologs at synonymous sites (Ks) between Compositae crops and their wild relatives and found greater divergence when the progenitors were self-incompatible. We also found greater divergence between pairs of taxa that had some evidence of postzygotic isolation. For several more distantly related congeners, such as chicory and endive, we identified a signature of introgression in the distribution of Ks values. © 2013 John Wiley & Sons Ltd.

De novo assembly of the transcriptome of the non-model plant Streptocarpus rexii employing a novel heuristic to recover locus-specific transcript clusters.

PubMed

Chiara, Matteo; Horner, David S; Spada, Alberto

2013-01-01

De novo transcriptome characterization from Next Generation Sequencing data has become an important approach in the study of non-model plants. Despite notable advances in the assembly of short reads, the clustering of transcripts into unigene-like (locus-specific) clusters remains a somewhat neglected subject. Indeed, closely related paralogous transcripts are often merged into single clusters by current approaches. Here, a novel heuristic method for locus-specific clustering is compared to that implemented in the de novo assembler Oases, using the same initial transcript collections, derived from Arabidopsis thaliana and the developmental model Streptocarpus rexii. We show that the proposed approach improves cluster specificity in the A. thaliana dataset for which the reference genome is available. Furthermore, for the S. rexii data our filtered transcript collection matches a larger number of distinct annotated loci in reference genomes than the Oases set, while containing a reduced overall number of loci. A detailed discussion of advantages and limitations of our approach in processing de novo transcriptome reconstructions is presented. The proposed method should be widely applicable to other organisms, irrespective of the transcript assembly method employed. The S. rexii transcriptome is available as a sophisticated and augmented publicly available online database.

Genetics and fine mapping of a purple leaf gene, BoPr, in ornamental kale (Brassica oleracea L. var. acephala).

PubMed

Liu, Xiao-Ping; Gao, Bao-Zhen; Han, Feng-Qing; Fang, Zhi-Yuan; Yang, Li-Mei; Zhuang, Mu; Lv, Hong-Hao; Liu, Yu-Mei; Li, Zhan-Sheng; Cai, Cheng-Cheng; Yu, Hai-Long; Li, Zhi-Yuan; Zhang, Yang-Yong

2017-03-14

Due to its variegated and colorful leaves, ornamental kale (Brassica oleracea L. var. acephala) has become a popular ornamental plant. In this study, we report the fine mapping and analysis of a candidate purple leaf gene using a backcross population and an F 2 population derived from two parental lines: W1827 (with white leaves) and P1835 (with purple leaves). Genetic analysis indicated that the purple leaf trait is controlled by a single dominant gene, which we named BoPr. Using markers developed based on the reference genome '02-12', the BoPr gene was preliminarily mapped to a 280-kb interval of chromosome C09, with flanking markers M17 and BoID4714 at genetic distances of 4.3 cM and 1.5 cM, respectively. The recombination rate within this interval is almost 12 times higher than the usual level, which could be caused by assembly error for reference genome '02-12' at this interval. Primers were designed based on 'TO1000', another B. oleracea reference genome. Among the newly designed InDel markers, BRID485 and BRID490 were found to be the closest to BoPr, flanking the gene at genetic distances of 0.1 cM and 0.2 cM, respectively; the interval between the two markers is 44.8 kb (reference genome 'TO1000'). Seven annotated genes are located within the 44.8 kb genomic region, of which only Bo9g058630 shows high homology to AT5G42800 (dihydroflavonol reductase), which was identified as a candidate gene for BoPr. Blast analysis revealed that this 44.8 kb interval is located on an unanchored scaffold (Scaffold000035_P2) of '02-12', confirming the existence of assembly error at the interval between M17 and BoID4714 for reference genome '02-12'. This study identified a candidate gene for BoPr and lays a foundation for the cloning and functional analysis of this gene.

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

PubMed Central

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

2018-01-01

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

Consensus coding sequence (CCDS) database: a standardized set of human and mouse protein-coding regions supported by expert curation

PubMed Central

Pujar, Shashikant; O’Leary, Nuala A; Farrell, Catherine M; Mudge, Jonathan M; Wallin, Craig; Diekhans, Mark; Barnes, If; Bennett, Ruth; Berry, Andrew E; Cox, Eric; Davidson, Claire; Goldfarb, Tamara; Gonzalez, Jose M; Hunt, Toby; Jackson, John; Joardar, Vinita; Kay, Mike P; Kodali, Vamsi K; McAndrews, Monica; McGarvey, Kelly M; Murphy, Michael; Rajput, Bhanu; Rangwala, Sanjida H; Riddick, Lillian D; Seal, Ruth L; Webb, David; Zhu, Sophia; Aken, Bronwen L; Bult, Carol J; Frankish, Adam; Pruitt, Kim D

2018-01-01

Abstract The Consensus Coding Sequence (CCDS) project provides a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assembly in genome annotations produced independently by NCBI and the Ensembl group at EMBL-EBI. This dataset is the product of an international collaboration that includes NCBI, Ensembl, HUGO Gene Nomenclature Committee, Mouse Genome Informatics and University of California, Santa Cruz. Identically annotated coding regions, which are generated using an automated pipeline and pass multiple quality assurance checks, are assigned a stable and tracked identifier (CCDS ID). Additionally, coordinated manual review by expert curators from the CCDS collaboration helps in maintaining the integrity and high quality of the dataset. The CCDS data are available through an interactive web page (https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi) and an FTP site (ftp://ftp.ncbi.nlm.nih.gov/pub/CCDS/). In this paper, we outline the ongoing work, growth and stability of the CCDS dataset and provide updates on new collaboration members and new features added to the CCDS user interface. We also present expert curation scenarios, with specific examples highlighting the importance of an accurate reference genome assembly and the crucial role played by input from the research community. PMID:29126148

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

PubMed Central

2013-01-01

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

Assembly of the draft genome of buckwheat and its applications in identifying agronomically useful genes

PubMed Central

Yasui, Yasuo; Hirakawa, Hideki; Ueno, Mariko; Matsui, Katsuhiro; Katsube-Tanaka, Tomoyuki; Yang, Soo Jung; Aii, Jotaro; Sato, Shingo; Mori, Masashi

2016-01-01

Buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is a nutritionally dense annual crop widely grown in temperate zones. To accelerate molecular breeding programmes of this important crop, we generated a draft assembly of the buckwheat genome using short reads obtained by next-generation sequencing (NGS), and constructed the Buckwheat Genome DataBase. After assembling short reads, we determined 387,594 scaffolds as the draft genome sequence (FES_r1.0). The total length of FES_r1.0 was 1,177,687,305 bp, and the N50 of the scaffolds was 25,109 bp. Gene prediction analysis revealed 286,768 coding sequences (CDSs; FES_r1.0_cds) including those related to transposable elements. The total length of FES_r1.0_cds was 212,917,911 bp, and the N50 was 1,101 bp. Of these, the functions of 35,816 CDSs excluding those for transposable elements were annotated by BLAST analysis. To demonstrate the utility of the database, we conducted several test analyses using BLAST and keyword searches. Furthermore, we used the draft genome as a reference sequence for NGS-based markers, and successfully identified novel candidate genes controlling heteromorphic self-incompatibility of buckwheat. The database and draft genome sequence provide a valuable resource that can be used in efforts to develop buckwheat cultivars with superior agronomic traits. PMID:27037832

A computational genomics pipeline for prokaryotic sequencing projects

PubMed Central

Kislyuk, Andrey O.; Katz, Lee S.; Agrawal, Sonia; Hagen, Matthew S.; Conley, Andrew B.; Jayaraman, Pushkala; Nelakuditi, Viswateja; Humphrey, Jay C.; Sammons, Scott A.; Govil, Dhwani; Mair, Raydel D.; Tatti, Kathleen M.; Tondella, Maria L.; Harcourt, Brian H.; Mayer, Leonard W.; Jordan, I. King

2010-01-01

Motivation: New sequencing technologies have accelerated research on prokaryotic genomes and have made genome sequencing operations outside major genome sequencing centers routine. However, no off-the-shelf solution exists for the combined assembly, gene prediction, genome annotation and data presentation necessary to interpret sequencing data. The resulting requirement to invest significant resources into custom informatics support for genome sequencing projects remains a major impediment to the accessibility of high-throughput sequence data. Results: We present a self-contained, automated high-throughput open source genome sequencing and computational genomics pipeline suitable for prokaryotic sequencing projects. The pipeline has been used at the Georgia Institute of Technology and the Centers for Disease Control and Prevention for the analysis of Neisseria meningitidis and Bordetella bronchiseptica genomes. The pipeline is capable of enhanced or manually assisted reference-based assembly using multiple assemblers and modes; gene predictor combining; and functional annotation of genes and gene products. Because every component of the pipeline is executed on a local machine with no need to access resources over the Internet, the pipeline is suitable for projects of a sensitive nature. Annotation of virulence-related features makes the pipeline particularly useful for projects working with pathogenic prokaryotes. Availability and implementation: The pipeline is licensed under the open-source GNU General Public License and available at the Georgia Tech Neisseria Base (http://nbase.biology.gatech.edu/). The pipeline is implemented with a combination of Perl, Bourne Shell and MySQL and is compatible with Linux and other Unix systems. Contact: king.jordan@biology.gatech.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20519285

The Genomic HyperBrowser: an analysis web server for genome-scale data

PubMed Central

Sandve, Geir K.; Gundersen, Sveinung; Johansen, Morten; Glad, Ingrid K.; Gunathasan, Krishanthi; Holden, Lars; Holden, Marit; Liestøl, Knut; Nygård, Ståle; Nygaard, Vegard; Paulsen, Jonas; Rydbeck, Halfdan; Trengereid, Kai; Clancy, Trevor; Drabløs, Finn; Ferkingstad, Egil; Kalaš, Matúš; Lien, Tonje; Rye, Morten B.; Frigessi, Arnoldo; Hovig, Eivind

2013-01-01

The immense increase in availability of genomic scale datasets, such as those provided by the ENCODE and Roadmap Epigenomics projects, presents unprecedented opportunities for individual researchers to pose novel falsifiable biological questions. With this opportunity, however, researchers are faced with the challenge of how to best analyze and interpret their genome-scale datasets. A powerful way of representing genome-scale data is as feature-specific coordinates relative to reference genome assemblies, i.e. as genomic tracks. The Genomic HyperBrowser (http://hyperbrowser.uio.no) is an open-ended web server for the analysis of genomic track data. Through the provision of several highly customizable components for processing and statistical analysis of genomic tracks, the HyperBrowser opens for a range of genomic investigations, related to, e.g., gene regulation, disease association or epigenetic modifications of the genome. PMID:23632163

The Genomic HyperBrowser: an analysis web server for genome-scale data.

PubMed

Sandve, Geir K; Gundersen, Sveinung; Johansen, Morten; Glad, Ingrid K; Gunathasan, Krishanthi; Holden, Lars; Holden, Marit; Liestøl, Knut; Nygård, Ståle; Nygaard, Vegard; Paulsen, Jonas; Rydbeck, Halfdan; Trengereid, Kai; Clancy, Trevor; Drabløs, Finn; Ferkingstad, Egil; Kalas, Matús; Lien, Tonje; Rye, Morten B; Frigessi, Arnoldo; Hovig, Eivind

2013-07-01

The immense increase in availability of genomic scale datasets, such as those provided by the ENCODE and Roadmap Epigenomics projects, presents unprecedented opportunities for individual researchers to pose novel falsifiable biological questions. With this opportunity, however, researchers are faced with the challenge of how to best analyze and interpret their genome-scale datasets. A powerful way of representing genome-scale data is as feature-specific coordinates relative to reference genome assemblies, i.e. as genomic tracks. The Genomic HyperBrowser (http://hyperbrowser.uio.no) is an open-ended web server for the analysis of genomic track data. Through the provision of several highly customizable components for processing and statistical analysis of genomic tracks, the HyperBrowser opens for a range of genomic investigations, related to, e.g., gene regulation, disease association or epigenetic modifications of the genome.

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

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

Complete genome assemblies and methylome characterization in infectious diseases

USDA-ARS?s Scientific Manuscript database

Understanding the genetic basis of infectious diseases is a critical component to effective treatments. Because of the rapid evolution of bacterial strains and frequent horizontal transfer of DNA between them, resequencing of new isolates against known reference strains often provides an incomplete ...

Zseq: An Approach for Preprocessing Next-Generation Sequencing Data.

PubMed

Alkhateeb, Abedalrhman; Rueda, Luis

2017-08-01

Next-generation sequencing technology generates a huge number of reads (short sequences), which contain a vast amount of genomic data. The sequencing process, however, comes with artifacts. Preprocessing of sequences is mandatory for further downstream analysis. We present Zseq, a linear method that identifies the most informative genomic sequences and reduces the number of biased sequences, sequence duplications, and ambiguous nucleotides. Zseq finds the complexity of the sequences by counting the number of unique k-mers in each sequence as its corresponding score and also takes into the account other factors such as ambiguous nucleotides or high GC-content percentage in k-mers. Based on a z-score threshold, Zseq sweeps through the sequences again and filters those with a z-score less than the user-defined threshold. Zseq algorithm is able to provide a better mapping rate; it reduces the number of ambiguous bases significantly in comparison with other methods. Evaluation of the filtered reads has been conducted by aligning the reads and assembling the transcripts using the reference genome as well as de novo assembly. The assembled transcripts show a better discriminative ability to separate cancer and normal samples in comparison with another state-of-the-art method. Moreover, de novo assembled transcripts from the reads filtered by Zseq have longer genomic sequences than other tested methods. Estimating the threshold of the cutoff point is introduced using labeling rules with optimistic results.

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

De Novo Assembly and Phasing of Dikaryotic Genomes from Two Isolates of Puccinia coronata f. sp. avenae, the Causal Agent of Oat Crown Rust

PubMed Central

Miller, Marisa E.; Zhang, Ying; Omidvar, Vahid; Sperschneider, Jana; Raley, Castle; Palmer, Jonathan M.; Garnica, Diana; Upadhyaya, Narayana; Rathjen, John; Taylor, Jennifer M.; Park, Robert F.; Dodds, Peter N.; Hirsch, Cory D.

2018-01-01

ABSTRACT Oat crown rust, caused by the fungus Pucinnia coronata f. sp. avenae, is a devastating disease that impacts worldwide oat production. For much of its life cycle, P. coronata f. sp. avenae is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two P. coronata f. sp. avenae isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for 12SD80, for a total assembly length of 99.16 Mbp, and 777 primary contigs for 12NC29, for a total length of 105.25 Mbp; approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of coexpressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in P. coronata f. sp. avenae. PMID:29463655

Transcriptome sequencing of different narrow-leafed lupin tissue types provides a comprehensive uni-gene assembly and extensive gene-based molecular markers

PubMed Central

Kamphuis, Lars G; Hane, James K; Nelson, Matthew N; Gao, Lingling; Atkins, Craig A; Singh, Karam B

2015-01-01

Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is an important grain legume crop that is valuable for sustainable farming and is becoming recognized as a human health food. NLL breeding is directed at improving grain production, disease resistance, drought tolerance and health benefits. However, genetic and genomic studies have been hindered by a lack of extensive genomic resources for the species. Here, the generation, de novo assembly and annotation of transcriptome datasets derived from five different NLL tissue types of the reference accession cv. Tanjil are described. The Tanjil transcriptome was compared to transcriptomes of an early domesticated cv. Unicrop, a wild accession P27255, as well as accession 83A:476, together being the founding parents of two recombinant inbred line (RIL) populations. In silico predictions for transcriptome-derived gene-based length and SNP polymorphic markers were conducted and corroborated using a survey assembly sequence for NLL cv. Tanjil. This yielded extensive indel and SNP polymorphic markers for the two RIL populations. A total of 335 transcriptome-derived markers and 66 BAC-end sequence-derived markers were evaluated, and 275 polymorphic markers were selected to genotype the reference NLL 83A:476 × P27255 RIL population. This significantly improved the completeness, marker density and quality of the reference NLL genetic map. PMID:25060816

Gramene 2016: comparative plant genomics and pathway resources

PubMed Central

Tello-Ruiz, Marcela K.; Stein, Joshua; Wei, Sharon; Preece, Justin; Olson, Andrew; Naithani, Sushma; Amarasinghe, Vindhya; Dharmawardhana, Palitha; Jiao, Yinping; Mulvaney, Joseph; Kumari, Sunita; Chougule, Kapeel; Elser, Justin; Wang, Bo; Thomason, James; Bolser, Daniel M.; Kerhornou, Arnaud; Walts, Brandon; Fonseca, Nuno A.; Huerta, Laura; Keays, Maria; Tang, Y. Amy; Parkinson, Helen; Fabregat, Antonio; McKay, Sheldon; Weiser, Joel; D'Eustachio, Peter; Stein, Lincoln; Petryszak, Robert; Kersey, Paul J.; Jaiswal, Pankaj; Ware, Doreen

2016-01-01

Gramene (http://www.gramene.org) is an online resource for comparative functional genomics in crops and model plant species. Its two main frameworks are genomes (collaboration with Ensembl Plants) and pathways (The Plant Reactome and archival BioCyc databases). Since our last NAR update, the database website adopted a new Drupal management platform. The genomes section features 39 fully assembled reference genomes that are integrated using ontology-based annotation and comparative analyses, and accessed through both visual and programmatic interfaces. Additional community data, such as genetic variation, expression and methylation, are also mapped for a subset of genomes. The Plant Reactome pathway portal (http://plantreactome.gramene.org) provides a reference resource for analyzing plant metabolic and regulatory pathways. In addition to ∼200 curated rice reference pathways, the portal hosts gene homology-based pathway projections for 33 plant species. Both the genome and pathway browsers interface with the EMBL-EBI's Expression Atlas to enable the projection of baseline and differential expression data from curated expression studies in plants. Gramene's archive website (http://archive.gramene.org) continues to provide previously reported resources on comparative maps, markers and QTL. To further aid our users, we have also introduced a live monthly educational webinar series and a Gramene YouTube channel carrying video tutorials. PMID:26553803

Status of the rainbow trout genome reference sequence assembly

USDA-ARS?s Scientific Manuscript database

Rainbow trout (Oncorhynchus mykiss) are the most cultivated cold water fish in the U.S. In addition to interests associated with aquaculture and sport fisheries, the rainbow trout serves as a model research organism for studies related to carcinogenesis, toxicology, comparative immunology, disease ...

Scanning the human genome at kilobase resolution.

PubMed

Chen, Jun; Kim, Yeong C; Jung, Yong-Chul; Xuan, Zhenyu; Dworkin, Geoff; Zhang, Yanming; Zhang, Michael Q; Wang, San Ming

2008-05-01

Normal genome variation and pathogenic genome alteration frequently affect small regions in the genome. Identifying those genomic changes remains a technical challenge. We report here the development of the DGS (Ditag Genome Scanning) technique for high-resolution analysis of genome structure. The basic features of DGS include (1) use of high-frequent restriction enzymes to fractionate the genome into small fragments; (2) collection of two tags from two ends of a given DNA fragment to form a ditag to represent the fragment; (3) application of the 454 sequencing system to reach a comprehensive ditag sequence collection; (4) determination of the genome origin of ditags by mapping to reference ditags from known genome sequences; (5) use of ditag sequences directly as the sense and antisense PCR primers to amplify the original DNA fragment. To study the relationship between ditags and genome structure, we performed a computational study by using the human genome reference sequences as a model, and analyzed the ditags experimentally collected from the well-characterized normal human DNA GM15510 and the leukemic human DNA of Kasumi-1 cells. Our studies show that DGS provides a kilobase resolution for studying genome structure with high specificity and high genome coverage. DGS can be applied to validate genome assembly, to compare genome similarity and variation in normal populations, and to identify genomic abnormality including insertion, inversion, deletion, translocation, and amplification in pathological genomes such as cancer genomes.

Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver.

PubMed

Wymant, Chris; Blanquart, François; Golubchik, Tanya; Gall, Astrid; Bakker, Margreet; Bezemer, Daniela; Croucher, Nicholas J; Hall, Matthew; Hillebregt, Mariska; Ong, Swee Hoe; Ratmann, Oliver; Albert, Jan; Bannert, Norbert; Fellay, Jacques; Fransen, Katrien; Gourlay, Annabelle; Grabowski, M Kate; Gunsenheimer-Bartmeyer, Barbara; Günthard, Huldrych F; Kivelä, Pia; Kouyos, Roger; Laeyendecker, Oliver; Liitsola, Kirsi; Meyer, Laurence; Porter, Kholoud; Ristola, Matti; van Sighem, Ard; Berkhout, Ben; Cornelissen, Marion; Kellam, Paul; Reiss, Peter; Fraser, Christophe

2018-01-01

Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver's constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied shiver to whole-genome samples of Hepatitis C Virus and Respiratory Syncytial Virus. shiver is publicly available from https://github.com/ChrisHIV/shiver.

Integration of Genomic and Other Epidemiologic Data to Investigate and Control a Cross-Institutional Outbreak of Streptococcus pyogenes.

PubMed

Chalker, Victoria J; Smith, Alyson; Al-Shahib, Ali; Botchway, Stella; Macdonald, Emily; Daniel, Roger; Phillips, Sarah; Platt, Steven; Doumith, Michel; Tewolde, Rediat; Coelho, Juliana; Jolley, Keith A; Underwood, Anthony; McCarthy, Noel D

2016-06-01

Single-strain outbreaks of Streptococcus pyogenes infections are common and often go undetected. In 2013, two clusters of invasive group A Streptococcus (iGAS) infection were identified in independent but closely located care homes in Oxfordshire, United Kingdom. Investigation included visits to each home, chart review, staff survey, microbiologic sampling, and genome sequencing. S. pyogenes emm type 1.0, the most common circulating type nationally, was identified from all cases yielding GAS isolates. A tailored whole-genome reference population comprising epidemiologically relevant contemporaneous isolates and published isolates was assembled. Data were analyzed independently using whole-genome multilocus sequencing and single-nucleotide polymorphism analyses. Six isolates from staff and residents of the homes formed a single cluster that was separated from the reference population by both analytical approaches. No further cases occurred after mass chemoprophylaxis and enhanced infection control. Our findings demonstrate the ability of 2 independent analytical approaches to enable robust conclusions from nonstandardized whole-genome analysis to support public health practice.

Piggy: a rapid, large-scale pan-genome analysis tool for intergenic regions in bacteria.

PubMed

Thorpe, Harry A; Bayliss, Sion C; Sheppard, Samuel K; Feil, Edward J

2018-04-01

The concept of the "pan-genome," which refers to the total complement of genes within a given sample or species, is well established in bacterial genomics. Rapid and scalable pipelines are available for managing and interpreting pan-genomes from large batches of annotated assemblies. However, despite overwhelming evidence that variation in intergenic regions in bacteria can directly influence phenotypes, most current approaches for analyzing pan-genomes focus exclusively on protein-coding sequences. To address this we present Piggy, a novel pipeline that emulates Roary except that it is based only on intergenic regions. A key utility provided by Piggy is the detection of highly divergent ("switched") intergenic regions (IGRs) upstream of genes. We demonstrate the use of Piggy on large datasets of clinically important lineages of Staphylococcus aureus and Escherichia coli. For S. aureus, we show that highly divergent (switched) IGRs are associated with differences in gene expression and we establish a multilocus reference database of IGR alleles (igMLST; implemented in BIGSdb).

GenPlay Multi-Genome, a tool to compare and analyze multiple human genomes in a graphical interface.

PubMed

Lajugie, Julien; Fourel, Nicolas; Bouhassira, Eric E

2015-01-01

Parallel visualization of multiple individual human genomes is a complex endeavor that is rapidly gaining importance with the increasing number of personal, phased and cancer genomes that are being generated. It requires the display of variants such as SNPs, indels and structural variants that are unique to specific genomes and the introduction of multiple overlapping gaps in the reference sequence. Here, we describe GenPlay Multi-Genome, an application specifically written to visualize and analyze multiple human genomes in parallel. GenPlay Multi-Genome is ideally suited for the comparison of allele-specific expression and functional genomic data obtained from multiple phased genomes in a graphical interface with access to multiple-track operation. It also allows the analysis of data that have been aligned to custom genomes rather than to a standard reference and can be used as a variant calling format file browser and as a tool to compare different genome assembly, such as hg19 and hg38. GenPlay is available under the GNU public license (GPL-3) from http://genplay.einstein.yu.edu. The source code is available at https://github.com/JulienLajugie/GenPlay. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

Constructing a 'Chromonome' of Yellowtail (Seriola quinqueradiata) for Comparative Analysis of Chromosomal Rearrangements

PubMed Central

Kawase, Junya; Aoki, Jun-ya; Araki, Kazuo

2018-01-01

To investigate chromosome evolution in fish species, we newly mapped 181 markers that allowed us to construct a yellowtail (Seriola quinqueradiata) radiation hybrid (RH) physical map with 1,713 DNA markers, which was far denser than a previous map, and we anchored the de novo assembled sequences onto the RH physical map. Finally, we mapped a total of 13,977 expressed sequence tags (ESTs) on a genome sequence assembly aligned with the physical map. Using the high-density physical map and anchored genome sequences, we accurately compared the yellowtail genome structure with the genome structures of five model fishes to identify characteristics of the yellowtail genome. Between yellowtail and Japanese medaka (Oryzias latipes), almost all regions of the chromosomes were conserved and some blocks comprising several markers were translocated. Using the genome information of the spotted gar (Lepisosteus oculatus) as a reference, we further documented syntenic relationships and chromosomal rearrangements that occurred during evolution in four other acanthopterygian species (Japanese medaka, zebrafish, spotted green pufferfish and three-spined stickleback). The evolutionary chromosome translocation frequency was 1.5-2-times higher in yellowtail than in medaka, pufferfish, and stickleback. PMID:29290830

SNP Identification from RNA Sequencing and Linkage Map Construction of Rubber Tree for Anchoring the Draft Genome

PubMed Central

Shearman, Jeremy R.; Sangsrakru, Duangjai; Jomchai, Nukoon; Ruang-areerate, Panthita; Sonthirod, Chutima; Naktang, Chaiwat; Theerawattanasuk, Kanikar; Tragoonrung, Somvong; Tangphatsornruang, Sithichoke

2015-01-01

Hevea brasiliensis, or rubber tree, is an important crop species that accounts for the majority of natural latex production. The rubber tree nuclear genome consists of 18 chromosomes and is roughly 2.15 Gb. The current rubber tree reference genome assembly consists of 1,150,326 scaffolds ranging from 200 to 531,465 bp and totalling 1.1 Gb. Only 143 scaffolds, totalling 7.6 Mb, have been placed into linkage groups. We have performed RNA-seq on 6 varieties of rubber tree to identify SNPs and InDels and used this information to perform target sequence enrichment and high throughput sequencing to genotype a set of SNPs in 149 rubber tree offspring from a cross between RRIM 600 and RRII 105 rubber tree varieties. We used this information to generate a linkage map allowing for the anchoring of 24,424 contigs from 3,009 scaffolds, totalling 115 Mb or 10.4% of the published sequence, into 18 linkage groups. Each linkage group contains between 319 and 1367 SNPs, or 60 to 194 non-redundant marker positions, and ranges from 156 to 336 cM in length. This linkage map includes 20,143 of the 69,300 predicted genes from rubber tree and will be useful for mapping studies and improving the reference genome assembly. PMID:25831195

Consensus coding sequence (CCDS) database: a standardized set of human and mouse protein-coding regions supported by expert curation.

PubMed

Pujar, Shashikant; O'Leary, Nuala A; Farrell, Catherine M; Loveland, Jane E; Mudge, Jonathan M; Wallin, Craig; Girón, Carlos G; Diekhans, Mark; Barnes, If; Bennett, Ruth; Berry, Andrew E; Cox, Eric; Davidson, Claire; Goldfarb, Tamara; Gonzalez, Jose M; Hunt, Toby; Jackson, John; Joardar, Vinita; Kay, Mike P; Kodali, Vamsi K; Martin, Fergal J; McAndrews, Monica; McGarvey, Kelly M; Murphy, Michael; Rajput, Bhanu; Rangwala, Sanjida H; Riddick, Lillian D; Seal, Ruth L; Suner, Marie-Marthe; Webb, David; Zhu, Sophia; Aken, Bronwen L; Bruford, Elspeth A; Bult, Carol J; Frankish, Adam; Murphy, Terence; Pruitt, Kim D

2018-01-04

The Consensus Coding Sequence (CCDS) project provides a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assembly in genome annotations produced independently by NCBI and the Ensembl group at EMBL-EBI. This dataset is the product of an international collaboration that includes NCBI, Ensembl, HUGO Gene Nomenclature Committee, Mouse Genome Informatics and University of California, Santa Cruz. Identically annotated coding regions, which are generated using an automated pipeline and pass multiple quality assurance checks, are assigned a stable and tracked identifier (CCDS ID). Additionally, coordinated manual review by expert curators from the CCDS collaboration helps in maintaining the integrity and high quality of the dataset. The CCDS data are available through an interactive web page (https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi) and an FTP site (ftp://ftp.ncbi.nlm.nih.gov/pub/CCDS/). In this paper, we outline the ongoing work, growth and stability of the CCDS dataset and provide updates on new collaboration members and new features added to the CCDS user interface. We also present expert curation scenarios, with specific examples highlighting the importance of an accurate reference genome assembly and the crucial role played by input from the research community. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

SNP identification from RNA sequencing and linkage map construction of rubber tree for anchoring the draft genome.

PubMed

Shearman, Jeremy R; Sangsrakru, Duangjai; Jomchai, Nukoon; Ruang-Areerate, Panthita; Sonthirod, Chutima; Naktang, Chaiwat; Theerawattanasuk, Kanikar; Tragoonrung, Somvong; Tangphatsornruang, Sithichoke

2015-01-01

Hevea brasiliensis, or rubber tree, is an important crop species that accounts for the majority of natural latex production. The rubber tree nuclear genome consists of 18 chromosomes and is roughly 2.15 Gb. The current rubber tree reference genome assembly consists of 1,150,326 scaffolds ranging from 200 to 531,465 bp and totalling 1.1 Gb. Only 143 scaffolds, totalling 7.6 Mb, have been placed into linkage groups. We have performed RNA-seq on 6 varieties of rubber tree to identify SNPs and InDels and used this information to perform target sequence enrichment and high throughput sequencing to genotype a set of SNPs in 149 rubber tree offspring from a cross between RRIM 600 and RRII 105 rubber tree varieties. We used this information to generate a linkage map allowing for the anchoring of 24,424 contigs from 3,009 scaffolds, totalling 115 Mb or 10.4% of the published sequence, into 18 linkage groups. Each linkage group contains between 319 and 1367 SNPs, or 60 to 194 non-redundant marker positions, and ranges from 156 to 336 cM in length. This linkage map includes 20,143 of the 69,300 predicted genes from rubber tree and will be useful for mapping studies and improving the reference genome assembly.

Assembly of the draft genome of buckwheat and its applications in identifying agronomically useful genes.

PubMed

Yasui, Yasuo; Hirakawa, Hideki; Ueno, Mariko; Matsui, Katsuhiro; Katsube-Tanaka, Tomoyuki; Yang, Soo Jung; Aii, Jotaro; Sato, Shingo; Mori, Masashi

2016-06-01

Buckwheat (Fagopyrum esculentum Moench; 2n = 2x = 16) is a nutritionally dense annual crop widely grown in temperate zones. To accelerate molecular breeding programmes of this important crop, we generated a draft assembly of the buckwheat genome using short reads obtained by next-generation sequencing (NGS), and constructed the Buckwheat Genome DataBase. After assembling short reads, we determined 387,594 scaffolds as the draft genome sequence (FES_r1.0). The total length of FES_r1.0 was 1,177,687,305 bp, and the N50 of the scaffolds was 25,109 bp. Gene prediction analysis revealed 286,768 coding sequences (CDSs; FES_r1.0_cds) including those related to transposable elements. The total length of FES_r1.0_cds was 212,917,911 bp, and the N50 was 1,101 bp. Of these, the functions of 35,816 CDSs excluding those for transposable elements were annotated by BLAST analysis. To demonstrate the utility of the database, we conducted several test analyses using BLAST and keyword searches. Furthermore, we used the draft genome as a reference sequence for NGS-based markers, and successfully identified novel candidate genes controlling heteromorphic self-incompatibility of buckwheat. The database and draft genome sequence provide a valuable resource that can be used in efforts to develop buckwheat cultivars with superior agronomic traits. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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

PubMed Central

2011-01-01

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

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

PubMed

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

2011-08-08

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

A Comprehensive Linkage Map of the Dog Genome

PubMed Central

Wong, Aaron K.; Ruhe, Alison L.; Dumont, Beth L.; Robertson, Kathryn R.; Guerrero, Giovanna; Shull, Sheila M.; Ziegle, Janet S.; Millon, Lee V.; Broman, Karl W.; Payseur, Bret A.; Neff, Mark W.

2010-01-01

We have leveraged the reference sequence of a boxer to construct the first complete linkage map for the domestic dog. The new map improves access to the dog's unique biology, from human disease counterparts to fascinating evolutionary adaptations. The map was constructed with ∼3000 microsatellite markers developed from the reference sequence. Familial resources afforded 450 mostly phase-known meioses for map assembly. The genotype data supported a framework map with ∼1500 loci. An additional ∼1500 markers served as map validators, contributing modestly to estimates of recombination rate but supporting the framework content. Data from ∼22,000 SNPs informing on a subset of meioses supported map integrity. The sex-averaged map extended 21 M and revealed marked region- and sex-specific differences in recombination rate. The map will enable empiric coverage estimates and multipoint linkage analysis. Knowledge of the variation in recombination rate will also inform on genomewide patterns of linkage disequilibrium (LD), and thus benefit association, selective sweep, and phylogenetic mapping approaches. The computational and wet-bench strategies can be applied to the reference genome of any nonmodel organism to assemble a de novo linkage map. PMID:19966068

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

PubMed

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

2016-10-21

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

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

Error Correcting Optical Mapping Data.

PubMed

Mukherjee, Kingshuk; Washimkar, Darshan; Muggli, Martin D; Salmela, Leena; Boucher, Christina

2018-05-26

Optical mapping is a unique system that is capable of producing high-resolution, high-throughput genomic map data that gives information about the structure of a genome [21]. Recently it has been used for scaffolding contigs and assembly validation for large-scale sequencing projects, including the maize [32], goat [6], and amborella [4] genomes. However, a major impediment in the use of this data is the variety and quantity of errors in the raw optical mapping data, which are called Rmaps. The challenges associated with using Rmap data are analogous to dealing with insertions and deletions in the alignment of long reads. Moreover, they are arguably harder to tackle since the data is numerical and susceptible to inaccuracy. We develop cOMET to error correct Rmap data, which to the best of our knowledge is the only optical mapping error correction method. Our experimental results demonstrate that cOMET has high prevision and corrects 82.49% of insertion errors and 77.38% of deletion errors in Rmap data generated from the E. coli K-12 reference genome. Out of the deletion errors corrected, 98.26% are true errors. Similarly, out of the insertion errors corrected, 82.19% are true errors. It also successfully scales to large genomes, improving the quality of 78% and 99% of the Rmaps in the plum and goat genomes, respectively. Lastly, we show the utility of error correction by demonstrating how it improves the assembly of Rmap data. Error corrected Rmap data results in an assembly that is more contiguous, and covers a larger fraction of the genome.

Assembly: a resource for assembled genomes at NCBI

PubMed Central

Kitts, Paul A.; Church, Deanna M.; Thibaud-Nissen, Françoise; Choi, Jinna; Hem, Vichet; Sapojnikov, Victor; Smith, Robert G.; Tatusova, Tatiana; Xiang, Charlie; Zherikov, Andrey; DiCuccio, Michael; Murphy, Terence D.; Pruitt, Kim D.; Kimchi, Avi

2016-01-01

The NCBI Assembly database (www.ncbi.nlm.nih.gov/assembly/) provides stable accessioning and data tracking for genome assembly data. The model underlying the database can accommodate a range of assembly structures, including sets of unordered contig or scaffold sequences, bacterial genomes consisting of a single complete chromosome, or complex structures such as a human genome with modeled allelic variation. The database provides an assembly accession and version to unambiguously identify the set of sequences that make up a particular version of an assembly, and tracks changes to updated genome assemblies. The Assembly database reports metadata such as assembly names, simple statistical reports of the assembly (number of contigs and scaffolds, contiguity metrics such as contig N50, total sequence length and total gap length) as well as the assembly update history. The Assembly database also tracks the relationship between an assembly submitted to the International Nucleotide Sequence Database Consortium (INSDC) and the assembly represented in the NCBI RefSeq project. Users can find assemblies of interest by querying the Assembly Resource directly or by browsing available assemblies for a particular organism. Links in the Assembly Resource allow users to easily download sequence and annotations for current versions of genome assemblies from the NCBI genomes FTP site. PMID:26578580

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.

Draft genome sequence of non-shiga toxin-producing Escherichia coli O157 NCCP15738.

PubMed

Kwon, Taesoo; Kim, Jung-Beom; Bak, Young-Seok; Yu, Young-Bin; Kwon, Ki Sung; Kim, Won; Cho, Seung-Hak

2016-01-01

The non-shiga toxin-producing Escherichia coli (non-STEC) O157 is a pathogenic strain that cause diarrhea but does not cause hemolytic-uremic syndrome, or hemorrhagic colitis. Here, we present the 5-Mb draft genome sequence of non-STEC O157 NCCP15738, which was isolated from the feces of a Korean patient with diarrhea, and describe its features and the structural basis for its genome evolution. A total of 565-Mbp paired-end reads were generated using the Illumina-HiSeq 2000 platform. The reads were assembled into 135 scaffolds throughout the de novo assembly. The assembled genome size of NCCP15738 was 5,005,278 bp with an N50 value of 142,450 bp and 50.65 % G+C content. Using Rapid Annotation using Subsystem Technology analysis, we predicted 4780 ORFs and 31 RNA genes. The evolutionary tree was inferred from multiple sequence alignment of 45 E. coli species. The most closely related neighbor of NCCP15738 indicated by whole-genome phylogeny was E. coli UMNK88, but that indicated by multilocus sequence analysis was E. coli DH1(ME8569). A comparison between the NCCP15738 genome and those of reference strains, E. coli K-12 substr. MG1655 and EHEC O157:H7 EDL933 by bioinformatics analyses revealed unique genes in NCCP15738 associated with lysis protein S, two-component signal transduction system, conjugation, the flagellum, nucleotide-binding proteins, and metal-ion binding proteins. Notably, NCCP15738 has a dual flagella system like that in Vibrio parahaemolyticus, Aeromonas spp., and Rhodospirillum centenum. The draft genome sequence and the results of bioinformatics analysis of NCCP15738 provide the basis for understanding the genomic evolution of this strain.

The physical map of wheat chromosome 1BS provides insights into its gene space organization and evolution

PubMed Central

2013-01-01

Background The wheat genome sequence is an essential tool for advanced genomic research and improvements. The generation of a high-quality wheat genome sequence is challenging due to its complex 17 Gb polyploid genome. To overcome these difficulties, sequencing through the construction of BAC-based physical maps of individual chromosomes is employed by the wheat genomics community. Here, we present the construction of the first comprehensive physical map of chromosome 1BS, and illustrate its unique gene space organization and evolution. Results Fingerprinted BAC clones were assembled into 57 long scaffolds, anchored and ordered with 2,438 markers, covering 83% of chromosome 1BS. The BAC-based chromosome 1BS physical map and gene order of the orthologous regions of model grass species were consistent, providing strong support for the reliability of the chromosome 1BS assembly. The gene space for chromosome 1BS spans the entire length of the chromosome arm, with 76% of the genes organized in small gene islands, accompanied by a two-fold increase in gene density from the centromere to the telomere. Conclusions This study provides new evidence on common and chromosome-specific features in the organization and evolution of the wheat genome, including a non-uniform distribution of gene density along the centromere-telomere axis, abundance of non-syntenic genes, the degree of colinearity with other grass genomes and a non-uniform size expansion along the centromere-telomere axis compared with other model cereal genomes. The high-quality physical map constructed in this study provides a solid basis for the assembly of a reference sequence of chromosome 1BS and for breeding applications. PMID:24359668

GFinisher: a new strategy to refine and finish bacterial genome assemblies

NASA Astrophysics Data System (ADS)

Guizelini, Dieval; Raittz, Roberto T.; Cruz, Leonardo M.; Souza, Emanuel M.; Steffens, Maria B. R.; Pedrosa, Fabio O.

2016-10-01

Despite the development in DNA sequencing technology, improving the number and the length of reads, the process of reconstruction of complete genome sequences, the so called genome assembly, is still complex. Only 13% of the prokaryotic genome sequencing projects have been completed. Draft genome sequences deposited in public databases are fragmented in contigs and may lack the full gene complement. The aim of the present work is to identify assembly errors and improve the assembly process of bacterial genomes. The biological patterns observed in genomic sequences and the application of a priori information can allow the identification of misassembled regions, and the reorganization and improvement of the overall de novo genome assembly. GFinisher starts generating a Fuzzy GC skew graphs for each contig in an assembly and follows breaking down the contigs in critical points in order to reassemble and close them using jFGap. This has been successfully applied to dataset from 96 genome assemblies, decreasing the number of contigs by up to 86%. GFinisher can easily optimize assemblies of prokaryotic draft genomes and can be used to improve the assembly programs based on nucleotide sequence patterns in the genome. The software and source code are available at http://gfinisher.sourceforge.net/.

GFinisher: a new strategy to refine and finish bacterial genome assemblies.

PubMed

Guizelini, Dieval; Raittz, Roberto T; Cruz, Leonardo M; Souza, Emanuel M; Steffens, Maria B R; Pedrosa, Fabio O

2016-10-10

Despite the development in DNA sequencing technology, improving the number and the length of reads, the process of reconstruction of complete genome sequences, the so called genome assembly, is still complex. Only 13% of the prokaryotic genome sequencing projects have been completed. Draft genome sequences deposited in public databases are fragmented in contigs and may lack the full gene complement. The aim of the present work is to identify assembly errors and improve the assembly process of bacterial genomes. The biological patterns observed in genomic sequences and the application of a priori information can allow the identification of misassembled regions, and the reorganization and improvement of the overall de novo genome assembly. GFinisher starts generating a Fuzzy GC skew graphs for each contig in an assembly and follows breaking down the contigs in critical points in order to reassemble and close them using jFGap. This has been successfully applied to dataset from 96 genome assemblies, decreasing the number of contigs by up to 86%. GFinisher can easily optimize assemblies of prokaryotic draft genomes and can be used to improve the assembly programs based on nucleotide sequence patterns in the genome. The software and source code are available at http://gfinisher.sourceforge.net/.

Survey of gene splicing algorithms based on reads.

PubMed

Si, Xiuhua; Wang, Qian; Zhang, Lei; Wu, Ruo; Ma, Jiquan

2017-11-02

Gene splicing is the process of assembling a large number of unordered short sequence fragments to the original genome sequence as accurately as possible. Several popular splicing algorithms based on reads are reviewed in this article, including reference genome algorithms and de novo splicing algorithms (Greedy-extension, Overlap-Layout-Consensus graph, De Bruijn graph). We also discuss a new splicing method based on the MapReduce strategy and Hadoop. By comparing these algorithms, some conclusions are drawn and some suggestions on gene splicing research are made.

RefSeq microbial genomes database: new representation and annotation strategy.

PubMed

Tatusova, Tatiana; Ciufo, Stacy; Fedorov, Boris; O'Neill, Kathleen; Tolstoy, Igor

2014-01-01

The source of the microbial genomic sequences in the RefSeq collection is the set of primary sequence records submitted to the International Nucleotide Sequence Database public archives. These can be accessed through the Entrez search and retrieval system at http://www.ncbi.nlm.nih.gov/genome. Next-generation sequencing has enabled researchers to perform genomic sequencing at rates that were unimaginable in the past. Microbial genomes can now be sequenced in a matter of hours, which has led to a significant increase in the number of assembled genomes deposited in the public archives. This huge increase in DNA sequence data presents new challenges for the annotation, analysis and visualization bioinformatics tools. New strategies have been developed for the annotation and representation of reference genomes and sequence variations derived from population studies and clinical outbreaks.

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.

Characterizing polymorphic inversions in human genomes by single-cell sequencing

PubMed Central

Sanders, Ashley D.; Hills, Mark; Porubský, David; Guryev, Victor; Falconer, Ester; Lansdorp, Peter M.

2016-01-01

Identifying genomic features that differ between individuals and cells can help uncover the functional variants that drive phenotypes and disease susceptibilities. For this, single-cell studies are paramount, as it becomes increasingly clear that the contribution of rare but functional cellular subpopulations is important for disease prognosis, management, and progression. Until now, studying these associations has been challenged by our inability to map structural rearrangements accurately and comprehensively. To overcome this, we coupled single-cell sequencing of DNA template strands (Strand-seq) with custom analysis software to rapidly discover, map, and genotype genomic rearrangements at high resolution. This allowed us to explore the distribution and frequency of inversions in a heterogeneous cell population, identify several polymorphic domains in complex regions of the genome, and locate rare alleles in the reference assembly. We then mapped the entire genomic complement of inversions within two unrelated individuals to characterize their distinct inversion profiles and built a nonredundant global reference of structural rearrangements in the human genome. The work described here provides a powerful new framework to study structural variation and genomic heterogeneity in single-cell samples, whether from individuals for population studies or tissue types for biomarker discovery. PMID:27472961

Reducing assembly complexity of microbial genomes with single-molecule sequencing.

PubMed

Koren, Sergey; Harhay, Gregory P; Smith, Timothy P L; Bono, James L; Harhay, Dayna M; Mcvey, Scott D; Radune, Diana; Bergman, Nicholas H; Phillippy, Adam M

2013-01-01

The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem. To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads. Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

De novo transcript sequence reconstruction from RNA-Seq: reference generation and analysis with Trinity

PubMed Central

Yassour, Moran; Grabherr, Manfred; Blood, Philip D.; Bowden, Joshua; Couger, Matthew Brian; Eccles, David; Li, Bo; Lieber, Matthias; MacManes, Matthew D.; Ott, Michael; Orvis, Joshua; Pochet, Nathalie; Strozzi, Francesco; Weeks, Nathan; Westerman, Rick; William, Thomas; Dewey, Colin N.; Henschel, Robert; LeDuc, Richard D.; Friedman, Nir; Regev, Aviv

2013-01-01

De novo assembly of RNA-Seq data allows us to study transcriptomes without the need for a genome sequence, such as in non-model organisms of ecological and evolutionary importance, cancer samples, or the microbiome. In this protocol, we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-Seq data in non-model organisms. We also present Trinity’s supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples, and approaches to identify protein coding genes. In an included tutorial we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sf.net. PMID:23845962

Rice Annotation Project Database (RAP-DB): an integrative and interactive database for rice genomics.

PubMed

Sakai, Hiroaki; Lee, Sung Shin; Tanaka, Tsuyoshi; Numa, Hisataka; Kim, Jungsok; Kawahara, Yoshihiro; Wakimoto, Hironobu; Yang, Ching-chia; Iwamoto, Masao; Abe, Takashi; Yamada, Yuko; Muto, Akira; Inokuchi, Hachiro; Ikemura, Toshimichi; Matsumoto, Takashi; Sasaki, Takuji; Itoh, Takeshi

2013-02-01

The Rice Annotation Project Database (RAP-DB, http://rapdb.dna.affrc.go.jp/) has been providing a comprehensive set of gene annotations for the genome sequence of rice, Oryza sativa (japonica group) cv. Nipponbare. Since the first release in 2005, RAP-DB has been updated several times along with the genome assembly updates. Here, we present our newest RAP-DB based on the latest genome assembly, Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0), which was released in 2011. We detected 37,869 loci by mapping transcript and protein sequences of 150 monocot species. To provide plant researchers with highly reliable and up to date rice gene annotations, we have been incorporating literature-based manually curated data, and 1,626 loci currently incorporate literature-based annotation data, including commonly used gene names or gene symbols. Transcriptional activities are shown at the nucleotide level by mapping RNA-Seq reads derived from 27 samples. We also mapped the Illumina reads of a Japanese leading japonica cultivar, Koshihikari, and a Chinese indica cultivar, Guangluai-4, to the genome and show alignments together with the single nucleotide polymorphisms (SNPs) and gene functional annotations through a newly developed browser, Short-Read Assembly Browser (S-RAB). We have developed two satellite databases, Plant Gene Family Database (PGFD) and Integrative Database of Cereal Gene Phylogeny (IDCGP), which display gene family and homologous gene relationships among diverse plant species. RAP-DB and the satellite databases offer simple and user-friendly web interfaces, enabling plant and genome researchers to access the data easily and facilitating a broad range of plant research topics.

De novo sequencing, assembly and analysis of the genome of the laboratory strain Saccharomyces cerevisiae CEN.PK113-7D, a model for modern industrial biotechnology

PubMed Central

2012-01-01

Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. We sequenced, assembled, annotated and analyzed its genome. Single-nucleotide variations (SNV), insertions/deletions (indels) and differences in genome organization compared to the reference strain S. cerevisiae S288C were analyzed. In addition to a few large deletions and duplications, nearly 3000 indels were identified in the CEN.PK113-7D genome relative to S288C. These differences were overrepresented in genes whose functions are related to transcriptional regulation and chromatin remodelling. Some of these variations were caused by unstable tandem repeats, suggesting an innate evolvability of the corresponding genes. Besides a previously characterized mutation in adenylate cyclase, the CEN.PK113-7D genome sequence revealed a significant enrichment of non-synonymous mutations in genes encoding for components of the cAMP signalling pathway. Some phenotypic characteristics of the CEN.PK113-7D strains were explained by the presence of additional specific metabolic genes relative to S288C. In particular, the presence of the BIO1 and BIO6 genes correlated with a biotin prototrophy of CEN.PK113-7D. Furthermore, the copy number, chromosomal location and sequences of the MAL loci were resolved. The assembled sequence reveals that CEN.PK113-7D has a mosaic genome that combines characteristics of laboratory strains and wild-industrial strains. PMID:22448915

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

PubMed

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

2017-11-28

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

Whole-genome analyses of DS-1-like human G2P[4] and G8P[4] rotavirus strains from Eastern, Western and Southern Africa

PubMed Central

Nyaga, Martin M.; Stucker, Karla M.; Esona, Mathew D.; Jere, Khuzwayo C.; Mwinyi, Bakari; Shonhai, Annie; Tsolenyanu, Enyonam; Mulindwa, Augustine; Chibumbya, Julia N.; Adolfine, Hokororo; Halpin, Rebecca A.; Roy, Sunando; Stockwell, Timothy B.; Berejena, Chipo; Seheri, Mapaseka L.; Mwenda, Jason M.; Steele, A. Duncan; Wentworth, David E.

2018-01-01

Group A rotaviruses (RVAs) with distinct G and P genotype combinations have been reported globally. We report the genome composition and possible origin of seven G8P[4] and five G2P[4] human RVA strains based on the genetic evolution of all 11 genome segments at the nucleotide level. Twelve RVA ELISA positive stool samples collected in the representative countries of Eastern, Southern and West Africa during the 2007–2012 surveillance seasons were subjected to sequencing using the Ion Torrent PGM and Illumina MiSeq platforms. A reference-based assembly was performed using CLC Bio’s clc_ref_assemble_long program, and full-genome consensus sequences were obtained. With the exception of the neutralising antigen, VP7, all study strains exhibited the DS-1-like genome constellation (P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2) and clustered phylogenetically with reference strains having a DS-1-like genetic backbone. Comparison of the nucleotide and amino acid sequences with selected global cognate genome segments revealed nucleotide and amino acid sequence identities of 81.7–100 % and 90.6–100 %, respectively, with NSP4 gene segment showing the most diversity among the strains. Bayesian analyses of all gene sequences to estimate the time of divergence of the lineage indicated that divergence times ranged from 16 to 44 years, except for the NSP4 gene where the lineage seemed to arise in the more distant past at an estimated 203 years ago. However, the long-term effects of changes found within the NSP4 genome segment should be further explored, and thus we recommend continued whole-genome analyses from larger sample sets to determine the evolutionary mechanisms of the DS-1-like strains collected in Africa. PMID:24952422

Hawkeye and AMOS: visualizing and assessing the quality of genome assemblies

PubMed Central

Schatz, Michael C.; Phillippy, Adam M.; Sommer, Daniel D.; Delcher, Arthur L.; Puiu, Daniela; Narzisi, Giuseppe; Salzberg, Steven L.; Pop, Mihai

2013-01-01

Since its launch in 2004, the open-source AMOS project has released several innovative DNA sequence analysis applications including: Hawkeye, a visual analytics tool for inspecting the structure of genome assemblies; the Assembly Forensics and FRCurve pipelines for systematically evaluating the quality of a genome assembly; and AMOScmp, the first comparative genome assembler. These applications have been used to assemble and analyze dozens of genomes ranging in complexity from simple microbial species through mammalian genomes. Recent efforts have been focused on enhancing support for new data characteristics brought on by second- and now third-generation sequencing. This review describes the major components of AMOS in light of these challenges, with an emphasis on methods for assessing assembly quality and the visual analytics capabilities of Hawkeye. These interactive graphical aspects are essential for navigating and understanding the complexities of a genome assembly, from the overall genome structure down to individual bases. Hawkeye and AMOS are available open source at http://amos.sourceforge.net. PMID:22199379

Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data.

PubMed

Desai, Aarti; Marwah, Veer Singh; Yadav, Akshay; Jha, Vineet; Dhaygude, Kishor; Bangar, Ujwala; Kulkarni, Vivek; Jere, Abhay

2013-01-01

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6-40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

Improving transcriptome de novo assembly by using a reference genome of a related species: Translational genomics from oil palm to coconut.

PubMed

Armero, Alix; Baudouin, Luc; Bocs, Stéphanie; This, Dominique

2017-01-01

The palms are a family of tropical origin and one of the main constituents of the ecosystems of these regions around the world. The two main species of palm represent different challenges: coconut (Cocos nucifera L.) is a source of multiple goods and services in tropical communities, while oil palm (Elaeis guineensis Jacq) is the main protagonist of the oil market. In this study, we present a workflow that exploits the comparative genomics between a target species (coconut) and a reference species (oil palm) to improve the transcriptomic data, providing a proteome useful to answer functional or evolutionary questions. This workflow reduces redundancy and fragmentation, two inherent problems of transcriptomic data, while preserving the functional representation of the target species. Our approach was validated in Arabidopsis thaliana using Arabidopsis lyrata and Capsella rubella as references species. This analysis showed the high sensitivity and specificity of our strategy, relatively independent of the reference proteome. The workflow increased the length of proteins products in A. thaliana by 13%, allowing, often, to recover 100% of the protein sequence length. In addition redundancy was reduced by a factor greater than 3. In coconut, the approach generated 29,366 proteins, 1,246 of these proteins deriving from new contigs obtained with the BRANCH software. The coconut proteome presented a functional profile similar to that observed in rice and an important number of metabolic pathways related to secondary metabolism. The new sequences found with BRANCH software were enriched in functions related to biotic stress. Our strategy can be used as a complementary step to de novo transcriptome assembly to get a representative proteome of a target species. The results of the current analysis are available on the website PalmComparomics (http://palm-comparomics.southgreen.fr/).

The archetype-genome exemplar in molecular dynamics and continuum mechanics

NASA Astrophysics Data System (ADS)

Greene, M. Steven; Li, Ying; Chen, Wei; Liu, Wing Kam

2014-04-01

We argue that mechanics and physics of solids rely on a fundamental exemplar: the apparent properties of a system depend on the building blocks that comprise it. Building blocks are referred to as archetypes and apparent system properties as the system genome. Three entities are of importance: the archetype properties, the conformation of archetypes, and the properties of interactions activated by that conformation. The combination of these entities into the system genome is called assembly. To show the utility of the archetype-genome exemplar, this work presents the mathematical ingredients and computational implementation of theories in solid mechanics that are (1) molecular and (2) continuum manifestations of the assembly process. Both coarse-grained molecular dynamics (CGMD) and the archetype-blending continuum (ABC) theories are formulated then applied to polymer nanocomposites (PNCs) to demonstrate the impact the components of the assembly triplet have on a material genome. CGMD simulations demonstrate the sensitivity of nanocomposite viscosities and diffusion coefficients to polymer chain types (archetype), polymer-nanoparticle interaction potentials (interaction), and the structural configuration (conformation) of dispersed nanoparticles. ABC simulations show the contributions of bulk polymer (archetype) properties, occluded region of bound rubber (interaction) properties, and microstructural binary images (conformation) to predictions of linear damping properties, the Payne effect, and localization/size effects in the same class of PNC material. The paper is light on mathematics. Instead, the focus is on the usefulness of the archetype-genome exemplar to predict system behavior inaccessible to classical theories by transitioning mechanics away from heuristic laws to mechanism-based ones. There are two core contributions of this research: (1) presentation of a fundamental axiom—the archetype-genome exemplar—to guide theory development in computational mechanics, and (2) demonstrations of its utility in modern theoretical realms: CGMD, and generalized continuum mechanics.

The Genome of the Self-Fertilizing Mangrove Rivulus Fish, Kryptolebias marmoratus: A Model for Studying Phenotypic Plasticity and Adaptations to Extreme Environments.

PubMed

Kelley, Joanna L; Yee, Muh-Ching; Brown, Anthony P; Richardson, Rhea R; Tatarenkov, Andrey; Lee, Clarence C; Harkins, Timothy T; Bustamante, Carlos D; Earley, Ryan L

2016-08-16

The mangrove rivulus (Kryptolebias marmoratus) is one of two preferentially self-fertilizing hermaphroditic vertebrates. This mode of reproduction makes mangrove rivulus an important model for evolutionary and biomedical studies because long periods of self-fertilization result in naturally homozygous genotypes that can produce isogenic lineages without significant limitations associated with inbreeding depression. Over 400 isogenic lineages currently held in laboratories across the globe show considerable among-lineage variation in physiology, behavior, and life history traits that is maintained under common garden conditions. Temperature mediates the development of primary males and also sex change between hermaphrodites and secondary males, which makes the system ideal for the study of sex determination and sexual plasticity. Mangrove rivulus also exhibit remarkable adaptations to living in extreme environments, and the system has great promise to shed light on the evolution of terrestrial locomotion, aerial respiration, and broad tolerances to hypoxia, salinity, temperature, and environmental pollutants. Genome assembly of the mangrove rivulus allows the study of genes and gene families associated with the traits described above. Here we present a de novo assembled reference genome for the mangrove rivulus, with an approximately 900 Mb genome, including 27,328 annotated, predicted, protein-coding genes. Moreover, we are able to place more than 50% of the assembled genome onto a recently published linkage map. The genome provides an important addition to the linkage map and transcriptomic tools recently developed for this species that together provide critical resources for epigenetic, transcriptomic, and proteomic analyses. Moreover, the genome will serve as the foundation for addressing key questions in behavior, physiology, toxicology, and evolutionary biology. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Creating reference gene annotation for the mouse C57BL6/J genome assembly.

PubMed

Mudge, Jonathan M; Harrow, Jennifer

2015-10-01

Annotation on the reference genome of the C57BL6/J mouse has been an ongoing project ever since the draft genome was first published. Initially, the principle focus was on the identification of all protein-coding genes, although today the importance of describing long non-coding RNAs, small RNAs, and pseudogenes is recognized. Here, we describe the progress of the GENCODE mouse annotation project, which combines manual annotation from the HAVANA group with Ensembl computational annotation, alongside experimental and in silico validation pipelines from other members of the consortium. We discuss the more recent incorporation of next-generation sequencing datasets into this workflow, including the usage of mass-spectrometry data to potentially identify novel protein-coding genes. Finally, we will outline how the C57BL6/J genebuild can be used to gain insights into the variant sites that distinguish different mouse strains and species.

Assembling the Marine Metagenome, One Cell at a Time

PubMed Central

Woyke, Tanja; Xie, Gary; Copeland, Alex; González, José M.; Han, Cliff; Kiss, Hajnalka; Saw, Jimmy H.; Senin, Pavel; Yang, Chi; Chatterji, Sourav; Cheng, Jan-Fang; Eisen, Jonathan A.; Sieracki, Michael E.; Stepanauskas, Ramunas

2009-01-01

The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa. PMID:19390573

Automated ensemble assembly and validation of microbial genomes.

PubMed

Koren, Sergey; Treangen, Todd J; Hill, Christopher M; Pop, Mihai; Phillippy, Adam M

2014-05-03

The continued democratization of DNA sequencing has sparked a new wave of development of genome assembly and assembly validation methods. As individual research labs, rather than centralized centers, begin to sequence the majority of new genomes, it is important to establish best practices for genome assembly. However, recent evaluations such as GAGE and the Assemblathon have concluded that there is no single best approach to genome assembly. Instead, it is preferable to generate multiple assemblies and validate them to determine which is most useful for the desired analysis; this is a labor-intensive process that is often impossible or unfeasible. To encourage best practices supported by the community, we present iMetAMOS, an automated ensemble assembly pipeline; iMetAMOS encapsulates the process of running, validating, and selecting a single assembly from multiple assemblies. iMetAMOS packages several leading open-source tools into a single binary that automates parameter selection and execution of multiple assemblers, scores the resulting assemblies based on multiple validation metrics, and annotates the assemblies for genes and contaminants. We demonstrate the utility of the ensemble process on 225 previously unassembled Mycobacterium tuberculosis genomes as well as a Rhodobacter sphaeroides benchmark dataset. On these real data, iMetAMOS reliably produces validated assemblies and identifies potential contamination without user intervention. In addition, intelligent parameter selection produces assemblies of R. sphaeroides comparable to or exceeding the quality of those from the GAGE-B evaluation, affecting the relative ranking of some assemblers. Ensemble assembly with iMetAMOS provides users with multiple, validated assemblies for each genome. Although computationally limited to small or mid-sized genomes, this approach is the most effective and reproducible means for generating high-quality assemblies and enables users to select an assembly best tailored to their specific needs.

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.

The genome of Eucalyptus grandis.

PubMed

Myburg, Alexander A; Grattapaglia, Dario; Tuskan, Gerald A; Hellsten, Uffe; Hayes, Richard D; Grimwood, Jane; Jenkins, Jerry; Lindquist, Erika; Tice, Hope; Bauer, Diane; Goodstein, David M; Dubchak, Inna; Poliakov, Alexandre; Mizrachi, Eshchar; Kullan, Anand R K; Hussey, Steven G; Pinard, Desre; van der Merwe, Karen; Singh, Pooja; van Jaarsveld, Ida; Silva-Junior, Orzenil B; Togawa, Roberto C; Pappas, Marilia R; Faria, Danielle A; Sansaloni, Carolina P; Petroli, Cesar D; Yang, Xiaohan; Ranjan, Priya; Tschaplinski, Timothy J; Ye, Chu-Yu; Li, Ting; Sterck, Lieven; Vanneste, Kevin; Murat, Florent; Soler, Marçal; Clemente, Hélène San; Saidi, Naijib; Cassan-Wang, Hua; Dunand, Christophe; Hefer, Charles A; Bornberg-Bauer, Erich; Kersting, Anna R; Vining, Kelly; Amarasinghe, Vindhya; Ranik, Martin; Naithani, Sushma; Elser, Justin; Boyd, Alexander E; Liston, Aaron; Spatafora, Joseph W; Dharmwardhana, Palitha; Raja, Rajani; Sullivan, Christopher; Romanel, Elisson; Alves-Ferreira, Marcio; Külheim, Carsten; Foley, William; Carocha, Victor; Paiva, Jorge; Kudrna, David; Brommonschenkel, Sergio H; Pasquali, Giancarlo; Byrne, Margaret; Rigault, Philippe; Tibbits, Josquin; Spokevicius, Antanas; Jones, Rebecca C; Steane, Dorothy A; Vaillancourt, René E; Potts, Brad M; Joubert, Fourie; Barry, Kerrie; Pappas, Georgios J; Strauss, Steven H; Jaiswal, Pankaj; Grima-Pettenati, Jacqueline; Salse, Jérôme; Van de Peer, Yves; Rokhsar, Daniel S; Schmutz, Jeremy

2014-06-19

Eucalypts are the world's most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Gramene 2016: comparative plant genomics and pathway resources.

PubMed

Tello-Ruiz, Marcela K; Stein, Joshua; Wei, Sharon; Preece, Justin; Olson, Andrew; Naithani, Sushma; Amarasinghe, Vindhya; Dharmawardhana, Palitha; Jiao, Yinping; Mulvaney, Joseph; Kumari, Sunita; Chougule, Kapeel; Elser, Justin; Wang, Bo; Thomason, James; Bolser, Daniel M; Kerhornou, Arnaud; Walts, Brandon; Fonseca, Nuno A; Huerta, Laura; Keays, Maria; Tang, Y Amy; Parkinson, Helen; Fabregat, Antonio; McKay, Sheldon; Weiser, Joel; D'Eustachio, Peter; Stein, Lincoln; Petryszak, Robert; Kersey, Paul J; Jaiswal, Pankaj; Ware, Doreen

2016-01-04

Gramene (http://www.gramene.org) is an online resource for comparative functional genomics in crops and model plant species. Its two main frameworks are genomes (collaboration with Ensembl Plants) and pathways (The Plant Reactome and archival BioCyc databases). Since our last NAR update, the database website adopted a new Drupal management platform. The genomes section features 39 fully assembled reference genomes that are integrated using ontology-based annotation and comparative analyses, and accessed through both visual and programmatic interfaces. Additional community data, such as genetic variation, expression and methylation, are also mapped for a subset of genomes. The Plant Reactome pathway portal (http://plantreactome.gramene.org) provides a reference resource for analyzing plant metabolic and regulatory pathways. In addition to ∼ 200 curated rice reference pathways, the portal hosts gene homology-based pathway projections for 33 plant species. Both the genome and pathway browsers interface with the EMBL-EBI's Expression Atlas to enable the projection of baseline and differential expression data from curated expression studies in plants. Gramene's archive website (http://archive.gramene.org) continues to provide previously reported resources on comparative maps, markers and QTL. To further aid our users, we have also introduced a live monthly educational webinar series and a Gramene YouTube channel carrying video tutorials. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver

PubMed Central

Blanquart, François; Golubchik, Tanya; Gall, Astrid; Bakker, Margreet; Bezemer, Daniela; Croucher, Nicholas J; Hall, Matthew; Hillebregt, Mariska; Ratmann, Oliver; Albert, Jan; Bannert, Norbert; Fellay, Jacques; Fransen, Katrien; Gourlay, Annabelle; Grabowski, M Kate; Gunsenheimer-Bartmeyer, Barbara; Günthard, Huldrych F; Kivelä, Pia; Kouyos, Roger; Laeyendecker, Oliver; Liitsola, Kirsi; Meyer, Laurence; Porter, Kholoud; Ristola, Matti; van Sighem, Ard; Cornelissen, Marion; Kellam, Paul; Reiss, Peter

2018-01-01

Abstract Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user’s choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver’s constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied shiver to whole-genome samples of Hepatitis C Virus and Respiratory Syncytial Virus. shiver is publicly available from https://github.com/ChrisHIV/shiver. PMID:29876136

Extensive Error in the Number of Genes Inferred from Draft Genome Assemblies

PubMed Central

Denton, James F.; Lugo-Martinez, Jose; Tucker, Abraham E.; Schrider, Daniel R.; Warren, Wesley C.; Hahn, Matthew W.

2014-01-01

Current sequencing methods produce large amounts of data, but genome assemblies based on these data are often woefully incomplete. These incomplete and error-filled assemblies result in many annotation errors, especially in the number of genes present in a genome. In this paper we investigate the magnitude of the problem, both in terms of total gene number and the number of copies of genes in specific families. To do this, we compare multiple draft assemblies against higher-quality versions of the same genomes, using several new assemblies of the chicken genome based on both traditional and next-generation sequencing technologies, as well as published draft assemblies of chimpanzee. We find that upwards of 40% of all gene families are inferred to have the wrong number of genes in draft assemblies, and that these incorrect assemblies both add and subtract genes. Using simulated genome assemblies of Drosophila melanogaster, we find that the major cause of increased gene numbers in draft genomes is the fragmentation of genes onto multiple individual contigs. Finally, we demonstrate the usefulness of RNA-Seq in improving the gene annotation of draft assemblies, largely by connecting genes that have been fragmented in the assembly process. PMID:25474019

Extensive error in the number of genes inferred from draft genome assemblies.

PubMed

Denton, James F; Lugo-Martinez, Jose; Tucker, Abraham E; Schrider, Daniel R; Warren, Wesley C; Hahn, Matthew W

2014-12-01

Current sequencing methods produce large amounts of data, but genome assemblies based on these data are often woefully incomplete. These incomplete and error-filled assemblies result in many annotation errors, especially in the number of genes present in a genome. In this paper we investigate the magnitude of the problem, both in terms of total gene number and the number of copies of genes in specific families. To do this, we compare multiple draft assemblies against higher-quality versions of the same genomes, using several new assemblies of the chicken genome based on both traditional and next-generation sequencing technologies, as well as published draft assemblies of chimpanzee. We find that upwards of 40% of all gene families are inferred to have the wrong number of genes in draft assemblies, and that these incorrect assemblies both add and subtract genes. Using simulated genome assemblies of Drosophila melanogaster, we find that the major cause of increased gene numbers in draft genomes is the fragmentation of genes onto multiple individual contigs. Finally, we demonstrate the usefulness of RNA-Seq in improving the gene annotation of draft assemblies, largely by connecting genes that have been fragmented in the assembly process.

De Novo Genome and Transcriptome Assembly of the Canadian Beaver (Castor canadensis).

PubMed

Lok, Si; Paton, Tara A; Wang, Zhuozhi; Kaur, Gaganjot; Walker, Susan; Yuen, Ryan K C; Sung, Wilson W L; Whitney, Joseph; Buchanan, Janet A; Trost, Brett; Singh, Naina; Apresto, Beverly; Chen, Nan; Coole, Matthew; Dawson, Travis J; Ho, Karen; Hu, Zhizhou; Pullenayegum, Sanjeev; Samler, Kozue; Shipstone, Arun; Tsoi, Fiona; Wang, Ting; Pereira, Sergio L; Rostami, Pirooz; Ryan, Carol Ann; Tong, Amy Hin Yan; Ng, Karen; Sundaravadanam, Yogi; Simpson, Jared T; Lim, Burton K; Engstrom, Mark D; Dutton, Christopher J; Kerr, Kevin C R; Franke, Maria; Rapley, William; Wintle, Richard F; Scherer, Stephen W

2017-02-09

The Canadian beaver ( Castor canadensis ) is the largest indigenous rodent in North America. We report a draft annotated assembly of the beaver genome, the first for a large rodent and the first mammalian genome assembled directly from uncorrected and moderate coverage (< 30 ×) long reads generated by single-molecule sequencing. The genome size is 2.7 Gb estimated by k-mer analysis. We assembled the beaver genome using the new Canu assembler optimized for noisy reads. The resulting assembly was refined using Pilon supported by short reads (80 ×) and checked for accuracy by congruency against an independent short read assembly. We scaffolded the assembly using the exon-gene models derived from 9805 full-length open reading frames (FL-ORFs) constructed from the beaver leukocyte and muscle transcriptomes. The final assembly comprised 22,515 contigs with an N50 of 278,680 bp and an N50-scaffold of 317,558 bp. Maximum contig and scaffold lengths were 3.3 and 4.2 Mb, respectively, with a combined scaffold length representing 92% of the estimated genome size. The completeness and accuracy of the scaffold assembly was demonstrated by the precise exon placement for 91.1% of the 9805 assembled FL-ORFs and 83.1% of the BUSCO (Benchmarking Universal Single-Copy Orthologs) gene set used to assess the quality of genome assemblies. Well-represented were genes involved in dentition and enamel deposition, defining characteristics of rodents with which the beaver is well-endowed. The study provides insights for genome assembly and an important genomics resource for Castoridae and rodent evolutionary biology. Copyright © 2017 Lok et al.

De Novo Genome and Transcriptome Assembly of the Canadian Beaver (Castor canadensis)

PubMed Central

Lok, Si; Paton, Tara A.; Wang, Zhuozhi; Kaur, Gaganjot; Walker, Susan; Yuen, Ryan K. C.; Sung, Wilson W. L.; Whitney, Joseph; Buchanan, Janet A.; Trost, Brett; Singh, Naina; Apresto, Beverly; Chen, Nan; Coole, Matthew; Dawson, Travis J.; Ho, Karen; Hu, Zhizhou; Pullenayegum, Sanjeev; Samler, Kozue; Shipstone, Arun; Tsoi, Fiona; Wang, Ting; Pereira, Sergio L.; Rostami, Pirooz; Ryan, Carol Ann; Tong, Amy Hin Yan; Ng, Karen; Sundaravadanam, Yogi; Simpson, Jared T.; Lim, Burton K.; Engstrom, Mark D.; Dutton, Christopher J.; Kerr, Kevin C. R.; Franke, Maria; Rapley, William; Wintle, Richard F.; Scherer, Stephen W.

2017-01-01

The Canadian beaver (Castor canadensis) is the largest indigenous rodent in North America. We report a draft annotated assembly of the beaver genome, the first for a large rodent and the first mammalian genome assembled directly from uncorrected and moderate coverage (< 30 ×) long reads generated by single-molecule sequencing. The genome size is 2.7 Gb estimated by k-mer analysis. We assembled the beaver genome using the new Canu assembler optimized for noisy reads. The resulting assembly was refined using Pilon supported by short reads (80 ×) and checked for accuracy by congruency against an independent short read assembly. We scaffolded the assembly using the exon–gene models derived from 9805 full-length open reading frames (FL-ORFs) constructed from the beaver leukocyte and muscle transcriptomes. The final assembly comprised 22,515 contigs with an N50 of 278,680 bp and an N50-scaffold of 317,558 bp. Maximum contig and scaffold lengths were 3.3 and 4.2 Mb, respectively, with a combined scaffold length representing 92% of the estimated genome size. The completeness and accuracy of the scaffold assembly was demonstrated by the precise exon placement for 91.1% of the 9805 assembled FL-ORFs and 83.1% of the BUSCO (Benchmarking Universal Single-Copy Orthologs) gene set used to assess the quality of genome assemblies. Well-represented were genes involved in dentition and enamel deposition, defining characteristics of rodents with which the beaver is well-endowed. The study provides insights for genome assembly and an important genomics resource for Castoridae and rodent evolutionary biology. PMID:28087693

Comparative genomic analysis of novel bacteriophages infecting Vibrio parahaemolyticus isolated from western and southern coastal areas of Korea.

PubMed

Yu, Junhyeok; Lim, Jeong-A; Kwak, Su-Jin; Park, Jong-Hyun; Chang, Hyun-Joo

2018-05-01

Vibrio parahaemolyticus, a foodborne pathogen, has become resistant to antibiotics. Therefore, alternative bio-control agents such bacteriophage are urgently needed for its control. Six novel bacteriophages specific to V. parahaemolyticus (vB_VpaP_KF1~2, vB_VpaS_KF3~6) were characterized at the molecular level in this study. Genomic similarity analysis revealed that these six bacteriophages could be divided into two groups with different genomic features, phylogenetic grouping, and morphologies. Two groups of bacteriophages had their own genes with different mechanisms for infection, assembly, and metabolism. Our results could be used as a future reference to study phage genomics or apply phages in future bio-control studies.

Ensembl core software resources: storage and programmatic access for DNA sequence and genome annotation.

PubMed

Ruffier, Magali; Kähäri, Andreas; Komorowska, Monika; Keenan, Stephen; Laird, Matthew; Longden, Ian; Proctor, Glenn; Searle, Steve; Staines, Daniel; Taylor, Kieron; Vullo, Alessandro; Yates, Andrew; Zerbino, Daniel; Flicek, Paul

2017-01-01

The Ensembl software resources are a stable infrastructure to store, access and manipulate genome assemblies and their functional annotations. The Ensembl 'Core' database and Application Programming Interface (API) was our first major piece of software infrastructure and remains at the centre of all of our genome resources. Since its initial design more than fifteen years ago, the number of publicly available genomic, transcriptomic and proteomic datasets has grown enormously, accelerated by continuous advances in DNA-sequencing technology. Initially intended to provide annotation for the reference human genome, we have extended our framework to support the genomes of all species as well as richer assembly models. Cross-referenced links to other informatics resources facilitate searching our database with a variety of popular identifiers such as UniProt and RefSeq. Our comprehensive and robust framework storing a large diversity of genome annotations in one location serves as a platform for other groups to generate and maintain their own tailored annotation. We welcome reuse and contributions: our databases and APIs are publicly available, all of our source code is released with a permissive Apache v2.0 licence at http://github.com/Ensembl and we have an active developer mailing list ( http://www.ensembl.org/info/about/contact/index.html ). http://www.ensembl.org. © The Author(s) 2017. Published by Oxford University Press.

MetaGenomic Assembly by Merging (MeGAMerge)

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

Scholz Chien-Chi Lo, Matthew B.

2015-08-03

"MetaGenomic Assembly by Merging" (MeGAMerge)Is a novel method of merging of multiple genomic assembly or long read data sources for assembly by use of internal trimming/filtering of data, followed by use of two 3rd party tools to merge data by overlap based assembly.

BG7: A New Approach for Bacterial Genome Annotation Designed for Next Generation Sequencing Data

PubMed Central

Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Pareja, Eduardo; Tobes, Raquel

2012-01-01

BG7 is a new system for de novo bacterial, archaeal and viral genome annotation based on a new approach specifically designed for annotating genomes sequenced with next generation sequencing technologies. The system is versatile and able to annotate genes even in the step of preliminary assembly of the genome. It is especially efficient detecting unexpected genes horizontally acquired from bacterial or archaeal distant genomes, phages, plasmids, and mobile elements. From the initial phases of the gene annotation process, BG7 exploits the massive availability of annotated protein sequences in databases. BG7 predicts ORFs and infers their function based on protein similarity with a wide set of reference proteins, integrating ORF prediction and functional annotation phases in just one step. BG7 is especially tolerant to sequencing errors in start and stop codons, to frameshifts, and to assembly or scaffolding errors. The system is also tolerant to the high level of gene fragmentation which is frequently found in not fully assembled genomes. BG7 current version – which is developed in Java, takes advantage of Amazon Web Services (AWS) cloud computing features, but it can also be run locally in any operating system. BG7 is a fast, automated and scalable system that can cope with the challenge of analyzing the huge amount of genomes that are being sequenced with NGS technologies. Its capabilities and efficiency were demonstrated in the 2011 EHEC Germany outbreak in which BG7 was used to get the first annotations right the next day after the first entero-hemorrhagic E. coli genome sequences were made publicly available. The suitability of BG7 for genome annotation has been proved for Illumina, 454, Ion Torrent, and PacBio sequencing technologies. Besides, thanks to its plasticity, our system could be very easily adapted to work with new technologies in the future. PMID:23185310

Recovery and characterization of a Citrus clementina Hort. ex Tan. 'Clemenules' haploid plant selected to establish the reference whole Citrus genome sequence.

PubMed

Aleza, Pablo; Juárez, José; Hernández, María; Pina, José A; Ollitrault, Patrick; Navarro, Luis

2009-08-22

In recent years, the development of structural genomics has generated a growing interest in obtaining haploid plants. The use of homozygous lines presents a significant advantage for the accomplishment of sequencing projects. Commercial citrus species are characterized by high heterozygosity, making it difficult to assemble large genome sequences. Thus, the International Citrus Genomic Consortium (ICGC) decided to establish a reference whole citrus genome sequence from a homozygous plant. Due to the existence of important molecular resources and previous success in obtaining haploid clementine plants, haploid clementine was selected as the target for the implementation of the reference whole genome citrus sequence. To obtain haploid clementine lines we used the technique of in situ gynogenesis induced by irradiated pollen. Flow cytometry, chromosome counts and SSR marker (Simple Sequence Repeats) analysis facilitated the identification of six different haploid lines (2n = x = 9), one aneuploid line (2n = 2x+4 = 22) and one doubled haploid plant (2n = 2x = 18) of 'Clemenules' clementine. One of the haploids, obtained directly from an original haploid embryo, grew vigorously and produced flowers after four years. This is the first haploid plant of clementine that has bloomed and we have, for the first time, characterized the histology of haploid and diploid flowers of clementine. Additionally a double haploid plant was obtained spontaneously from this haploid line. The first haploid plant of 'Clemenules' clementine produced directly by germination of a haploid embryo, which grew vigorously and produced flowers, has been obtained in this work. This haploid line has been selected and it is being used by the ICGC to establish the reference sequence of the nuclear genome of citrus.

DangerTrack: A scoring system to detect difficult-to-assess regions.

PubMed

Dolgalev, Igor; Sedlazeck, Fritz; Busby, Ben

2017-01-01

Over recent years, multiple groups have shown that a large number of structural variants, repeats, or problems with the underlying genome assembly have dramatic effects on the mapping, calling, and overall reliability of single nucleotide polymorphism calls. This project endeavored to develop an easy-to-use track for looking at structural variant and repeat regions. This track, DangerTrack, can be displayed alongside the existing Genome Reference Consortium assembly tracks to warn clinicians and biologists when variants of interest may be incorrectly called, of dubious quality, or on an insertion or copy number expansion. While mapping and variant calling can be automated, it is our opinion that when these regions are of interest to a particular clinical or research group, they warrant a careful examination, potentially involving localized reassembly. DangerTrack is available at https://github.com/DCGenomics/DangerTrack.

snpTree--a web-server to identify and construct SNP trees from whole genome sequence data.

PubMed

Leekitcharoenphon, Pimlapas; Kaas, Rolf S; Thomsen, Martin Christen Frølund; Friis, Carsten; Rasmussen, Simon; Aarestrup, Frank M

2012-01-01

The advances and decreasing economical cost of whole genome sequencing (WGS), will soon make this technology available for routine infectious disease epidemiology. In epidemiological studies, outbreak isolates have very little diversity and require extensive genomic analysis to differentiate and classify isolates. One of the successfully and broadly used methods is analysis of single nucletide polymorphisms (SNPs). Currently, there are different tools and methods to identify SNPs including various options and cut-off values. Furthermore, all current methods require bioinformatic skills. Thus, we lack a standard and simple automatic tool to determine SNPs and construct phylogenetic tree from WGS data. Here we introduce snpTree, a server for online-automatic SNPs analysis. This tool is composed of different SNPs analysis suites, perl and python scripts. snpTree can identify SNPs and construct phylogenetic trees from WGS as well as from assembled genomes or contigs. WGS data in fastq format are aligned to reference genomes by BWA while contigs in fasta format are processed by Nucmer. SNPs are concatenated based on position on reference genome and a tree is constructed from concatenated SNPs using FastTree and a perl script. The online server was implemented by HTML, Java and python script.The server was evaluated using four published bacterial WGS data sets (V. cholerae, S. aureus CC398, S. Typhimurium and M. tuberculosis). The evaluation results for the first three cases was consistent and concordant for both raw reads and assembled genomes. In the latter case the original publication involved extensive filtering of SNPs, which could not be repeated using snpTree. The snpTree server is an easy to use option for rapid standardised and automatic SNP analysis in epidemiological studies also for users with limited bioinformatic experience. The web server is freely accessible at http://www.cbs.dtu.dk/services/snpTree-1.0/.

Comprehensive evaluation of non-hybrid genome assembly tools for third-generation PacBio long-read sequence data.

PubMed

Jayakumar, Vasanthan; Sakakibara, Yasubumi

2017-11-03

Long reads obtained from third-generation sequencing platforms can help overcome the long-standing challenge of the de novo assembly of sequences for the genomic analysis of non-model eukaryotic organisms. Numerous long-read-aided de novo assemblies have been published recently, which exhibited superior quality of the assembled genomes in comparison with those achieved using earlier second-generation sequencing technologies. Evaluating assemblies is important in guiding the appropriate choice for specific research needs. In this study, we evaluated 10 long-read assemblers using a variety of metrics on Pacific Biosciences (PacBio) data sets from different taxonomic categories with considerable differences in genome size. The results allowed us to narrow down the list to a few assemblers that can be effectively applied to eukaryotic assembly projects. Moreover, we highlight how best to use limited genomic resources for effectively evaluating the genome assemblies of non-model organisms. © The Author 2017. Published by Oxford University Press.

Comparing memory-efficient genome assemblers on stand-alone and cloud infrastructures.

PubMed

Kleftogiannis, Dimitrios; Kalnis, Panos; Bajic, Vladimir B

2013-01-01

A fundamental problem in bioinformatics is genome assembly. Next-generation sequencing (NGS) technologies produce large volumes of fragmented genome reads, which require large amounts of memory to assemble the complete genome efficiently. With recent improvements in DNA sequencing technologies, it is expected that the memory footprint required for the assembly process will increase dramatically and will emerge as a limiting factor in processing widely available NGS-generated reads. In this report, we compare current memory-efficient techniques for genome assembly with respect to quality, memory consumption and execution time. Our experiments prove that it is possible to generate draft assemblies of reasonable quality on conventional multi-purpose computers with very limited available memory by choosing suitable assembly methods. Our study reveals the minimum memory requirements for different assembly programs even when data volume exceeds memory capacity by orders of magnitude. By combining existing methodologies, we propose two general assembly strategies that can improve short-read assembly approaches and result in reduction of the memory footprint. Finally, we discuss the possibility of utilizing cloud infrastructures for genome assembly and we comment on some findings regarding suitable computational resources for assembly.

Minimus: a fast, lightweight genome assembler.

PubMed

Sommer, Daniel D; Delcher, Arthur L; Salzberg, Steven L; Pop, Mihai

2007-02-26

Genome assemblers have grown very large and complex in response to the need for algorithms to handle the challenges of large whole-genome sequencing projects. Many of the most common uses of assemblers, however, are best served by a simpler type of assembler that requires fewer software components, uses less memory, and is far easier to install and run. We have developed the Minimus assembler to address these issues, and tested it on a range of assembly problems. We show that Minimus performs well on several small assembly tasks, including the assembly of viral genomes, individual genes, and BAC clones. In addition, we evaluate Minimus' performance in assembling bacterial genomes in order to assess its suitability as a component of a larger assembly pipeline. We show that, unlike other software currently used for these tasks, Minimus produces significantly fewer assembly errors, at the cost of generating a more fragmented assembly. We find that for small genomes and other small assembly tasks, Minimus is faster and far more flexible than existing tools. Due to its small size and modular design Minimus is perfectly suited to be a component of complex assembly pipelines. Minimus is released as an open-source software project and the code is available as part of the AMOS project at Sourceforge.

Improvement of the Threespine Stickleback Genome Using a Hi-C-Based Proximity-Guided Assembly.

PubMed

Peichel, Catherine L; Sullivan, Shawn T; Liachko, Ivan; White, Michael A

2017-09-01

Scaffolding genomes into complete chromosome assemblies remains challenging even with the rapidly increasing sequence coverage generated by current next-generation sequence technologies. Even with scaffolding information, many genome assemblies remain incomplete. The genome of the threespine stickleback (Gasterosteus aculeatus), a fish model system in evolutionary genetics and genomics, is not completely assembled despite scaffolding with high-density linkage maps. Here, we first test the ability of a Hi-C based proximity-guided assembly (PGA) to perform a de novo genome assembly from relatively short contigs. Using Hi-C based PGA, we generated complete chromosome assemblies from a distribution of short contigs (20-100 kb). We found that 96.40% of contigs were correctly assigned to linkage groups (LGs), with ordering nearly identical to the previous genome assembly. Using available bacterial artificial chromosome (BAC) end sequences, we provide evidence that some of the few discrepancies between the Hi-C assembly and the existing assembly are due to structural variation between the populations used for the 2 assemblies or errors in the existing assembly. This Hi-C assembly also allowed us to improve the existing assembly, assigning over 60% (13.35 Mb) of the previously unassigned (~21.7 Mb) contigs to LGs. Together, our results highlight the potential of the Hi-C based PGA method to be used in combination with short read data to perform relatively inexpensive de novo genome assemblies. This approach will be particularly useful in organisms in which it is difficult to perform linkage mapping or to obtain high molecular weight DNA required for other scaffolding methods. © The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

U50: A New Metric for Measuring Assembly Output Based on Non-Overlapping, Target-Specific Contigs.

PubMed

Castro, Christina J; Ng, Terry Fei Fan

2017-11-01

Advances in next-generation sequencing technologies enable routine genome sequencing, generating millions of short reads. A crucial step for full genome analysis is the de novo assembly, and currently, performance of different assembly methods is measured by a metric called N 50 . However, the N 50 value can produce skewed, inaccurate results when complex data are analyzed, especially for viral and microbial datasets. To provide a better assessment of assembly output, we developed a new metric called U 50 . The U 50 identifies unique, target-specific contigs by using a reference genome as baseline, aiming at circumventing some limitations that are inherent to the N 50 metric. Specifically, the U 50 program removes overlapping sequence of multiple contigs by utilizing a mask array, so the performance of the assembly is only measured by unique contigs. We compared simulated and real datasets by using U 50 and N 50 , and our results demonstrated that U 50 has the following advantages over N 50 : (1) reducing erroneously large N 50 values due to a poor assembly, (2) eliminating overinflated N 50 values caused by large measurements from overlapping contigs, (3) eliminating diminished N 50 values caused by an abundance of small contigs, and (4) allowing comparisons across different platforms or samples based on the new percentage-based metric UG 50 %. The use of the U 50 metric allows for a more accurate measure of assembly performance by analyzing only the unique, non-overlapping contigs. In addition, most viral and microbial sequencing have high background noise (i.e., host and other non-targets), which contributes to having a skewed, misrepresented N 50 value-this is corrected by U 50 . Also, the UG 50 % can be used to compare assembly results from different samples or studies, the cross-comparisons of which cannot be performed with N 50 .

Scoring-and-unfolding trimmed tree assembler: concepts, constructs and comparisons.

PubMed

Narzisi, Giuseppe; Mishra, Bud

2011-01-15

Mired by its connection to a well-known -complete combinatorial optimization problem-namely, the Shortest Common Superstring Problem (SCSP)-historically, the whole-genome sequence assembly (WGSA) problem has been assumed to be amenable only to greedy and heuristic methods. By placing efficiency as their first priority, these methods opted to rely only on local searches, and are thus inherently approximate, ambiguous or error prone, especially, for genomes with complex structures. Furthermore, since choice of the best heuristics depended critically on the properties of (e.g. errors in) the input data and the available long range information, these approaches hindered designing an error free WGSA pipeline. We dispense with the idea of limiting the solutions to just the approximated ones, and instead favor an approach that could potentially lead to an exhaustive (exponential-time) search of all possible layouts. Its computational complexity thus must be tamed through a constrained search (Branch-and-Bound) and quick identification and pruning of implausible overlays. For his purpose, such a method necessarily relies on a set of score functions (oracles) that can combine different structural properties (e.g. transitivity, coverage, physical maps, etc.). We give a detailed description of this novel assembly framework, referred to as Scoring-and-Unfolding Trimmed Tree Assembler (SUTTA), and present experimental results on several bacterial genomes using next-generation sequencing technology data. We also report experimental evidence that the assembly quality strongly depends on the choice of the minimum overlap parameter k. SUTTA's binaries are freely available to non-profit institutions for research and educational purposes at http://www.bioinformatics.nyu.edu.

Efficient Graph Based Assembly of Short-Read Sequences on Hybrid Core Architecture

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

Sczyrba, Alex; Pratap, Abhishek; Canon, Shane

2011-03-22

Advanced architectures can deliver dramatically increased throughput for genomics and proteomics applications, reducing time-to-completion in some cases from days to minutes. One such architecture, hybrid-core computing, marries a traditional x86 environment with a reconfigurable coprocessor, based on field programmable gate array (FPGA) technology. In addition to higher throughput, increased performance can fundamentally improve research quality by allowing more accurate, previously impractical approaches. We will discuss the approach used by Convey?s de Bruijn graph constructor for short-read, de-novo assembly. Bioinformatics applications that have random access patterns to large memory spaces, such as graph-based algorithms, experience memory performance limitations on cache-based x86more » servers. Convey?s highly parallel memory subsystem allows application-specific logic to simultaneously access 8192 individual words in memory, significantly increasing effective memory bandwidth over cache-based memory systems. Many algorithms, such as Velvet and other de Bruijn graph based, short-read, de-novo assemblers, can greatly benefit from this type of memory architecture. Furthermore, small data type operations (four nucleotides can be represented in two bits) make more efficient use of logic gates than the data types dictated by conventional programming models.JGI is comparing the performance of Convey?s graph constructor and Velvet on both synthetic and real data. We will present preliminary results on memory usage and run time metrics for various data sets with different sizes, from small microbial and fungal genomes to very large cow rumen metagenome. For genomes with references we will also present assembly quality comparisons between the two assemblers.« less

Genome Assembly Forensics: Metrics for Assessing Assembly Correctness (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

ScienceCinema

Pop, Mihai

2018-04-27

University of Maryland's Mihai Pop on Genome Assembly Forensics: Metrics for Assessing Assembly Correctness at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Semi-Automatic In Silico Gap Closure Enabled De Novo Assembly of Two Dehalobacter Genomes from Metagenomic Data

PubMed Central

Tang, Shuiquan; Gong, Yunchen; Edwards, Elizabeth A.

2012-01-01

Typically, the assembly and closure of a complete bacterial genome requires substantial additional effort spent in a wet lab for gap resolution and genome polishing. Assembly is further confounded by subspecies polymorphism when starting from metagenome sequence data. In this paper, we describe an in silico gap-resolution strategy that can substantially improve assembly. This strategy resolves assembly gaps in scaffolds using pre-assembled contigs, followed by verification with read mapping. It is capable of resolving assembly gaps caused by repetitive elements and subspecies polymorphisms. Using this strategy, we realized the de novo assembly of the first two Dehalobacter genomes from the metagenomes of two anaerobic mixed microbial cultures capable of reductive dechlorination of chlorinated ethanes and chloroform. Only four additional PCR reactions were required even though the initial assembly with Newbler v. 2.5 produced 101 contigs within 9 scaffolds belonging to two Dehalobacter strains. By applying this strategy to the re-assembly of a recently published genome of Bacteroides, we demonstrate its potential utility for other sequencing projects, both metagenomic and genomic. PMID:23284863

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome

DOE PAGES

Chapman, Jarrod A.; Mascher, Martin; Buluc, Aydin; ...

2015-01-31

We report that polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible tomore » construct a mapping population.« less

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome

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

Chapman, Jarrod A.; Mascher, Martin; Buluc, Aydin

We report that polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible tomore » construct a mapping population.« less

AFRESh: an adaptive framework for compression of reads and assembled sequences with random access functionality.

PubMed

Paridaens, Tom; Van Wallendael, Glenn; De Neve, Wesley; Lambert, Peter

2017-05-15

The past decade has seen the introduction of new technologies that lowered the cost of genomic sequencing increasingly. We can even observe that the cost of sequencing is dropping significantly faster than the cost of storage and transmission. The latter motivates a need for continuous improvements in the area of genomic data compression, not only at the level of effectiveness (compression rate), but also at the level of functionality (e.g. random access), configurability (effectiveness versus complexity, coding tool set …) and versatility (support for both sequenced reads and assembled sequences). In that regard, we can point out that current approaches mostly do not support random access, requiring full files to be transmitted, and that current approaches are restricted to either read or sequence compression. We propose AFRESh, an adaptive framework for no-reference compression of genomic data with random access functionality, targeting the effective representation of the raw genomic symbol streams of both reads and assembled sequences. AFRESh makes use of a configurable set of prediction and encoding tools, extended by a Context-Adaptive Binary Arithmetic Coding scheme (CABAC), to compress raw genetic codes. To the best of our knowledge, our paper is the first to describe an effective implementation CABAC outside of its' original application. By applying CABAC, the compression effectiveness improves by up to 19% for assembled sequences and up to 62% for reads. By applying AFRESh to the genomic symbols of the MPEG genomic compression test set for reads, a compression gain is achieved of up to 51% compared to SCALCE, 42% compared to LFQC and 44% compared to ORCOM. When comparing to generic compression approaches, a compression gain is achieved of up to 41% compared to GNU Gzip and 22% compared to 7-Zip at the Ultra setting. Additionaly, when compressing assembled sequences of the Human Genome, a compression gain is achieved up to 34% compared to GNU Gzip and 16% compared to 7-Zip at the Ultra setting. A Windows executable version can be downloaded at https://github.com/tparidae/AFresh . tom.paridaens@ugent.be. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Functional Immunomics of the Squash Bug, Anasa tristis (De Geer) (Heteroptera: Coreidae)

PubMed Central

Shelby, Kent S.

2013-01-01

The Squash bug, Anasa tristis (De Geer), is a major piercing/sucking pest of cucurbits, causing extensive damage to plants and fruits, and transmitting phytopathogens. No genomic resources to facilitate field and laboratory studies of this pest were available; therefore the first de novo exome for this destructive pest was assembled. RNA was extracted from insects challenged with bacterial and fungal immunoelicitors, insects fed on different cucurbit species, and insects from all life stages from egg to adult. All treatments and replicates were separately barcoded for subsequent analyses, then pooled for sequencing in a single lane using the Illumina HiSeq2000 platform. Over 211 million 100-base tags generated in this manner were trimmed, filtered, and cleaned, then assembled into a de novo reference transcriptome using the Broad Institute Trinity assembly algorithm. The assembly was annotated using NCBIx NR, BLAST2GO, KEGG and other databases. Of the >130,000 total assemblies 37,327 were annotated identifying the sequences of candidate gene silencing targets from immune, endocrine, reproductive, cuticle, and other physiological systems. Expression profiling of the adult immune response was accomplished by aligning the 100-base tags from each biological replicate from each treatment and controls to the annotated reference assembly of the A. tristis transcriptome. PMID:26462532

Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species.

PubMed

Kim, Seungill; Park, Minkyu; Yeom, Seon-In; Kim, Yong-Min; Lee, Je Min; Lee, Hyun-Ah; Seo, Eunyoung; Choi, Jaeyoung; Cheong, Kyeongchae; Kim, Ki-Tae; Jung, Kyongyong; Lee, Gir-Won; Oh, Sang-Keun; Bae, Chungyun; Kim, Saet-Byul; Lee, Hye-Young; Kim, Shin-Young; Kim, Myung-Shin; Kang, Byoung-Cheorl; Jo, Yeong Deuk; Yang, Hee-Bum; Jeong, Hee-Jin; Kang, Won-Hee; Kwon, Jin-Kyung; Shin, Chanseok; Lim, Jae Yun; Park, June Hyun; Huh, Jin Hoe; Kim, June-Sik; Kim, Byung-Dong; Cohen, Oded; Paran, Ilan; Suh, Mi Chung; Lee, Saet Buyl; Kim, Yeon-Ki; Shin, Younhee; Noh, Seung-Jae; Park, Junhyung; Seo, Young Sam; Kwon, Suk-Yoon; Kim, Hyun A; Park, Jeong Mee; Kim, Hyun-Jin; Choi, Sang-Bong; Bosland, Paul W; Reeves, Gregory; Jo, Sung-Hwan; Lee, Bong-Woo; Cho, Hyung-Taeg; Choi, Hee-Seung; Lee, Min-Soo; Yu, Yeisoo; Do Choi, Yang; Park, Beom-Seok; van Deynze, Allen; Ashrafi, Hamid; Hill, Theresa; Kim, Woo Taek; Pai, Hyun-Sook; Ahn, Hee Kyung; Yeam, Inhwa; Giovannoni, James J; Rose, Jocelyn K C; Sørensen, Iben; Lee, Sang-Jik; Kim, Ryan W; Choi, Ik-Young; Choi, Beom-Soon; Lim, Jong-Sung; Lee, Yong-Hwan; Choi, Doil

2014-03-01

Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species.

De Novo Assembly and Phasing of Dikaryotic Genomes from Two Isolates of Puccinia coronata f. sp. avenae, the Causal Agent of Oat Crown Rust.

PubMed

Miller, Marisa E; Zhang, Ying; Omidvar, Vahid; Sperschneider, Jana; Schwessinger, Benjamin; Raley, Castle; Palmer, Jonathan M; Garnica, Diana; Upadhyaya, Narayana; Rathjen, John; Taylor, Jennifer M; Park, Robert F; Dodds, Peter N; Hirsch, Cory D; Kianian, Shahryar F; Figueroa, Melania

2018-02-20

Oat crown rust, caused by the fungus Pucinnia coronata f. sp. avenae , is a devastating disease that impacts worldwide oat production. For much of its life cycle, P. coronata f. sp. avenae is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two P. coronata f. sp. avenae isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for 12SD80, for a total assembly length of 99.16 Mbp, and 777 primary contigs for 12NC29, for a total length of 105.25 Mbp; approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of coexpressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in P. coronata f. sp. avenae IMPORTANCE Disease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae , which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding P. coronata f. sp. avenae , resources to study the molecular mechanisms underpinning pathogenicity and the emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of P. coronata f. sp. avenae as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of P. coronata f. sp. avenae . Copyright © 2018 Miller et al.

A de novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi.

PubMed

Tan, Qian-Qian; Zhu, Li; Li, Yi; Liu, Wen; Ma, Wei-Hua; Lei, Chao-Liang; Wang, Xiao-Ping

2015-01-01

The cabbage beetle Colaphellus bowringi Baly is a serious insect pest of crucifers and undergoes reproductive diapause in soil. An understanding of the molecular mechanisms of diapause regulation, insecticide resistance, and other physiological processes is helpful for developing new management strategies for this beetle. However, the lack of genomic information and valid reference genes limits knowledge on the molecular bases of these physiological processes in this species. Using Illumina sequencing, we obtained more than 57 million sequence reads derived from C. bowringi, which were assembled into 39,390 unique sequences. A Clusters of Orthologous Groups classification was obtained for 9,048 of these sequences, covering 25 categories, and 16,951 were assigned to 255 Kyoto Encyclopedia of Genes and Genomes pathways. Eleven candidate reference gene sequences from the transcriptome were then identified through reverse transcriptase polymerase chain reaction. Among these candidate genes, EF1α, ACT1, and RPL19 proved to be the most stable reference genes for different reverse transcriptase quantitative polymerase chain reaction experiments in C. bowringi. Conversely, aTUB and GAPDH were the least stable reference genes. The abundant putative C. bowringi transcript sequences reported enrich the genomic resources of this beetle. Importantly, the larger number of gene sequences and valid reference genes provide a valuable platform for future gene expression studies, especially with regard to exploring the molecular mechanisms of different physiological processes in this species.

Extreme-Scale De Novo Genome Assembly

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

Georganas, Evangelos; Hofmeyr, Steven; Egan, Rob

De novo whole genome assembly reconstructs genomic sequence from short, overlapping, and potentially erroneous DNA segments and is one of the most important computations in modern genomics. This work presents HipMER, a high-quality end-to-end de novo assembler designed for extreme scale analysis, via efficient parallelization of the Meraculous code. Genome assembly software has many components, each of which stresses different components of a computer system. This chapter explains the computational challenges involved in each step of the HipMer pipeline, the key distributed data structures, and communication costs in detail. We present performance results of assembling the human genome and themore » large hexaploid wheat genome on large supercomputers up to tens of thousands of cores.« less

Reptilian Transcriptomes v2.0: An Extensive Resource for Sauropsida Genomics and Transcriptomics

PubMed Central

Tzika, Athanasia C.; Ullate-Agote, Asier; Grbic, Djordje; Milinkovitch, Michel C.

2015-01-01

Despite the availability of deep-sequencing techniques, genomic and transcriptomic data remain unevenly distributed across phylogenetic groups. For example, reptiles are poorly represented in sequence databases, hindering functional evolutionary and developmental studies in these lineages substantially more diverse than mammals. In addition, different studies use different assembly and annotation protocols, inhibiting meaningful comparisons. Here, we present the “Reptilian Transcriptomes Database 2.0,” which provides extensive annotation of transcriptomes and genomes from species covering the major reptilian lineages. To this end, we sequenced normalized complementary DNA libraries of multiple adult tissues and various embryonic stages of the leopard gecko and the corn snake and gathered published reptilian sequence data sets from representatives of the four extant orders of reptiles: Squamata (snakes and lizards), the tuatara, crocodiles, and turtles. The LANE runner 2.0 software was implemented to annotate all assemblies within a single integrated pipeline. We show that this approach increases the annotation completeness of the assembled transcriptomes/genomes. We then built large concatenated protein alignments of single-copy genes and inferred phylogenetic trees that support the positions of turtles and the tuatara as sister groups of Archosauria and Squamata, respectively. The Reptilian Transcriptomes Database 2.0 resource will be updated to include selected new data sets as they become available, thus making it a reference for differential expression studies, comparative genomics and transcriptomics, linkage mapping, molecular ecology, and phylogenomic analyses involving reptiles. The database is available at www.reptilian-transcriptomes.org and can be enquired using a wwwblast server installed at the University of Geneva. PMID:26133641

SNPchiMp: a database to disentangle the SNPchip jungle in bovine livestock.

PubMed

Nicolazzi, Ezequiel Luis; Picciolini, Matteo; Strozzi, Francesco; Schnabel, Robert David; Lawley, Cindy; Pirani, Ali; Brew, Fiona; Stella, Alessandra

2014-02-11

Currently, six commercial whole-genome SNP chips are available for cattle genotyping, produced by two different genotyping platforms. Technical issues need to be addressed to combine data that originates from the different platforms, or different versions of the same array generated by the manufacturer. For example: i) genome coordinates for SNPs may refer to different genome assemblies; ii) reference genome sequences are updated over time changing the positions, or even removing sequences which contain SNPs; iii) not all commercial SNP ID's are searchable within public databases; iv) SNPs can be coded using different formats and referencing different strands (e.g. A/B or A/C/T/G alleles, referencing forward/reverse, top/bottom or plus/minus strand); v) Due to new information being discovered, higher density chips do not necessarily include all the SNPs present in the lower density chips; and, vi) SNP IDs may not be consistent across chips and platforms. Most researchers and breed associations manage SNP data in real-time and thus require tools to standardise data in a user-friendly manner. Here we present SNPchiMp, a MySQL database linked to an open access web-based interface. Features of this interface include, but are not limited to, the following functions: 1) referencing the SNP mapping information to the latest genome assembly, 2) extraction of information contained in dbSNP for SNPs present in all commercially available bovine chips, and 3) identification of SNPs in common between two or more bovine chips (e.g. for SNP imputation from lower to higher density). In addition, SNPchiMp can retrieve this information on subsets of SNPs, accessing such data either via physical position on a supported assembly, or by a list of SNP IDs, rs or ss identifiers. This tool combines many different sources of information, that otherwise are time consuming to obtain and difficult to integrate. The SNPchiMp not only provides the information in a user-friendly format, but also enables researchers to perform a large number of operations with a few clicks of the mouse. This significantly reduces the time needed to execute the large number of operations required to manage SNP data.

Improved de novo genomic assembly for the domestic donkey.

PubMed

Renaud, Gabriel; Petersen, Bent; Seguin-Orlando, Andaine; Bertelsen, Mads Frost; Waller, Andrew; Newton, Richard; Paillot, Romain; Bryant, Neil; Vaudin, Mark; Librado, Pablo; Orlando, Ludovic

2018-04-01

Donkeys and horses share a common ancestor dating back to about 4 million years ago. Although a high-quality genome assembly at the chromosomal level is available for the horse, current assemblies available for the donkey are limited to moderately sized scaffolds. The absence of a better-quality assembly for the donkey has hampered studies involving the characterization of patterns of genetic variation at the genome-wide scale. These range from the application of genomic tools to selective breeding and conservation to the more fundamental characterization of the genomic loci underlying speciation and domestication. We present a new high-quality donkey genome assembly obtained using the Chicago HiRise assembly technology, providing scaffolds of subchromosomal size. We make use of this new assembly to obtain more accurate measures of heterozygosity for equine species other than the horse, both genome-wide and locally, and to detect runs of homozygosity potentially pertaining to positive selection in domestic donkeys. Finally, this new assembly allowed us to identify fine-scale chromosomal rearrangements between the horse and the donkey that likely played an active role in their divergence and, ultimately, speciation.

Improved de novo genomic assembly for the domestic donkey

PubMed Central

Newton, Richard; Paillot, Romain; Bryant, Neil; Vaudin, Mark

2018-01-01

Donkeys and horses share a common ancestor dating back to about 4 million years ago. Although a high-quality genome assembly at the chromosomal level is available for the horse, current assemblies available for the donkey are limited to moderately sized scaffolds. The absence of a better-quality assembly for the donkey has hampered studies involving the characterization of patterns of genetic variation at the genome-wide scale. These range from the application of genomic tools to selective breeding and conservation to the more fundamental characterization of the genomic loci underlying speciation and domestication. We present a new high-quality donkey genome assembly obtained using the Chicago HiRise assembly technology, providing scaffolds of subchromosomal size. We make use of this new assembly to obtain more accurate measures of heterozygosity for equine species other than the horse, both genome-wide and locally, and to detect runs of homozygosity potentially pertaining to positive selection in domestic donkeys. Finally, this new assembly allowed us to identify fine-scale chromosomal rearrangements between the horse and the donkey that likely played an active role in their divergence and, ultimately, speciation. PMID:29740610

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

A High-Quality Reference Genome for the Invasive Mosquitofish Gambusia affinis Using a Chicago Library.

PubMed

Hoffberg, Sandra L; Troendle, Nicholas J; Glenn, Travis C; Mahmud, Ousman; Louha, Swarnali; Chalopin, Domitille; Bennetzen, Jeffrey L; Mauricio, Rodney

2018-04-27

The western mosquitofish, Gambusia affinis, is a freshwater poecilid fish native to the southeastern United States but with a global distribution due to widespread human introduction. Gambusia affinis has been used as a model species for a broad range of evolutionary and ecological studies. We sequenced the genome of a male G. affinis to facilitate genetic studies in diverse fields including invasion biology and comparative genetics. We generated Illumina short read data from paired-end libraries and in vitro proximity-ligation libraries. We obtained 54.9× coverage, N50 contig length of 17.6 kb, and N50 scaffold length of 6.65 Mb. Compared to two other species in the Poeciliidae family, G. affinis has slightly fewer genes that have shorter total, exon, and intron length on average. Using a set of universal single-copy orthologs in fish genomes, we found 95.5% of these genes were complete in the G. affinis assembly. The number of transposable elements in the G. affinis assembly is similar to those of closely related species. The high-quality genome sequence and annotations we report will be valuable resources for scientists to map the genetic architecture of traits of interest in this species. Copyright © 2018, G3: Genes, Genomes, Genetics.

Survey of endosymbionts in the Diaphorina citri metagenome and assembly of a Wolbachia wDi draft genome.

PubMed

Saha, Surya; Hunter, Wayne B; Reese, Justin; Morgan, J Kent; Marutani-Hert, Mizuri; Huang, Hong; Lindeberg, Magdalen

2012-01-01

Diaphorina citri (Hemiptera: Psyllidae), the Asian citrus psyllid, is the insect vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening disease. Sequencing of the D. citri metagenome has been initiated to gain better understanding of the biology of this organism and the potential roles of its bacterial endosymbionts. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the D. citri metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Genome alignment with the complete sequences reveals membership of Wolbachia wDi in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida D. citri isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese D. citri isolates, supporting the hypothesis that the D. citri introduced into Florida did not originate from China.

Survey of Endosymbionts in the Diaphorina citri Metagenome and Assembly of a Wolbachia wDi Draft Genome

PubMed Central

Saha, Surya; Hunter, Wayne B.; Reese, Justin; Morgan, J. Kent; Marutani-Hert, Mizuri; Huang, Hong; Lindeberg, Magdalen

2012-01-01

Diaphorina citri (Hemiptera: Psyllidae), the Asian citrus psyllid, is the insect vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening disease. Sequencing of the D. citri metagenome has been initiated to gain better understanding of the biology of this organism and the potential roles of its bacterial endosymbionts. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the D. citri metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Genome alignment with the complete sequences reveals membership of Wolbachia wDi in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida D. citri isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese D. citri isolates, supporting the hypothesis that the D. citri introduced into Florida did not originate from China. PMID:23166822

De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds.

PubMed

Dudchenko, Olga; Batra, Sanjit S; Omer, Arina D; Nyquist, Sarah K; Hoeger, Marie; Durand, Neva C; Shamim, Muhammad S; Machol, Ido; Lander, Eric S; Aiden, Aviva Presser; Aiden, Erez Lieberman

2017-04-07

The Zika outbreak, spread by the Aedes aegypti mosquito, highlights the need to create high-quality assemblies of large genomes in a rapid and cost-effective way. Here we combine Hi-C data with existing draft assemblies to generate chromosome-length scaffolds. We validate this method by assembling a human genome, de novo, from short reads alone (67× coverage). We then combine our method with draft sequences to create genome assemblies of the mosquito disease vectors Ae aegypti and Culex quinquefasciatus , each consisting of three scaffolds corresponding to the three chromosomes in each species. These assemblies indicate that almost all genomic rearrangements among these species occur within, rather than between, chromosome arms. The genome assembly procedure we describe is fast, inexpensive, and accurate, and can be applied to many species. Copyright © 2017, American Association for the Advancement of Science.

Discovery of a Novel Periodontal Disease-Associated Bacterium.

PubMed

Torres, Pedro J; Thompson, John; McLean, Jeffrey S; Kelley, Scott T; Edlund, Anna

2018-06-02

One of the world's most common infectious disease, periodontitis (PD), derives from largely uncharacterized communities of oral bacteria growing as biofilms (a.k.a. plaque) on teeth and gum surfaces in periodontal pockets. Bacteria associated with periodontal disease trigger inflammatory responses in immune cells, which in later stages of the disease cause loss of both soft and hard tissue structures supporting teeth. Thus far, only a handful of bacteria have been characterized as infectious agents of PD. Although deep sequencing technologies, such as whole community shotgun sequencing have the potential to capture a detailed picture of highly complex bacterial communities in any given environment, we still lack major reference genomes for the oral microbiome associated with PD and other diseases. In recent work, by using a combination of supervised machine learning and genome assembly, we identified a genome from a novel member of the Bacteroidetes phylum in periodontal samples. Here, by applying a comparative metagenomics read-classification approach, including 272 metagenomes from various human body sites, and our previously assembled draft genome of the uncultivated Candidatus Bacteroides periocalifornicus (CBP) bacterium, we show CBP's ubiquitous distribution in dental plaque, as well as its strong association with the well-known pathogenic "red complex" that resides in deep periodontal pockets.

SIMBA: a web tool for managing bacterial genome assembly generated by Ion PGM sequencing technology.

PubMed

Mariano, Diego C B; Pereira, Felipe L; Aguiar, Edgar L; Oliveira, Letícia C; Benevides, Leandro; Guimarães, Luís C; Folador, Edson L; Sousa, Thiago J; Ghosh, Preetam; Barh, Debmalya; Figueiredo, Henrique C P; Silva, Artur; Ramos, Rommel T J; Azevedo, Vasco A C

2016-12-15

The evolution of Next-Generation Sequencing (NGS) has considerably reduced the cost per sequenced-base, allowing a significant rise of sequencing projects, mainly in prokaryotes. However, the range of available NGS platforms requires different strategies and software to correctly assemble genomes. Different strategies are necessary to properly complete an assembly project, in addition to the installation or modification of various software. This requires users to have significant expertise in these software and command line scripting experience on Unix platforms, besides possessing the basic expertise on methodologies and techniques for genome assembly. These difficulties often delay the complete genome assembly projects. In order to overcome this, we developed SIMBA (SImple Manager for Bacterial Assemblies), a freely available web tool that integrates several component tools for assembling and finishing bacterial genomes. SIMBA provides a friendly and intuitive user interface so bioinformaticians, even with low computational expertise, can work under a centralized administrative control system of assemblies managed by the assembly center head. SIMBA guides the users to execute assembly process through simple and interactive pages. SIMBA workflow was divided in three modules: (i) projects: allows a general vision of genome sequencing projects, in addition to data quality analysis and data format conversions; (ii) assemblies: allows de novo assemblies with the software Mira, Minia, Newbler and SPAdes, also assembly quality validations using QUAST software; and (iii) curation: presents methods to finishing assemblies through tools for scaffolding contigs and close gaps. We also presented a case study that validated the efficacy of SIMBA to manage bacterial assemblies projects sequenced using Ion Torrent PGM. Besides to be a web tool for genome assembly, SIMBA is a complete genome assemblies project management system, which can be useful for managing of several projects in laboratories. SIMBA source code is available to download and install in local webservers at http://ufmg-simba.sourceforge.net .

Evaluation and Validation of Assembling Corrected PacBio Long Reads for Microbial Genome Completion via Hybrid Approaches.

PubMed

Lin, Hsin-Hung; Liao, Yu-Chieh

2015-01-01

Despite the ever-increasing output of next-generation sequencing data along with developing assemblers, dozens to hundreds of gaps still exist in de novo microbial assemblies due to uneven coverage and large genomic repeats. Third-generation single-molecule, real-time (SMRT) sequencing technology avoids amplification artifacts and generates kilobase-long reads with the potential to complete microbial genome assembly. However, due to the low accuracy (~85%) of third-generation sequences, a considerable amount of long reads (>50X) are required for self-correction and for subsequent de novo assembly. Recently-developed hybrid approaches, using next-generation sequencing data and as few as 5X long reads, have been proposed to improve the completeness of microbial assembly. In this study we have evaluated the contemporary hybrid approaches and demonstrated that assembling corrected long reads (by runCA) produced the best assembly compared to long-read scaffolding (e.g., AHA, Cerulean and SSPACE-LongRead) and gap-filling (SPAdes). For generating corrected long reads, we further examined long-read correction tools, such as ECTools, LSC, LoRDEC, PBcR pipeline and proovread. We have demonstrated that three microbial genomes including Escherichia coli K12 MG1655, Meiothermus ruber DSM1279 and Pdeobacter heparinus DSM2366 were successfully hybrid assembled by runCA into near-perfect assemblies using ECTools-corrected long reads. In addition, we developed a tool, Patch, which implements corrected long reads and pre-assembled contigs as inputs, to enhance microbial genome assemblies. With the additional 20X long reads, short reads of S. cerevisiae W303 were hybrid assembled into 115 contigs using the verified strategy, ECTools + runCA. Patch was subsequently applied to upgrade the assembly to a 35-contig draft genome. Our evaluation of the hybrid approaches shows that assembling the ECTools-corrected long reads via runCA generates near complete microbial genomes, suggesting that genome assembly could benefit from re-analyzing the available hybrid datasets that were not assembled in an optimal fashion.

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

PubMed

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

2015-02-01

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

De Novo Assembly and Annotation of the Transcriptome of the Agricultural Weed Ipomoea purpurea Uncovers Gene Expression Changes Associated with Herbicide Resistance

PubMed Central

Leslie, Trent; Baucom, Regina S.

2014-01-01

Human-mediated selection can lead to rapid evolution in very short time scales, and the evolution of herbicide resistance in agricultural weeds is an excellent example of this phenomenon. The common morning glory, Ipomoea purpurea, is resistant to the herbicide glyphosate, but genetic investigations of this trait have been hampered by the lack of genomic resources for this species. Here, we present the annotated transcriptome of the common morning glory, Ipomoea purpurea, along with an examination of whole genome expression profiling to assess potential gene expression differences between three artificially selected herbicide resistant lines and three susceptible lines. The assembled Ipomoea transcriptome reported in this work contains 65,459 assembled transcripts, ~28,000 of which were functionally annotated by assignment to Gene Ontology categories. Our RNA-seq survey using this reference transcriptome identified 19 differentially expressed genes associated with resistance—one of which, a cytochrome P450, belongs to a large plant family of genes involved in xenobiotic detoxification. The differentially expressed genes also broadly implicated receptor-like kinases, which were down-regulated in the resistant lines, and other growth and defense genes, which were up-regulated in resistant lines. Interestingly, the target of glyphosate—EPSP synthase—was not overexpressed in the resistant Ipomoea lines as in other glyphosate resistant weeds. Overall, this work identifies potential candidate resistance loci for future investigations and dramatically increases genomic resources for this species. The assembled transcriptome presented herein will also provide a valuable resource to the Ipomoea community, as well as to those interested in utilizing the close relationship between the Convolvulaceae and the Solanaceae for phylogenetic and comparative genomics examinations. PMID:25155274

De novo assembly and annotation of the transcriptome of the agricultural weed Ipomoea purpurea uncovers gene expression changes associated with herbicide resistance.

PubMed

Leslie, Trent; Baucom, Regina S

2014-08-25

Human-mediated selection can lead to rapid evolution in very short time scales, and the evolution of herbicide resistance in agricultural weeds is an excellent example of this phenomenon. The common morning glory, Ipomoea purpurea, is resistant to the herbicide glyphosate, but genetic investigations of this trait have been hampered by the lack of genomic resources for this species. Here, we present the annotated transcriptome of the common morning glory, Ipomoea purpurea, along with an examination of whole genome expression profiling to assess potential gene expression differences between three artificially selected herbicide resistant lines and three susceptible lines. The assembled Ipomoea transcriptome reported in this work contains 65,459 assembled transcripts, ~28,000 of which were functionally annotated by assignment to Gene Ontology categories. Our RNA-seq survey using this reference transcriptome identified 19 differentially expressed genes associated with resistance-one of which, a cytochrome P450, belongs to a large plant family of genes involved in xenobiotic detoxification. The differentially expressed genes also broadly implicated receptor-like kinases, which were down-regulated in the resistant lines, and other growth and defense genes, which were up-regulated in resistant lines. Interestingly, the target of glyphosate-EPSP synthase-was not overexpressed in the resistant Ipomoea lines as in other glyphosate resistant weeds. Overall, this work identifies potential candidate resistance loci for future investigations and dramatically increases genomic resources for this species. The assembled transcriptome presented herein will also provide a valuable resource to the Ipomoea community, as well as to those interested in utilizing the close relationship between the Convolvulaceae and the Solanaceae for phylogenetic and comparative genomics examinations. Copyright © 2014 Leslie and Baucom.

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

Integrative structural annotation of de novo RNA-Seq provides an accurate reference gene set of the enormous genome of the onion (Allium cepa L.)

PubMed Central

Kim, Seungill; Kim, Myung-Shin; Kim, Yong-Min; Yeom, Seon-In; Cheong, Kyeongchae; Kim, Ki-Tae; Jeon, Jongbum; Kim, Sunggil; Kim, Do-Sun; Sohn, Seong-Han; Lee, Yong-Hwan; Choi, Doil

2015-01-01

The onion (Allium cepa L.) is one of the most widely cultivated and consumed vegetable crops in the world. Although a considerable amount of onion transcriptome data has been deposited into public databases, the sequences of the protein-coding genes are not accurate enough to be used, owing to non-coding sequences intermixed with the coding sequences. We generated a high-quality, annotated onion transcriptome from de novo sequence assembly and intensive structural annotation using the integrated structural gene annotation pipeline (ISGAP), which identified 54,165 protein-coding genes among 165,179 assembled transcripts totalling 203.0 Mb by eliminating the intron sequences. ISGAP performed reliable annotation, recognizing accurate gene structures based on reference proteins, and ab initio gene models of the assembled transcripts. Integrative functional annotation and gene-based SNP analysis revealed a whole biological repertoire of genes and transcriptomic variation in the onion. The method developed in this study provides a powerful tool for the construction of reference gene sets for organisms based solely on de novo transcriptome data. Furthermore, the reference genes and their variation described here for the onion represent essential tools for molecular breeding and gene cloning in Allium spp. PMID:25362073

In Silico Identification of Protein Disulfide Isomerase Gene Families in the De Novo Assembled Transcriptomes of Four Different Species of the Genus Conus.

PubMed

Figueroa-Montiel, Andrea; Ramos, Marco A; Mares, Rosa E; Dueñas, Salvador; Pimienta, Genaro; Ortiz, Ernesto; Possani, Lourival D; Licea-Navarro, Alexei F

2016-01-01

Small peptides isolated from the venom of the marine snails belonging to the genus Conus have been largely studied because of their therapeutic value. These peptides can be classified in two groups. The largest one is composed by peptides rich in disulfide bonds, and referred to as conotoxins. Despite the importance of conotoxins given their pharmacology value, little is known about the protein disulfide isomerase (PDI) enzymes that are required to catalyze their correct folding. To discover the PDIs that may participate in the folding and structural maturation of conotoxins, the transcriptomes of the venom duct of four different species of Conus from the peninsula of Baja California (Mexico) were assembled. Complementary DNA (cDNA) libraries were constructed for each species and sequenced using a Genome Analyzer Illumina platform. The raw RNA-seq data was converted into transcript sequences using Trinity, a de novo assembler that allows the grouping of reads into contigs without a reference genome. An N50 value of 605 was established as a reference for future assemblies of Conus transcriptomes using this software. Transdecoder was used to extract likely coding sequences from Trinity transcripts, and PDI-specific sequence motif "APWCGHCK" was used to capture potential PDIs. An in silico analysis was performed to characterize the group of PDI protein sequences encoded by the duct-transcriptome of each species. The computational approach entailed a structural homology characterization, based on the presence of functional Thioredoxin-like domains. Four different PDI families were characterized, which are constituted by a total of 41 different gene sequences. The sequences had an average of 65% identity with other PDIs. Using MODELLER 9.14, the homology-based three-dimensional structure prediction of a subset of the sequences reported, showed the expected thioredoxin fold which was confirmed by a "simulated annealing" method.

Genome Sequencing of the Phytoseiid Predatory Mite Metaseiulus occidentalis Reveals Completely Atomized Hox Genes and Superdynamic Intron Evolution

PubMed Central

Hoy, Marjorie A.; Waterhouse, Robert M.; Wu, Ke; Estep, Alden S.; Ioannidis, Panagiotis; Palmer, William J.; Pomerantz, Aaron F.; Simão, Felipe A.; Thomas, Jainy; Jiggins, Francis M.; Murphy, Terence D.; Pritham, Ellen J.; Robertson, Hugh M.; Zdobnov, Evgeny M.; Gibbs, Richard A.; Richards, Stephen

2016-01-01

Metaseiulus occidentalis is an eyeless phytoseiid predatory mite employed for the biological control of agricultural pests including spider mites. Despite appearances, these predator and prey mites are separated by some 400 Myr of evolution and radically different lifestyles. We present a 152-Mb draft assembly of the M. occidentalis genome: Larger than that of its favored prey, Tetranychus urticae, but considerably smaller than those of many other chelicerates, enabling an extremely contiguous and complete assembly to be built—the best arachnid to date. Aided by transcriptome data, genome annotation cataloged 18,338 protein-coding genes and identified large numbers of Helitron transposable elements. Comparisons with other arthropods revealed a particularly dynamic and turbulent genomic evolutionary history. Its genes exhibit elevated molecular evolution, with strikingly high numbers of intron gains and losses, in stark contrast to the deer tick Ixodes scapularis. Uniquely among examined arthropods, this predatory mite’s Hox genes are completely atomized, dispersed across the genome, and it encodes five copies of the normally single-copy RNA processing Dicer-2 gene. Examining gene families linked to characteristic biological traits of this tiny predator provides initial insights into processes of sex determination, development, immune defense, and how it detects, disables, and digests its prey. As the first reference genome for the Phytoseiidae, and for any species with the rare sex determination system of parahaploidy, the genome of the western orchard predatory mite improves genomic sampling of chelicerates and provides invaluable new resources for functional genomic analyses of this family of agriculturally important mites. PMID:26951779

Genome resolved analysis of a premature infant gut microbial community reveals a Varibaculum cambriense genome and a shift towards fermentation-based metabolism during the third week of life.

PubMed

Brown, Christopher T; Sharon, Itai; Thomas, Brian C; Castelle, Cindy J; Morowitz, Michael J; Banfield, Jillian F

2013-12-17

The premature infant gut has low individual but high inter-individual microbial diversity compared with adults. Based on prior 16S rRNA gene surveys, many species from this environment are expected to be similar to those previously detected in the human microbiota. However, the level of genomic novelty and metabolic variation of strains found in the infant gut remains relatively unexplored. To study the stability and function of early microbial colonizers of the premature infant gut, nine stool samples were taken during the third week of life of a premature male infant delivered via Caesarean section. Metagenomic sequences were assembled and binned into near-complete and partial genomes, enabling strain-level genomic analysis of the microbial community.We reconstructed eleven near-complete and six partial bacterial genomes representative of the key members of the microbial community. Twelve of these genomes share >90% putative ortholog amino acid identity with reference genomes. Manual curation of the assembly of one particularly novel genome resulted in the first essentially complete genome sequence (in three pieces, the order of which could not be determined due to a repeat) for Varibaculum cambriense (strain Dora), a medically relevant species that has been implicated in abscess formation.During the period studied, the microbial community undergoes a compositional shift, in which obligate anaerobes (fermenters) overtake Escherichia coli as the most abundant species. Other species remain stable, probably due to their ability to either respire anaerobically or grow by fermentation, and their capacity to tolerate fluctuating levels of oxygen. Metabolic predictions for V. cambriense suggest that, like other members of the microbial community, this organism is able to process various sugar substrates and make use of multiple different electron acceptors during anaerobic respiration. Genome comparisons within the family Actinomycetaceae reveal important differences related to respiratory metabolism and motility. Genome-based analysis provided direct insight into strain-specific potential for anaerobic respiration and yielded the first genome for the genus Varibaculum. Importantly, comparison of these de novo assembled genomes with closely related isolate genomes supported the accuracy of the metagenomic methodology. Over a one-week period, the early gut microbial community transitioned to a community with a higher representation of obligate anaerobes, emphasizing both taxonomic and metabolic instability during colonization.

Genome resolved analysis of a premature infant gut microbial community reveals a Varibaculum cambriense genome and a shift towards fermentation-based metabolism during the third week of life

PubMed Central

2013-01-01

Background The premature infant gut has low individual but high inter-individual microbial diversity compared with adults. Based on prior 16S rRNA gene surveys, many species from this environment are expected to be similar to those previously detected in the human microbiota. However, the level of genomic novelty and metabolic variation of strains found in the infant gut remains relatively unexplored. Results To study the stability and function of early microbial colonizers of the premature infant gut, nine stool samples were taken during the third week of life of a premature male infant delivered via Caesarean section. Metagenomic sequences were assembled and binned into near-complete and partial genomes, enabling strain-level genomic analysis of the microbial community. We reconstructed eleven near-complete and six partial bacterial genomes representative of the key members of the microbial community. Twelve of these genomes share >90% putative ortholog amino acid identity with reference genomes. Manual curation of the assembly of one particularly novel genome resulted in the first essentially complete genome sequence (in three pieces, the order of which could not be determined due to a repeat) for Varibaculum cambriense (strain Dora), a medically relevant species that has been implicated in abscess formation. During the period studied, the microbial community undergoes a compositional shift, in which obligate anaerobes (fermenters) overtake Escherichia coli as the most abundant species. Other species remain stable, probably due to their ability to either respire anaerobically or grow by fermentation, and their capacity to tolerate fluctuating levels of oxygen. Metabolic predictions for V. cambriense suggest that, like other members of the microbial community, this organism is able to process various sugar substrates and make use of multiple different electron acceptors during anaerobic respiration. Genome comparisons within the family Actinomycetaceae reveal important differences related to respiratory metabolism and motility. Conclusions Genome-based analysis provided direct insight into strain-specific potential for anaerobic respiration and yielded the first genome for the genus Varibaculum. Importantly, comparison of these de novo assembled genomes with closely related isolate genomes supported the accuracy of the metagenomic methodology. Over a one-week period, the early gut microbial community transitioned to a community with a higher representation of obligate anaerobes, emphasizing both taxonomic and metabolic instability during colonization. PMID:24451181

Allele Identification for Transcriptome-Based Population Genomics in the Invasive Plant Centaurea solstitialis

PubMed Central

Dlugosch, Katrina M.; Lai, Zhao; Bonin, Aurélie; Hierro, José; Rieseberg, Loren H.

2013-01-01

Transcriptome sequences are becoming more broadly available for multiple individuals of the same species, providing opportunities to derive population genomic information from these datasets. Using the 454 Life Science Genome Sequencer FLX and FLX-Titanium next-generation platforms, we generated 11−430 Mbp of sequence for normalized cDNA for 40 wild genotypes of the invasive plant Centaurea solstitialis, yellow starthistle, from across its worldwide distribution. We examined the impact of sequencing effort on transcriptome recovery and overlap among individuals. To do this, we developed two novel publicly available software pipelines: SnoWhite for read cleaning before assembly, and AllelePipe for clustering of loci and allele identification in assembled datasets with or without a reference genome. AllelePipe is designed specifically for cases in which read depth information is not appropriate or available to assist with disentangling closely related paralogs from allelic variation, as in transcriptome or previously assembled libraries. We find that modest applications of sequencing effort recover most of the novel sequences present in the transcriptome of this species, including single-copy loci and a representative distribution of functional groups. In contrast, the coverage of variable sites, observation of heterozygosity, and overlap among different libraries are all highly dependent on sequencing effort. Nevertheless, the information gained from overlapping regions was informative regarding coarse population structure and variation across our small number of population samples, providing the first genetic evidence in support of hypothesized invasion scenarios. PMID:23390612

Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice.

PubMed

Brozynska, Marta; Copetti, Dario; Furtado, Agnelo; Wing, Rod A; Crayn, Darren; Fox, Glen; Ishikawa, Ryuji; Henry, Robert J

2017-06-01

The related A genome species of the Oryza genus are the effective gene pool for rice. Here, we report draft genomes for two Australian wild A genome taxa: O. rufipogon-like population, referred to as Taxon A, and O. meridionalis-like population, referred to as Taxon B. These two taxa were sequenced and assembled by integration of short- and long-read next-generation sequencing (NGS) data to create a genomic platform for a wider rice gene pool. Here, we report that, despite the distinct chloroplast genome, the nuclear genome of the Australian Taxon A has a sequence that is much closer to that of domesticated rice (O. sativa) than to the other Australian wild populations. Analysis of 4643 genes in the A genome clade showed that the Australian annual, O. meridionalis, and related perennial taxa have the most divergent (around 3 million years) genome sequences relative to domesticated rice. A test for admixture showed possible introgression into the Australian Taxon A (diverged around 1.6 million years ago) especially from the wild indica/O. nivara clade in Asia. These results demonstrate that northern Australia may be the centre of diversity of the A genome Oryza and suggest the possibility that this might also be the centre of origin of this group and represent an important resource for rice improvement. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Mutation Detection with Next-Generation Resequencing through a Mediator Genome

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

Wurtzel, Omri; Dori-Bachash, Mally; Pietrokovski, Shmuel

2010-12-31

The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WTmore » and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.« less

Assembly of cucumber (Cucumis sativus L.) somaclones

NASA Astrophysics Data System (ADS)

Skarzyńska, Agnieszka; Kuśmirek, Wiktor; Pawełkowicz, Magdalena; PlÄ der, Wojciech; Nowak, Robert M.

2017-08-01

The development of next generation sequencing opens the possibility of using sequencing in various plant studies, such as finding structural changes and small polymorphisms between species and within them. Most analyzes rely on genomic sequences and it is crucial to use well-assembled genomes of high quality and completeness. Herein we compare commonly available programs for genomic assembling and newly developed software - dnaasm. Assemblies were tested on cucumber (Cucumis sativus L.) lines obtained by in vitro regeneration (somaclones), showing different phenotypes. Obtained results shows that dnaasm assembler is a good tool for short read assembly, which allows obtaining genomes of high quality and completeness.

A whole-genome assembly of the domestic cow, Bos taurus

USDA-ARS?s Scientific Manuscript database

Background: The genome of the domestic cow, Bos taurus, was sequenced using a mixture of hierarchical and whole-genome shotgun sequencing methods. Results: We have assembled the 35 million sequence reads and applied a variety of assembly improvement techniques, creating an assembly of 2.86 billion b...

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

Hybrid De Novo Genome Assembly Using MiSeq and SOLiD Short Read Data

PubMed Central

Ikegami, Tsutomu; Inatsugi, Toyohiro; Kojima, Isao; Umemura, Myco; Hagiwara, Hiroko; Machida, Masayuki; Asai, Kiyoshi

2015-01-01

A hybrid de novo assembly pipeline was constructed to utilize both MiSeq and SOLiD short read data in combination in the assembly. The short read data were converted to a standard format of the pipeline, and were supplied to the pipeline components such as ABySS and SOAPdenovo. The assembly pipeline proceeded through several stages, and either MiSeq paired-end data, SOLiD mate-paired data, or both of them could be specified as input data at each stage separately. The pipeline was examined on the filamentous fungus Aspergillus oryzae RIB40, by aligning the assembly results against the reference sequences. Using both the MiSeq and the SOLiD data in the hybrid assembly, the alignment length was improved by a factor of 3 to 8, compared with the assemblies using either one of the data types. The number of the reproduced gene cluster regions encoding secondary metabolite biosyntheses (SMB) was also improved by the hybrid assemblies. These results imply that the MiSeq data with long read length are essential to construct accurate nucleotide sequences, while the SOLiD mate-paired reads with long insertion length enhance long-range arrangements of the sequences. The pipeline was also tested on the actinomycete Streptomyces avermitilis MA-4680, whose gene is known to have high-GC content. Although the quality of the SOLiD reads was too low to perform any meaningful assemblies by themselves, the alignment length to the reference was improved by a factor of 2, compared with the assembly using only the MiSeq data. PMID:25919614

Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships

PubMed Central

Booher, Nicholas J.; Carpenter, Sara C. D.; Sebra, Robert P.; Wang, Li; Salzberg, Steven L.; Leach, Jan E.

2015-01-01

Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33–35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution. PMID:27148456

An integrated pipeline for next generation sequencing and annotation of the complete mitochondrial genome of the giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss, 1899

PubMed Central

Biswal, Devendra Kumar; Ghatani, Sudeep; Shylla, Jollin A.; Sahu, Ranjana; Mullapudi, Nandita

2013-01-01

Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create an NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest trematode mitochondrial genome sequenced to date. The noncoding control regions are separated into two parts by the tRNA-Gly gene and don’t contain either tandem repeats or secondary structures, which are typical for trematode control regions. The gene content and arrangement are identical to that of F. hepatica. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The mtDNA for the intestinal fluke is reported herein for the first time by our group that would help investigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data. More so, it would serve as a resource for comparative mitochondrial genomics and systematic studies of trematode parasites. PMID:24255820

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.

Review of General Algorithmic Features for Genome Assemblers for Next Generation Sequencers

PubMed Central

Wajid, Bilal; Serpedin, Erchin

2012-01-01

In the realm of bioinformatics and computational biology, the most rudimentary data upon which all the analysis is built is the sequence data of genes, proteins and RNA. The sequence data of the entire genome is the solution to the genome assembly problem. The scope of this contribution is to provide an overview on the art of problem-solving applied within the domain of genome assembly in the next-generation sequencing (NGS) platforms. This article discusses the major genome assemblers that were proposed in the literature during the past decade by outlining their basic working principles. It is intended to act as a qualitative, not a quantitative, tutorial to all working on genome assemblers pertaining to the next generation of sequencers. We discuss the theoretical aspects of various genome assemblers, identifying their working schemes. We also discuss briefly the direction in which the area is headed towards along with discussing core issues on software simplicity. PMID:22768980

BAC sequencing using pooled methods.

PubMed

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

2015-01-01

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

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

Lapidus, Alla L.

From the date its role in heredity was discovered, DNA has been generating interest among scientists from different fields of knowledge: physicists have studied the three dimensional structure of the DNA molecule, biologists tried to decode the secrets of life hidden within these long molecules, and technologists invent and improve methods of DNA analysis. The analysis of the nucleotide sequence of DNA occupies a special place among the methods developed. Thanks to the variety of sequencing technologies available, the process of decoding the sequence of genomic DNA (or whole genome sequencing) has become robust and inexpensive. Meanwhile the assembly ofmore » whole genome sequences remains a challenging task. In addition to the need to assemble millions of DNA fragments of different length (from 35 bp (Solexa) to 800 bp (Sanger)), great interest in analysis of microbial communities (metagenomes) of different complexities raises new problems and pushes some new requirements for sequence assembly tools to the forefront. The genome assembly process can be divided into two steps: draft assembly and assembly improvement (finishing). Despite the fact that automatically performed assembly (or draft assembly) is capable of covering up to 98% of the genome, in most cases, it still contains incorrectly assembled reads. The error rate of the consensus sequence produced at this stage is about 1/2000 bp. A finished genome represents the genome assembly of much higher accuracy (with no gaps or incorrectly assembled areas) and quality ({approx}1 error/10,000 bp), validated through a number of computer and laboratory experiments.« less

Integrated studies of uncultured microbes in the global ocean (Invited)

NASA Astrophysics Data System (ADS)

Dupont, C.; Rusch, D.; Martiny, A.; Lasken, R.

2010-12-01

The Global Ocean Sampling (GOS) initiative at the J. Craig Venter Institute represents the most extensive metagenomic study of a single environment. Early findings highlighted the potential of shotgun metagenomics to expand our knowledge of marine microbial biodiversity and physiology. However, it also became clear that many of the abundant marine microbes remain uncultured, hindering a direct connection between phylogeny and ecophysiology. In two recent studies, a combination of single cell genomics and aggressive assembly of binned metagenomic data have resulted in the acquisition of multiple genomes for two uncultured but globally relevant organisms. Metabolic reconstructions of the whole genomes revealed unique physiological adaptations in marine Prochlorococcus to high nutrient, low Fe regions of the global ocean and illuminated the potential ecological role of the gamma-proteobacterial 16S clade SAR86. The internal reference genomes also facilitate fragment recruitment based biogeographical studies, both at the whole genome level and the protein level.

The genomic basis of adaptive evolution in threespine sticklebacks

PubMed Central

Jones, Felicity C; Grabherr, Manfred G; Chan, Yingguang Frank; Russell, Pamela; Mauceli, Evan; Johnson, Jeremy; Swofford, Ross; Pirun, Mono; Zody, Michael C; White, Simon; Birney, Ewan; Searle, Stephen; Schmutz, Jeremy; Grimwood, Jane; Dickson, Mark C; Myers, Richard M; Miller, Craig T; Summers, Brian R; Knecht, Anne K; Brady, Shannon D; Zhang, Haili; Pollen, Alex A; Howes, Timothy; Amemiya, Chris; Lander, Eric S; Di Palma, Federica

2012-01-01

Summary Marine stickleback fish have colonized and adapted to innumerable streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of 20 additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine-freshwater divergence. Our results suggest that reuse of globally-shared standing genetic variation, including chromosomal inversions, plays an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine-freshwater evolution, with regulatory changes likely predominating in this classic example of repeated adaptive evolution in nature. PMID:22481358

The genomic basis of adaptive evolution in threespine sticklebacks.

PubMed

Jones, Felicity C; Grabherr, Manfred G; Chan, Yingguang Frank; Russell, Pamela; Mauceli, Evan; Johnson, Jeremy; Swofford, Ross; Pirun, Mono; Zody, Michael C; White, Simon; Birney, Ewan; Searle, Stephen; Schmutz, Jeremy; Grimwood, Jane; Dickson, Mark C; Myers, Richard M; Miller, Craig T; Summers, Brian R; Knecht, Anne K; Brady, Shannon D; Zhang, Haili; Pollen, Alex A; Howes, Timothy; Amemiya, Chris; Baldwin, Jen; Bloom, Toby; Jaffe, David B; Nicol, Robert; Wilkinson, Jane; Lander, Eric S; Di Palma, Federica; Lindblad-Toh, Kerstin; Kingsley, David M

2012-04-04

Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine-freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine-freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature.

Review of general algorithmic features for genome assemblers for next generation sequencers.

PubMed

Wajid, Bilal; Serpedin, Erchin

2012-04-01

In the realm of bioinformatics and computational biology, the most rudimentary data upon which all the analysis is built is the sequence data of genes, proteins and RNA. The sequence data of the entire genome is the solution to the genome assembly problem. The scope of this contribution is to provide an overview on the art of problem-solving applied within the domain of genome assembly in the next-generation sequencing (NGS) platforms. This article discusses the major genome assemblers that were proposed in the literature during the past decade by outlining their basic working principles. It is intended to act as a qualitative, not a quantitative, tutorial to all working on genome assemblers pertaining to the next generation of sequencers. We discuss the theoretical aspects of various genome assemblers, identifying their working schemes. We also discuss briefly the direction in which the area is headed towards along with discussing core issues on software simplicity. Copyright © 2012 Beijing Institute of Genomics, Chinese Academy of Sciences. Published by Elsevier Ltd. All rights reserved.

The first near-complete assembly of the hexaploid bread wheat genome, Triticum aestivum.

PubMed

Zimin, Aleksey V; Puiu, Daniela; Hall, Richard; Kingan, Sarah; Clavijo, Bernardo J; Salzberg, Steven L

2017-11-01

Common bread wheat, Triticum aestivum, has one of the most complex genomes known to science, with 6 copies of each chromosome, enormous numbers of near-identical sequences scattered throughout, and an overall haploid size of more than 15 billion bases. Multiple past attempts to assemble the genome have produced assemblies that were well short of the estimated genome size. Here we report the first near-complete assembly of T. aestivum, using deep sequencing coverage from a combination of short Illumina reads and very long Pacific Biosciences reads. The final assembly contains 15 344 693 583 bases and has a weighted average (N50) contig size of 232 659 bases. This represents by far the most complete and contiguous assembly of the wheat genome to date, providing a strong foundation for future genetic studies of this important food crop. We also report how we used the recently published genome of Aegilops tauschii, the diploid ancestor of the wheat D genome, to identify 4 179 762 575 bp of T. aestivum that correspond to its D genome components. © The Author 2017. Published by Oxford University Press.

Comparing de novo assemblers for 454 transcriptome data

PubMed Central

2010-01-01

Background Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base) reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC) to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode Litomosoides sigmodontis. Results Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects), which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs. Conclusions Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies from different programs however gave a more credible final product, and this strategy is recommended. PMID:20950480

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

Draft sequencing and assembly of the genome of the world's largest fish, the whale shark: Rhincodon typus Smith 1828.

PubMed

Read, Timothy D; Petit, Robert A; Joseph, Sandeep J; Alam, Md Tauqeer; Weil, M Ryan; Ahmad, Maida; Bhimani, Ravila; Vuong, Jocelyn S; Haase, Chad P; Webb, D Harry; Tan, Milton; Dove, Alistair D M

2017-07-14

The whale shark (Rhincodon typus) has by far the largest body size of any elasmobranch (shark or ray) species. Therefore, it is also the largest extant species of the paraphyletic assemblage commonly referred to as fishes. As both a phenotypic extreme and a member of the group Chondrichthyes - the sister group to the remaining gnathostomes, which includes all tetrapods and therefore also humans - its genome is of substantial comparative interest. Whale sharks are also listed as an endangered species on the International Union for Conservation of Nature's Red List of threatened species and are of growing popularity as both a target of ecotourism and as a charismatic conservation ambassador for the pelagic ecosystem. A genome map for this species would aid in defining effective conservation units and understanding global population structure. We characterised the nuclear genome of the whale shark using next generation sequencing (454, Illumina) and de novo assembly and annotation methods, based on material collected from the Georgia Aquarium. The data set consisted of 878,654,233 reads, which yielded a draft assembly of 1,213,200 contigs and 997,976 scaffolds. The estimated genome size was 3.44Gb. As expected, the proteome of the whale shark was most closely related to the only other complete genome of a cartilaginous fish, the holocephalan elephant shark. The whale shark contained a novel Toll-like-receptor (TLR) protein with sequence similarity to both the TLR4 and TLR13 proteins of mammals and TLR21 of teleosts. The data are publicly available on GenBank, FigShare, and from the NCBI Short Read Archive under accession number SRP044374. This represents the first shotgun elasmobranch genome and will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this and other shark species, as well as providing comparative data for studies of evolutionary biology and immunology across the jawed vertebrate lineages.

The UCSC genome browser and associated tools

PubMed Central

Haussler, David; Kent, W. James

2013-01-01

The UCSC Genome Browser (http://genome.ucsc.edu) is a graphical viewer for genomic data now in its 13th year. Since the early days of the Human Genome Project, it has presented an integrated view of genomic data of many kinds. Now home to assemblies for 58 organisms, the Browser presents visualization of annotations mapped to genomic coordinates. The ability to juxtapose annotations of many types facilitates inquiry-driven data mining. Gene predictions, mRNA alignments, epigenomic data from the ENCODE project, conservation scores from vertebrate whole-genome alignments and variation data may be viewed at any scale from a single base to an entire chromosome. The Browser also includes many other widely used tools, including BLAT, which is useful for alignments from high-throughput sequencing experiments. Private data uploaded as Custom Tracks and Data Hubs in many formats may be displayed alongside the rich compendium of precomputed data in the UCSC database. The Table Browser is a full-featured graphical interface, which allows querying, filtering and intersection of data tables. The Saved Session feature allows users to store and share customized views, enhancing the utility of the system for organizing multiple trains of thought. Binary Alignment/Map (BAM), Variant Call Format and the Personal Genome Single Nucleotide Polymorphisms (SNPs) data formats are useful for visualizing a large sequencing experiment (whole-genome or whole-exome), where the differences between the data set and the reference assembly may be displayed graphically. Support for high-throughput sequencing extends to compact, indexed data formats, such as BAM, bigBed and bigWig, allowing rapid visualization of large datasets from RNA-seq and ChIP-seq experiments via local hosting. PMID:22908213

The UCSC genome browser and associated tools.

PubMed

Kuhn, Robert M; Haussler, David; Kent, W James

2013-03-01

The UCSC Genome Browser (http://genome.ucsc.edu) is a graphical viewer for genomic data now in its 13th year. Since the early days of the Human Genome Project, it has presented an integrated view of genomic data of many kinds. Now home to assemblies for 58 organisms, the Browser presents visualization of annotations mapped to genomic coordinates. The ability to juxtapose annotations of many types facilitates inquiry-driven data mining. Gene predictions, mRNA alignments, epigenomic data from the ENCODE project, conservation scores from vertebrate whole-genome alignments and variation data may be viewed at any scale from a single base to an entire chromosome. The Browser also includes many other widely used tools, including BLAT, which is useful for alignments from high-throughput sequencing experiments. Private data uploaded as Custom Tracks and Data Hubs in many formats may be displayed alongside the rich compendium of precomputed data in the UCSC database. The Table Browser is a full-featured graphical interface, which allows querying, filtering and intersection of data tables. The Saved Session feature allows users to store and share customized views, enhancing the utility of the system for organizing multiple trains of thought. Binary Alignment/Map (BAM), Variant Call Format and the Personal Genome Single Nucleotide Polymorphisms (SNPs) data formats are useful for visualizing a large sequencing experiment (whole-genome or whole-exome), where the differences between the data set and the reference assembly may be displayed graphically. Support for high-throughput sequencing extends to compact, indexed data formats, such as BAM, bigBed and bigWig, allowing rapid visualization of large datasets from RNA-seq and ChIP-seq experiments via local hosting.

Genome sequence and analysis of a stress-tolerant, wild-derived strain of Saccharomyces cerevisiae used in biofuels research

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

McIlwain, Sean J.; Peris, Davis; Sardi, Maria

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assemblymore » approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. Lastly, the Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.« less

Genome sequence and analysis of a stress-tolerant, wild-derived strain of Saccharomyces cerevisiae used in biofuels research

DOE PAGES

McIlwain, Sean J.; Peris, Davis; Sardi, Maria; ...

2016-04-20

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assemblymore » approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. Lastly, the Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.« less

Observation of quantum criticality with ultracold atoms in optical lattices

NASA Astrophysics Data System (ADS)

Zhang, Xibo

As biological problems are becoming more complex and data growing at a rate much faster than that of computer hardware, new and faster algorithms are required. This dissertation investigates computational problems arising in two of the fields: comparative genomics and epigenomics, and employs a variety of computational techniques to address the problems. One fundamental question in the studies of chromosome evolution is whether the rearrangement breakpoints are happening at random positions or along certain hotspots. We investigate the breakpoint reuse phenomenon, and show the analyses that support the more recently proposed fragile breakage model as opposed to the conventional random breakage models for chromosome evolution. The identification of syntenic regions between chromosomes forms the basis for studies of genome architectures, comparative genomics, and evolutionary genomics. The previous synteny block reconstruction algorithms could not be scaled to a large number of mammalian genomes being sequenced; neither did they address the issue of generating non-overlapping synteny blocks suitable for analyzing rearrangements and evolutionary history of large-scale duplications prevalent in plant genomes. We present a new unified synteny block generation algorithm based on A-Bruijn graph framework that overcomes these shortcomings. In the epigenome sequencing, a sample may contain a mixture of epigenomes and there is a need to resolve the distinct methylation patterns from the mixture. Many sequencing applications, such as haplotype inference for diploid or polyploid genomes, and metagenomic sequencing, share the similar objective: to infer a set of distinct assemblies from reads that are sequenced from a heterogeneous sample and subsequently aligned to a reference genome. We model the problem from both a combinatorial and a statistical angles. First, we describe a theoretical framework. A linear-time algorithm is then given to resolve a minimum number of assemblies that are consistent with all reads, substantially improving on previous algorithms. An efficient algorithm is also described to determine a set of assemblies that is consistent with a maximum subset of the reads, a previously untreated problem. We then prove that allowing nested reads or permitting mismatches between reads and their assemblies renders these problems NP-hard. Second, we describe a mixture model-based approach, and applied the model for the detection of allele-specific methylations.

The map-based genome sequence of Spirodela polyrhiza aligned with its chromosomes, a reference for karyotype evolution.

PubMed

Cao, Hieu Xuan; Vu, Giang Thi Ha; Wang, Wenqin; Appenroth, Klaus J; Messing, Joachim; Schubert, Ingo

2016-01-01

Duckweeds are aquatic monocotyledonous plants of potential economic interest with fast vegetative propagation, comprising 37 species with variable genome sizes (0.158-1.88 Gbp). The genomic sequence of Spirodela polyrhiza, the smallest and the most ancient duckweed genome, needs to be aligned to its chromosomes as a reference and prerequisite to study the genome and karyotype evolution of other duckweed species. We selected physically mapped bacterial artificial chromosomes (BACs) containing Spirodela DNA inserts with little or no repetitive elements as probes for multicolor fluorescence in situ hybridization (mcFISH), using an optimized BAC pooling strategy, to validate its physical map and correlate it with its chromosome complement. By consecutive mcFISH analyses, we assigned the originally assembled 32 pseudomolecules (supercontigs) of the genomic sequences to the 20 chromosomes of S. polyrhiza. A Spirodela cytogenetic map containing 96 BAC markers with an average distance of 0.89 Mbp was constructed. Using a cocktail of 41 BACs in three colors, all chromosome pairs could be individualized simultaneously. Seven ancestral blocks emerged from duplicated chromosome segments of 19 Spirodela chromosomes. The chromosomally integrated genome of S. polyrhiza and the established prerequisites for comparative chromosome painting enable future studies on the chromosome homoeology and karyotype evolution of duckweed species. © 2015 IPK Gatersleben. New Phytologist © 2015 New Phytologist Trust.

Genomic analyses of the CAM plant pineapple.

PubMed

Zhang, Jisen; Liu, Juan; Ming, Ray

2014-07-01

The innovation of crassulacean acid metabolism (CAM) photosynthesis in arid and/or low CO2 conditions is a remarkable case of adaptation in flowering plants. As the most important crop that utilizes CAM photosynthesis, the genetic and genomic resources of pineapple have been developed over many years. Genetic diversity studies using various types of DNA markers led to the reclassification of the two genera Ananas and Pseudananas and nine species into one genus Ananas and two species, A. comosus and A. macrodontes with five botanical varieties in A. comosus. Five genetic maps have been constructed using F1 or F2 populations, and high-density genetic maps generated by genotype sequencing are essential resources for sequencing and assembling the pineapple genome and for marker-assisted selection. There are abundant expression sequence tag resources but limited genomic sequences in pineapple. Genes involved in the CAM pathway has been analysed in several CAM plants but only a few of them are from pineapple. A reference genome of pineapple is being generated and will accelerate genetic and genomic research in this major CAM crop. This reference genome of pineapple provides the foundation for studying the origin and regulatory mechanism of CAM photosynthesis, and the opportunity to evaluate the classification of Ananas species and botanical cultivars. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Congruent Deep Relationships in the Grape Family (Vitaceae) Based on Sequences of Chloroplast Genomes and Mitochondrial Genes via Genome Skimming

PubMed Central

Zhang, Ning; Wen, Jun; Zimmer, Elizabeth A.

2015-01-01

Vitaceae is well-known for having one of the most economically important fruits, i.e., the grape (Vitis vinifera). The deep phylogeny of the grape family was not resolved until a recent phylogenomic analysis of 417 nuclear genes from transcriptome data. However, it has been reported extensively that topologies based on nuclear and organellar genes may be incongruent due to differences in their evolutionary histories. Therefore, it is important to reconstruct a backbone phylogeny of the grape family using plastomes and mitochondrial genes. In this study, next-generation sequencing data sets of 27 species were obtained using genome skimming with total DNAs from silica-gel preserved tissue samples on an Illumina HiSeq 2500 instrument. Plastomes were assembled using the combination of de novo and reference genome (of V. vinifera) methods. Sixteen mitochondrial genes were also obtained via genome skimming using the reference genome of V. vinifera. Extensive phylogenetic analyses were performed using maximum likelihood and Bayesian methods. The topology based on either plastome data or mitochondrial genes is congruent with the one using hundreds of nuclear genes, indicating that the grape family did not exhibit significant reticulation at the deep level. The results showcase the power of genome skimming in capturing extensive phylogenetic data: especially from chloroplast and mitochondrial DNAs. PMID:26656830

Congruent Deep Relationships in the Grape Family (Vitaceae) Based on Sequences of Chloroplast Genomes and Mitochondrial Genes via Genome Skimming.

PubMed

Zhang, Ning; Wen, Jun; Zimmer, Elizabeth A

2015-01-01

Vitaceae is well-known for having one of the most economically important fruits, i.e., the grape (Vitis vinifera). The deep phylogeny of the grape family was not resolved until a recent phylogenomic analysis of 417 nuclear genes from transcriptome data. However, it has been reported extensively that topologies based on nuclear and organellar genes may be incongruent due to differences in their evolutionary histories. Therefore, it is important to reconstruct a backbone phylogeny of the grape family using plastomes and mitochondrial genes. In this study,next-generation sequencing data sets of 27 species were obtained using genome skimming with total DNAs from silica-gel preserved tissue samples on an Illumina NextSeq 500 instrument [corrected]. Plastomes were assembled using the combination of de novo and reference genome (of V. vinifera) methods. Sixteen mitochondrial genes were also obtained via genome skimming using the reference genome of V. vinifera. Extensive phylogenetic analyses were performed using maximum likelihood and Bayesian methods. The topology based on either plastome data or mitochondrial genes is congruent with the one using hundreds of nuclear genes, indicating that the grape family did not exhibit significant reticulation at the deep level. The results showcase the power of genome skimming in capturing extensive phylogenetic data: especially from chloroplast and mitochondrial DNAs.

Effects of short read quality and quantity on a de novo vertebrate transcriptome assembly.

PubMed

Garcia, T I; Shen, Y; Catchen, J; Amores, A; Schartl, M; Postlethwait, J; Walter, R B

2012-01-01

For many researchers, next generation sequencing data holds the key to answering a category of questions previously unassailable. One of the important and challenging steps in achieving these goals is accurately assembling the massive quantity of short sequencing reads into full nucleic acid sequences. For research groups working with non-model or wild systems, short read assembly can pose a significant challenge due to the lack of pre-existing EST or genome reference libraries. While many publications describe the overall process of sequencing and assembly, few address the topic of how many and what types of reads are best for assembly. The goal of this project was use real world data to explore the effects of read quantity and short read quality scores on the resulting de novo assemblies. Using several samples of short reads of various sizes and qualities we produced many assemblies in an automated manner. We observe how the properties of read length, read quality, and read quantity affect the resulting assemblies and provide some general recommendations based on our real-world data set. Published by Elsevier Inc.

High-speed and high-ratio referential genome compression.

PubMed

Liu, Yuansheng; Peng, Hui; Wong, Limsoon; Li, Jinyan

2017-11-01

The rapidly increasing number of genomes generated by high-throughput sequencing platforms and assembly algorithms is accompanied by problems in data storage, compression and communication. Traditional compression algorithms are unable to meet the demand of high compression ratio due to the intrinsic challenging features of DNA sequences such as small alphabet size, frequent repeats and palindromes. Reference-based lossless compression, by which only the differences between two similar genomes are stored, is a promising approach with high compression ratio. We present a high-performance referential genome compression algorithm named HiRGC. It is based on a 2-bit encoding scheme and an advanced greedy-matching search on a hash table. We compare the performance of HiRGC with four state-of-the-art compression methods on a benchmark dataset of eight human genomes. HiRGC takes <30 min to compress about 21 gigabytes of each set of the seven target genomes into 96-260 megabytes, achieving compression ratios of 217 to 82 times. This performance is at least 1.9 times better than the best competing algorithm on its best case. Our compression speed is also at least 2.9 times faster. HiRGC is stable and robust to deal with different reference genomes. In contrast, the competing methods' performance varies widely on different reference genomes. More experiments on 100 human genomes from the 1000 Genome Project and on genomes of several other species again demonstrate that HiRGC's performance is consistently excellent. The C ++ and Java source codes of our algorithm are freely available for academic and non-commercial use. They can be downloaded from https://github.com/yuansliu/HiRGC. jinyan.li@uts.edu.au. 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

Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

ScienceCinema

Sczyrba, Alex

2018-02-13

DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

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

Sczyrba, Alex

2011-10-13

DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color.

PubMed

Motamayor, Juan C; Mockaitis, Keithanne; Schmutz, Jeremy; Haiminen, Niina; Livingstone, Donald; Cornejo, Omar; Findley, Seth D; Zheng, Ping; Utro, Filippo; Royaert, Stefan; Saski, Christopher; Jenkins, Jerry; Podicheti, Ram; Zhao, Meixia; Scheffler, Brian E; Stack, Joseph C; Feltus, Frank A; Mustiga, Guiliana M; Amores, Freddy; Phillips, Wilbert; Marelli, Jean Philippe; May, Gregory D; Shapiro, Howard; Ma, Jianxin; Bustamante, Carlos D; Schnell, Raymond J; Main, Dorrie; Gilbert, Don; Parida, Laxmi; Kuhn, David N

2013-06-03

Theobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders. We describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina 1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation. We report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits.

The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color

PubMed Central

2013-01-01

Background Theobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders. Results We describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina 1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation. Conclusions We report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits. PMID:23731509

The Genome of Winter Moth (Operophtera brumata) Provides a Genomic Perspective on Sexual Dimorphism and Phenology.

PubMed

Derks, Martijn F L; Smit, Sandra; Salis, Lucia; Schijlen, Elio; Bossers, Alex; Mateman, Christa; Pijl, Agata S; de Ridder, Dick; Groenen, Martien A M; Visser, Marcel E; Megens, Hendrik-Jan

2015-07-29

The winter moth (Operophtera brumata) belongs to one of the most species-rich families in Lepidoptera, the Geometridae (approximately 23,000 species). This family is of great economic importance as most species are herbivorous and capable of defoliating trees. Genome assembly of the winter moth allows the study of genes and gene families, such as the cytochrome P450 gene family, which is known to be vital in plant secondary metabolite detoxification and host-plant selection. It also enables exploration of the genomic basis for female brachyptery (wing reduction), a feature of sexual dimorphism in winter moth, and for seasonal timing, a trait extensively studied in this species. Here we present a reference genome for the winter moth, the first geometrid and largest sequenced Lepidopteran genome to date (638 Mb) including a set of 16,912 predicted protein-coding genes. This allowed us to assess the dynamics of evolution on a genome-wide scale using the P450 gene family. We also identified an expanded gene family potentially linked to female brachyptery, and annotated the genes involved in the circadian clock mechanism as main candidates for involvement in seasonal timing. The genome will contribute to Lepidopteran genomic resources and comparative genomics. In addition, the genome enhances our ability to understand the genetic and molecular basis of insect seasonal timing and thereby provides a reference for future evolutionary and population studies on the winter moth. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Gene-enriched draft genome of the cattle tick Rhipicephalus microplus: Assembly by the hybrid Pacific Biosciences/Illumina approach enabled analysis of the highly repetitive genome

USDA-ARS?s Scientific Manuscript database

The genome of the cattle tick R. microplus, an ectoparasite with global distribution, is estimated to be 7.1 Gbp and consists of ~70% repetitive DNA. We report the first assembly of a tick genome that utilized a hybrid sequencing and assembly approach to capture the repetitive fractions of the genom...

GigaTON: an extensive publicly searchable database providing a new reference transcriptome in the pacific oyster Crassostrea gigas.

PubMed

Riviere, Guillaume; Klopp, Christophe; Ibouniyamine, Nabihoudine; Huvet, Arnaud; Boudry, Pierre; Favrel, Pascal

2015-12-02

The Pacific oyster, Crassostrea gigas, is one of the most important aquaculture shellfish resources worldwide. Important efforts have been undertaken towards a better knowledge of its genome and transcriptome, which makes now C. gigas becoming a model organism among lophotrochozoans, the under-described sister clade of ecdysozoans within protostomes. These massive sequencing efforts offer the opportunity to assemble gene expression data and make such resource accessible and exploitable for the scientific community. Therefore, we undertook this assembly into an up-to-date publicly available transcriptome database: the GigaTON (Gigas TranscriptOme pipeliNe) database. We assembled 2204 million sequences obtained from 114 publicly available RNA-seq libraries that were realized using all embryo-larval development stages, adult organs, different environmental stressors including heavy metals, temperature, salinity and exposure to air, which were mostly performed as part of the Crassostrea gigas genome project. This data was analyzed in silico and resulted into 56621 newly assembled contigs that were deposited into a publicly available database, the GigaTON database. This database also provides powerful and user-friendly request tools to browse and retrieve information about annotation, expression level, UTRs, splice and polymorphism, and gene ontology associated to all the contigs into each, and between all libraries. The GigaTON database provides a convenient, potent and versatile interface to browse, retrieve, confront and compare massive transcriptomic information in an extensive range of conditions, tissues and developmental stages in Crassostrea gigas. To our knowledge, the GigaTON database constitutes the most extensive transcriptomic database to date in marine invertebrates, thereby a new reference transcriptome in the oyster, a highly valuable resource to physiologists and evolutionary biologists.

A SNP resource for Douglas-fir: de novo transcriptome assembly and SNP detection and validation.

PubMed

Howe, Glenn T; Yu, Jianbin; Knaus, Brian; Cronn, Richard; Kolpak, Scott; Dolan, Peter; Lorenz, W Walter; Dean, Jeffrey F D

2013-02-28

Douglas-fir (Pseudotsuga menziesii), one of the most economically and ecologically important tree species in the world, also has one of the largest tree breeding programs. Although the coastal and interior varieties of Douglas-fir (vars. menziesii and glauca) are native to North America, the coastal variety is also widely planted for timber production in Europe, New Zealand, Australia, and Chile. Our main goal was to develop a SNP resource large enough to facilitate genomic selection in Douglas-fir breeding programs. To accomplish this, we developed a 454-based reference transcriptome for coastal Douglas-fir, annotated and evaluated the quality of the reference, identified putative SNPs, and then validated a sample of those SNPs using the Illumina Infinium genotyping platform. We assembled a reference transcriptome consisting of 25,002 isogroups (unique gene models) and 102,623 singletons from 2.76 million 454 and Sanger cDNA sequences from coastal Douglas-fir. We identified 278,979 unique SNPs by mapping the 454 and Sanger sequences to the reference, and by mapping four datasets of Illumina cDNA sequences from multiple seed sources, genotypes, and tissues. The Illumina datasets represented coastal Douglas-fir (64.00 and 13.41 million reads), interior Douglas-fir (80.45 million reads), and a Yakima population similar to interior Douglas-fir (8.99 million reads). We assayed 8067 SNPs on 260 trees using an Illumina Infinium SNP genotyping array. Of these SNPs, 5847 (72.5%) were called successfully and were polymorphic. Based on our validation efficiency, our SNP database may contain as many as ~200,000 true SNPs, and as many as ~69,000 SNPs that could be genotyped at ~20,000 gene loci using an Infinium II array-more SNPs than are needed to use genomic selection in tree breeding programs. Ultimately, these genomic resources will enhance Douglas-fir breeding and allow us to better understand landscape-scale patterns of genetic variation and potential responses to climate change.

Improving transcriptome de novo assembly by using a reference genome of a related species: Translational genomics from oil palm to coconut

PubMed Central

Armero, Alix; Bocs, Stéphanie; This, Dominique

2017-01-01

The palms are a family of tropical origin and one of the main constituents of the ecosystems of these regions around the world. The two main species of palm represent different challenges: coconut (Cocos nucifera L.) is a source of multiple goods and services in tropical communities, while oil palm (Elaeis guineensis Jacq) is the main protagonist of the oil market. In this study, we present a workflow that exploits the comparative genomics between a target species (coconut) and a reference species (oil palm) to improve the transcriptomic data, providing a proteome useful to answer functional or evolutionary questions. This workflow reduces redundancy and fragmentation, two inherent problems of transcriptomic data, while preserving the functional representation of the target species. Our approach was validated in Arabidopsis thaliana using Arabidopsis lyrata and Capsella rubella as references species. This analysis showed the high sensitivity and specificity of our strategy, relatively independent of the reference proteome. The workflow increased the length of proteins products in A. thaliana by 13%, allowing, often, to recover 100% of the protein sequence length. In addition redundancy was reduced by a factor greater than 3. In coconut, the approach generated 29,366 proteins, 1,246 of these proteins deriving from new contigs obtained with the BRANCH software. The coconut proteome presented a functional profile similar to that observed in rice and an important number of metabolic pathways related to secondary metabolism. The new sequences found with BRANCH software were enriched in functions related to biotic stress. Our strategy can be used as a complementary step to de novo transcriptome assembly to get a representative proteome of a target species. The results of the current analysis are available on the website PalmComparomics (http://palm-comparomics.southgreen.fr/). PMID:28334050

A SNP resource for Douglas-fir: de novo transcriptome assembly and SNP detection and validation

PubMed Central

2013-01-01

Background Douglas-fir (Pseudotsuga menziesii), one of the most economically and ecologically important tree species in the world, also has one of the largest tree breeding programs. Although the coastal and interior varieties of Douglas-fir (vars. menziesii and glauca) are native to North America, the coastal variety is also widely planted for timber production in Europe, New Zealand, Australia, and Chile. Our main goal was to develop a SNP resource large enough to facilitate genomic selection in Douglas-fir breeding programs. To accomplish this, we developed a 454-based reference transcriptome for coastal Douglas-fir, annotated and evaluated the quality of the reference, identified putative SNPs, and then validated a sample of those SNPs using the Illumina Infinium genotyping platform. Results We assembled a reference transcriptome consisting of 25,002 isogroups (unique gene models) and 102,623 singletons from 2.76 million 454 and Sanger cDNA sequences from coastal Douglas-fir. We identified 278,979 unique SNPs by mapping the 454 and Sanger sequences to the reference, and by mapping four datasets of Illumina cDNA sequences from multiple seed sources, genotypes, and tissues. The Illumina datasets represented coastal Douglas-fir (64.00 and 13.41 million reads), interior Douglas-fir (80.45 million reads), and a Yakima population similar to interior Douglas-fir (8.99 million reads). We assayed 8067 SNPs on 260 trees using an Illumina Infinium SNP genotyping array. Of these SNPs, 5847 (72.5%) were called successfully and were polymorphic. Conclusions Based on our validation efficiency, our SNP database may contain as many as ~200,000 true SNPs, and as many as ~69,000 SNPs that could be genotyped at ~20,000 gene loci using an Infinium II array—more SNPs than are needed to use genomic selection in tree breeding programs. Ultimately, these genomic resources will enhance Douglas-fir breeding and allow us to better understand landscape-scale patterns of genetic variation and potential responses to climate change. PMID:23445355

AGAPE (Automated Genome Analysis PipelinE) for Pan-Genome Analysis of Saccharomyces cerevisiae

PubMed Central

Song, Giltae; Dickins, Benjamin J. A.; Demeter, Janos; Engel, Stacia; Dunn, Barbara; Cherry, J. Michael

2015-01-01

The characterization and public release of genome sequences from thousands of organisms is expanding the scope for genetic variation studies. However, understanding the phenotypic consequences of genetic variation remains a challenge in eukaryotes due to the complexity of the genotype-phenotype map. One approach to this is the intensive study of model systems for which diverse sources of information can be accumulated and integrated. Saccharomyces cerevisiae is an extensively studied model organism, with well-known protein functions and thoroughly curated phenotype data. To develop and expand the available resources linking genomic variation with function in yeast, we aim to model the pan-genome of S. cerevisiae. To initiate the yeast pan-genome, we newly sequenced or re-sequenced the genomes of 25 strains that are commonly used in the yeast research community using advanced sequencing technology at high quality. We also developed a pipeline for automated pan-genome analysis, which integrates the steps of assembly, annotation, and variation calling. To assign strain-specific functional annotations, we identified genes that were not present in the reference genome. We classified these according to their presence or absence across strains and characterized each group of genes with known functional and phenotypic features. The functional roles of novel genes not found in the reference genome and associated with strains or groups of strains appear to be consistent with anticipated adaptations in specific lineages. As more S. cerevisiae strain genomes are released, our analysis can be used to collate genome data and relate it to lineage-specific patterns of genome evolution. Our new tool set will enhance our understanding of genomic and functional evolution in S. cerevisiae, and will be available to the yeast genetics and molecular biology community. PMID:25781462

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.

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.

Comparative Genomics as a Foundation for Evo-Devo Studies in Birds.

PubMed

Grayson, Phil; Sin, Simon Y W; Sackton, Timothy B; Edwards, Scott V

2017-01-01

Developmental genomics is a rapidly growing field, and high-quality genomes are a useful foundation for comparative developmental studies. A high-quality genome forms an essential reference onto which the data from numerous assays and experiments, including ChIP-seq, ATAC-seq, and RNA-seq, can be mapped. A genome also streamlines and simplifies the development of primers used to amplify putative regulatory regions for enhancer screens, cDNA probes for in situ hybridization, microRNAs (miRNAs) or short hairpin RNAs (shRNA) for RNA interference (RNAi) knockdowns, mRNAs for misexpression studies, and even guide RNAs (gRNAs) for CRISPR knockouts. Finally, much can be gleaned from comparative genomics alone, including the identification of highly conserved putative regulatory regions. This chapter provides an overview of laboratory and bioinformatics protocols for DNA extraction, library preparation, library quantification, and genome assembly, from fresh or frozen tissue to a draft avian genome. Generating a high-quality draft genome can provide a developmental research group with excellent resources for their study organism, opening the doors to many additional assays and experiments.

Multiple hybrid de novo genome assembly of finger millet, an orphan allotetraploid crop

PubMed Central

Hatakeyama, Masaomi; Aluri, Sirisha; Balachadran, Mathi Thumilan; Sivarajan, Sajeevan Radha; Patrignani, Andrea; Grüter, Simon; Poveda, Lucy; Shimizu-Inatsugi, Rie; Baeten, John; Francoijs, Kees-Jan; Nataraja, Karaba N; Reddy, Yellodu A Nanja; Phadnis, Shamprasad; Ravikumar, Ramapura L; Schlapbach, Ralph; Sreeman, Sheshshayee M; Shimizu, Kentaro K

2018-01-01

Abstract Finger millet (Eleusine coracana (L.) Gaertn) is an important crop for food security because of its tolerance to drought, which is expected to be exacerbated by global climate changes. Nevertheless, it is often classified as an orphan/underutilized crop because of the paucity of scientific attention. Among several small millets, finger millet is considered as an excellent source of essential nutrient elements, such as iron and zinc; hence, it has potential as an alternate coarse cereal. However, high-quality genome sequence data of finger millet are currently not available. One of the major problems encountered in the genome assembly of this species was its polyploidy, which hampers genome assembly compared with a diploid genome. To overcome this problem, we sequenced its genome using diverse technologies with sufficient coverage and assembled it via a novel multiple hybrid assembly workflow that combines next-generation with single-molecule sequencing, followed by whole-genome optical mapping using the Bionano Irys® system. The total number of scaffolds was 1,897 with an N50 length >2.6 Mb and detection of 96% of the universal single-copy orthologs. The majority of the homeologs were assembled separately. This indicates that the proposed workflow is applicable to the assembly of other allotetraploid genomes. PMID:28985356

First de novo whole genome sequencing and assembly of the pink-footed goose.

PubMed

Pujolar, J M; Dalén, L; Olsen, R A; Hansen, M M; Madsen, J

2018-03-01

Annotated genomes can provide new perspectives on the biology of species. We present the first de novo whole genome sequencing for the pink-footed goose. In order to obtain a high-quality de novo assembly the strategy used was to combine one short insert paired-end library with two mate-pair libraries. The pink-footed goose genome was assembled de novo using three different assemblers and an assembly evaluation was subsequently performed in order to choose the best assembler. For our data, ALLPATHS-LG performed the best, since the assembly produced covers most of the genome, while introducing the fewest errors. A total of 26,134 genes were annotated, with bird species accounting for virtually all BLAST hits. We also estimated the substitution rate in the pink-footed goose, which can be of use in future demographic studies, by using a comparative approach with the genome of the chicken, the mallard and the swan goose. A substitution rate of 1.38×10 -7 per nucleotide per generation was obtained when comparing the genomes of the two closely-related goose species (the pink-footed and the swan goose). Altogether, we provide a valuable tool for future genomic studies aiming at particular genes and regions of the pink-footed goose genome as well as other bird species. Copyright © 2017 Elsevier Inc. All rights reserved.

The A, C, G, and T of Genome Assembly.

PubMed

Wajid, Bilal; Sohail, Muhammad U; Ekti, Ali R; Serpedin, Erchin

2016-01-01

Genome assembly in its two decades of history has produced significant research, in terms of both biotechnology and computational biology. This contribution delineates sequencing platforms and their characteristics, examines key steps involved in filtering and processing raw data, explains assembly frameworks, and discusses quality statistics for the assessment of the assembled sequence. Furthermore, the paper explores recent Ubuntu-based software environments oriented towards genome assembly as well as some avenues for future research.

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

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.

Genome-wide resequencing of KRICE_CORE reveals their potential for future breeding, as well as functional and evolutionary studies in the post-genomic era.

PubMed

Kim, Tae-Sung; He, Qiang; Kim, Kyu-Won; Yoon, Min-Young; Ra, Won-Hee; Li, Feng Peng; Tong, Wei; Yu, Jie; Oo, Win Htet; Choi, Buung; Heo, Eun-Beom; Yun, Byoung-Kook; Kwon, Soon-Jae; Kwon, Soon-Wook; Cho, Yoo-Hyun; Lee, Chang-Yong; Park, Beom-Seok; Park, Yong-Jin

2016-05-26

Rice germplasm collections continue to grow in number and size around the world. Since maintaining and screening such massive resources remains challenging, it is important to establish practical methods to manage them. A core collection, by definition, refers to a subset of the entire population that preserves the majority of genetic diversity, enhancing the efficiency of germplasm utilization. Here, we report whole-genome resequencing of the 137 rice mini core collection or Korean rice core set (KRICE_CORE) that represents 25,604 rice germplasms deposited in the Korean genebank of the Rural Development Administration (RDA). We implemented the Illumina HiSeq 2000 and 2500 platform to produce short reads and then assembled those with 9.8 depths using Nipponbare as a reference. Comparisons of the sequences with the reference genome yielded more than 15 million (M) single nucleotide polymorphisms (SNPs) and 1.3 M INDELs. Phylogenetic and population analyses using 2,046,529 high-quality SNPs successfully assigned rice accessions to the relevant rice subgroups, suggesting that these SNPs capture evolutionary signatures that have accumulated in rice subpopulations. Furthermore, genome-wide association studies (GWAS) for four exemplary agronomic traits in the KRIC_CORE manifest the utility of KRICE_CORE; that is, identifying previously defined genes or novel genetic factors that potentially regulate important phenotypes. This study provides strong evidence that the size of KRICE_CORE is small but contains high genetic and functional diversity across the genome. Thus, our resequencing results will be useful for future breeding, as well as functional and evolutionary studies, in the post-genomic era.

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease

PubMed Central

Owen, Joseph R.; Noyes, Noelle; Young, Amy E.; Prince, Daniel J.; Blanchard, Patricia C.; Lehenbauer, Terry W.; Aly, Sharif S.; Davis, Jessica H.; O’Rourke, Sean M.; Abdo, Zaid; Belk, Keith; Miller, Michael R.; Morley, Paul; Van Eenennaam, Alison L.

2017-01-01

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease–associated bacterial isolates (Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis. While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni, M. haemolytica, and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% (P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes. PMID:28739600

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease.

PubMed

Owen, Joseph R; Noyes, Noelle; Young, Amy E; Prince, Daniel J; Blanchard, Patricia C; Lehenbauer, Terry W; Aly, Sharif S; Davis, Jessica H; O'Rourke, Sean M; Abdo, Zaid; Belk, Keith; Miller, Michael R; Morley, Paul; Van Eenennaam, Alison L

2017-09-07

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease-associated bacterial isolates ( Histophilus somni , Mycoplasma bovis , Mannheimia haemolytica , and Pasteurella multocida ) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni , M. haemolytica , and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% ( P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease treatment and morbidity/mortality outcomes. Copyright © 2017 Owen et al.

Strategies and tools for whole genome alignments

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

Couronne, Olivier; Poliakov, Alexander; Bray, Nicolas

2002-11-25

The availability of the assembled mouse genome makespossible, for the first time, an alignment and comparison of two largevertebrate genomes. We have investigated different strategies ofalignment for the subsequent analysis of conservation of genomes that areeffective for different quality assemblies. These strategies were appliedto the comparison of the working draft of the human genome with the MouseGenome Sequencing Consortium assembly, as well as other intermediatemouse assemblies. Our methods are fast and the resulting alignmentsexhibit a high degree of sensitivity, covering more than 90 percent ofknown coding exons in the human genome. We have obtained such coveragewhile preserving specificity. With amore » view towards the end user, we havedeveloped a suite of tools and websites for automatically aligning, andsubsequently browsing and working with whole genome comparisons. Wedescribe the use of these tools to identify conserved non-coding regionsbetween the human and mouse genomes, some of which have not beenidentified by other methods.« less

Identification of a Recently Active Mammalian SINE Derived from Ribosomal RNA

PubMed Central

Longo, Mark S.; Brown, Judy D.; Zhang, Chu; O’Neill, Michael J.; O’Neill, Rachel J.

2015-01-01

Complex eukaryotic genomes are riddled with repeated sequences whose derivation does not coincide with phylogenetic history and thus is often unknown. Among such sequences, the capacity for transcriptional activity coupled with the adaptive use of reverse transcription can lead to a diverse group of genomic elements across taxa, otherwise known as selfish elements or mobile elements. Short interspersed nuclear elements (SINEs) are nonautonomous mobile elements found in eukaryotic genomes, typically derived from cellular RNAs such as tRNAs, 7SL or 5S rRNA. Here, we identify and characterize a previously unknown SINE derived from the 3′-end of the large ribosomal subunit (LSU or 28S rDNA) and transcribed via RNA polymerase III. This new element, SINE28, is represented in low-copy numbers in the human reference genome assembly, wherein we have identified 27 discrete loci. Phylogenetic analysis indicates these elements have been transpositionally active within primate lineages as recently as 6 MYA while modern humans still carry transcriptionally active copies. Moreover, we have identified SINE28s in all currently available assembled mammalian genome sequences. Phylogenetic comparisons indicate that these elements are frequently rederived from the highly conserved LSU rRNA sequences in a lineage-specific manner. We propose that this element has not been previously recognized as a SINE given its high identity to the canonical LSU, and that SINE28 likely represents one of possibly many unidentified, active transposable elements within mammalian genomes. PMID:25637222

An ultra-high density linkage map and QTL mapping for sex and growth-related traits of common carp (Cyprinus carpio)

PubMed Central

Peng, Wenzhu; Xu, Jian; Zhang, Yan; Feng, Jianxin; Dong, Chuanju; Jiang, Likun; Feng, Jingyan; Chen, Baohua; Gong, Yiwen; Chen, Lin; Xu, Peng

2016-01-01

High density genetic linkage maps are essential for QTL fine mapping, comparative genomics and high quality genome sequence assembly. In this study, we constructed a high-density and high-resolution genetic linkage map with 28,194 SNP markers on 14,146 distinct loci for common carp based on high-throughput genotyping with the carp 250 K single nucleotide polymorphism (SNP) array in a mapping family. The genetic length of the consensus map was 10,595.94 cM with an average locus interval of 0.75 cM and an average marker interval of 0.38 cM. Comparative genomic analysis revealed high level of conserved syntenies between common carp and the closely related model species zebrafish and medaka. The genome scaffolds were anchored to the high-density linkage map, spanning 1,357 Mb of common carp reference genome. QTL mapping and association analysis identified 22 QTLs for growth-related traits and 7 QTLs for sex dimorphism. Candidate genes underlying growth-related traits were identified, including important regulators such as KISS2, IGF1, SMTLB, NPFFR1 and CPE. Candidate genes associated with sex dimorphism were also identified including 3KSR and DMRT2b. The high-density and high-resolution genetic linkage map provides an important tool for QTL fine mapping and positional cloning of economically important traits, and improving common carp genome assembly. PMID:27225429

High-coverage sequencing and annotated assembly of the genome of the Australian dragon lizard Pogona vitticeps.

PubMed

Georges, Arthur; Li, Qiye; Lian, Jinmin; O'Meally, Denis; Deakin, Janine; Wang, Zongji; Zhang, Pei; Fujita, Matthew; Patel, Hardip R; Holleley, Clare E; Zhou, Yang; Zhang, Xiuwen; Matsubara, Kazumi; Waters, Paul; Graves, Jennifer A Marshall; Sarre, Stephen D; Zhang, Guojie

2015-01-01

The lizards of the family Agamidae are one of the most prominent elements of the Australian reptile fauna. Here, we present a genomic resource built on the basis of a wild-caught male ZZ central bearded dragon Pogona vitticeps. The genomic sequence for P. vitticeps, generated on the Illumina HiSeq 2000 platform, comprised 317 Gbp (179X raw read depth) from 13 insert libraries ranging from 250 bp to 40 kbp. After filtering for low-quality and duplicated reads, 146 Gbp of data (83X) was available for assembly. Exceptionally high levels of heterozygosity (0.85 % of single nucleotide polymorphisms plus sequence insertions or deletions) complicated assembly; nevertheless, 96.4 % of reads mapped back to the assembled scaffolds, indicating that the assembly included most of the sequenced genome. Length of the assembly was 1.8 Gbp in 545,310 scaffolds (69,852 longer than 300 bp), the longest being 14.68 Mbp. N50 was 2.29 Mbp. Genes were annotated on the basis of de novo prediction, similarity to the green anole Anolis carolinensis, Gallus gallus and Homo sapiens proteins, and P. vitticeps transcriptome sequence assemblies, to yield 19,406 protein-coding genes in the assembly, 63 % of which had intact open reading frames. Our assembly captured 99 % (246 of 248) of core CEGMA genes, with 93 % (231) being complete. The quality of the P. vitticeps assembly is comparable or superior to that of other published squamate genomes, and the annotated P. vitticeps genome can be accessed through a genome browser available at https://genomics.canberra.edu.au.

Draft Sequencing of the Heterozygous Diploid Genome of Satsuma (Citrus unshiu Marc.) Using a Hybrid Assembly Approach

PubMed Central

Shimizu, Tokurou; Tanizawa, Yasuhiro; Mochizuki, Takako; Nagasaki, Hideki; Yoshioka, Terutaka; Toyoda, Atsushi; Fujiyama, Asao; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

Satsuma (Citrus unshiu Marc.) is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma (“Miyagawa Wase”) was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome. PMID:29259619

Draft Sequencing of the Heterozygous Diploid Genome of Satsuma (Citrus unshiu Marc.) Using a Hybrid Assembly Approach.

PubMed

Shimizu, Tokurou; Tanizawa, Yasuhiro; Mochizuki, Takako; Nagasaki, Hideki; Yoshioka, Terutaka; Toyoda, Atsushi; Fujiyama, Asao; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

Satsuma ( Citrus unshiu Marc.) is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma ("Miyagawa Wase") was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N 50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome.

Reducing assembly complexity of microbial genomes with single-molecule sequencing

USDA-ARS?s Scientific Manuscript database

Genome assembly algorithms cannot fully reconstruct microbial chromosomes from the DNA reads output by first or second-generation sequencing instruments. Therefore, most genomes are left unfinished due to the significant resources required to manually close gaps left in the draft assemblies. Single-...

De Novo Transcriptomic Analysis of an Oleaginous Microalga: Pathway Description and Gene Discovery for Production of Next-Generation Biofuels

PubMed Central

Wan, LingLin; Han, Juan; Sang, Min; Li, AiFen; Wu, Hong; Yin, ShunJi; Zhang, ChengWu

2012-01-01

Background Eustigmatos cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte with high biomass and considerable production of triacylglycerols (TAGs) for biofuels, which is thus referred to as an oleaginous microalga. The paucity of microalgae genome sequences, however, limits development of gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for a non-model microalgae species, E. cf. polyphem, and identify pathways and genes of importance related to biofuel production. Results We performed the de novo assembly of E. cf. polyphem transcriptome using Illumina paired-end sequencing technology. In a single run, we produced 29,199,432 sequencing reads corresponding to 2.33 Gb total nucleotides. These reads were assembled into 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp, ranging from 100 bp to >3,000 bp. Assembled unigenes were subjected to BLAST similarity searches and annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology identifiers. These analyses identified the majority of carbohydrate, fatty acids, TAG and carotenoids biosynthesis and catabolism pathways in E. cf. polyphem. Conclusions Our data provides the construction of metabolic pathways involved in the biosynthesis and catabolism of carbohydrate, fatty acids, TAG and carotenoids in E. cf. polyphem and provides a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock. PMID:22536352

MEGAnnotator: a user-friendly pipeline for microbial genomes assembly and annotation.

PubMed

Lugli, Gabriele Andrea; Milani, Christian; Mancabelli, Leonardo; van Sinderen, Douwe; Ventura, Marco

2016-04-01

Genome annotation is one of the key actions that must be undertaken in order to decipher the genetic blueprint of organisms. Thus, a correct and reliable annotation is essential in rendering genomic data valuable. Here, we describe a bioinformatics pipeline based on freely available software programs coordinated by a multithreaded script named MEGAnnotator (Multithreaded Enhanced prokaryotic Genome Annotator). This pipeline allows the generation of multiple annotated formats fulfilling the NCBI guidelines for assembled microbial genome submission, based on DNA shotgun sequencing reads, and minimizes manual intervention, while also reducing waiting times between software program executions and improving final quality of both assembly and annotation outputs. MEGAnnotator provides an efficient way to pre-arrange the assembly and annotation work required to process NGS genome sequence data. The script improves the final quality of microbial genome annotation by reducing ambiguous annotations. Moreover, the MEGAnnotator platform allows the user to perform a partial annotation of pre-assembled genomes and includes an option to accomplish metagenomic data set assemblies. MEGAnnotator platform will be useful for microbiologists interested in genome analyses of bacteria as well as those investigating the complexity of microbial communities that do not possess the necessary skills to prepare their own bioinformatics pipeline. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The A, C, G, and T of Genome Assembly

PubMed Central

Wajid, Bilal; Sohail, Muhammad U.; Ekti, Ali R.; Serpedin, Erchin

2016-01-01

Genome assembly in its two decades of history has produced significant research, in terms of both biotechnology and computational biology. This contribution delineates sequencing platforms and their characteristics, examines key steps involved in filtering and processing raw data, explains assembly frameworks, and discusses quality statistics for the assessment of the assembled sequence. Furthermore, the paper explores recent Ubuntu-based software environments oriented towards genome assembly as well as some avenues for future research. PMID:27247941

Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly

PubMed Central

Austin, Christopher M; Hammer, Michael P; Lee, Yin Peng; Gan, Han Ming

2018-01-01

Abstract Background Some of the most widely recognized coral reef fishes are clownfish or anemonefish, members of the family Pomacentridae (subfamily: Amphiprioninae). They are popular aquarium species due to their bright colours, adaptability to captivity, and fascinating behavior. Their breeding biology (sequential hermaphrodites) and symbiotic mutualism with sea anemones have attracted much scientific interest. Moreover, there are some curious geographic-based phenotypes that warrant investigation. Leveraging on the advancement in Nanopore long read technology, we report the first hybrid assembly of the clown anemonefish (Amphiprion ocellaris) genome utilizing Illumina and Nanopore reads, further demonstrating the substantial impact of modest long read sequencing data sets on improving genome assembly statistics. Results We generated 43 Gb of short Illumina reads and 9 Gb of long Nanopore reads, representing approximate genome coverage of 54× and 11×, respectively, based on the range of estimated k-mer-predicted genome sizes of between 791 and 967 Mbp. The final assembled genome is contained in 6404 scaffolds with an accumulated length of 880 Mb (96.3% BUSCO-calculated genome completeness). Compared with the Illumina-only assembly, the hybrid approach generated 94% fewer scaffolds with an 18-fold increase in N50 length (401 kb) and increased the genome completeness by an additional 16%. A total of 27 240 high-quality protein-coding genes were predicted from the clown anemonefish, 26 211 (96%) of which were annotated functionally with information from either sequence homology or protein signature searches. Conclusions We present the first genome of any anemonefish and demonstrate the value of low coverage (∼11×) long Nanopore read sequencing in improving both genome assembly contiguity and completeness. The near-complete assembly of the A. ocellaris genome will be an invaluable molecular resource for supporting a range of genetic, genomic, and phylogenetic studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae. PMID:29342277

Finding Nemo: hybrid assembly with Oxford Nanopore and Illumina reads greatly improves the clownfish (Amphiprion ocellaris) genome assembly.

PubMed

Tan, Mun Hua; Austin, Christopher M; Hammer, Michael P; Lee, Yin Peng; Croft, Laurence J; Gan, Han Ming

2018-03-01

Some of the most widely recognized coral reef fishes are clownfish or anemonefish, members of the family Pomacentridae (subfamily: Amphiprioninae). They are popular aquarium species due to their bright colours, adaptability to captivity, and fascinating behavior. Their breeding biology (sequential hermaphrodites) and symbiotic mutualism with sea anemones have attracted much scientific interest. Moreover, there are some curious geographic-based phenotypes that warrant investigation. Leveraging on the advancement in Nanopore long read technology, we report the first hybrid assembly of the clown anemonefish (Amphiprion ocellaris) genome utilizing Illumina and Nanopore reads, further demonstrating the substantial impact of modest long read sequencing data sets on improving genome assembly statistics. We generated 43 Gb of short Illumina reads and 9 Gb of long Nanopore reads, representing approximate genome coverage of 54× and 11×, respectively, based on the range of estimated k-mer-predicted genome sizes of between 791 and 967 Mbp. The final assembled genome is contained in 6404 scaffolds with an accumulated length of 880 Mb (96.3% BUSCO-calculated genome completeness). Compared with the Illumina-only assembly, the hybrid approach generated 94% fewer scaffolds with an 18-fold increase in N50 length (401 kb) and increased the genome completeness by an additional 16%. A total of 27 240 high-quality protein-coding genes were predicted from the clown anemonefish, 26 211 (96%) of which were annotated functionally with information from either sequence homology or protein signature searches. We present the first genome of any anemonefish and demonstrate the value of low coverage (∼11×) long Nanopore read sequencing in improving both genome assembly contiguity and completeness. The near-complete assembly of the A. ocellaris genome will be an invaluable molecular resource for supporting a range of genetic, genomic, and phylogenetic studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae.

Integrative structural annotation of de novo RNA-Seq provides an accurate reference gene set of the enormous genome of the onion (Allium cepa L.).

PubMed

Kim, Seungill; Kim, Myung-Shin; Kim, Yong-Min; Yeom, Seon-In; Cheong, Kyeongchae; Kim, Ki-Tae; Jeon, Jongbum; Kim, Sunggil; Kim, Do-Sun; Sohn, Seong-Han; Lee, Yong-Hwan; Choi, Doil

2015-02-01

The onion (Allium cepa L.) is one of the most widely cultivated and consumed vegetable crops in the world. Although a considerable amount of onion transcriptome data has been deposited into public databases, the sequences of the protein-coding genes are not accurate enough to be used, owing to non-coding sequences intermixed with the coding sequences. We generated a high-quality, annotated onion transcriptome from de novo sequence assembly and intensive structural annotation using the integrated structural gene annotation pipeline (ISGAP), which identified 54,165 protein-coding genes among 165,179 assembled transcripts totalling 203.0 Mb by eliminating the intron sequences. ISGAP performed reliable annotation, recognizing accurate gene structures based on reference proteins, and ab initio gene models of the assembled transcripts. Integrative functional annotation and gene-based SNP analysis revealed a whole biological repertoire of genes and transcriptomic variation in the onion. The method developed in this study provides a powerful tool for the construction of reference gene sets for organisms based solely on de novo transcriptome data. Furthermore, the reference genes and their variation described here for the onion represent essential tools for molecular breeding and gene cloning in Allium spp. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Finding the missing honey bee genes: lessons learned from a genome upgrade.

PubMed

Elsik, Christine G; Worley, Kim C; Bennett, Anna K; Beye, Martin; Camara, Francisco; Childers, Christopher P; de Graaf, Dirk C; Debyser, Griet; Deng, Jixin; Devreese, Bart; Elhaik, Eran; Evans, Jay D; Foster, Leonard J; Graur, Dan; Guigo, Roderic; Hoff, Katharina Jasmin; Holder, Michael E; Hudson, Matthew E; Hunt, Greg J; Jiang, Huaiyang; Joshi, Vandita; Khetani, Radhika S; Kosarev, Peter; Kovar, Christie L; Ma, Jian; Maleszka, Ryszard; Moritz, Robin F A; Munoz-Torres, Monica C; Murphy, Terence D; Muzny, Donna M; Newsham, Irene F; Reese, Justin T; Robertson, Hugh M; Robinson, Gene E; Rueppell, Olav; Solovyev, Victor; Stanke, Mario; Stolle, Eckart; Tsuruda, Jennifer M; Vaerenbergh, Matthias Van; Waterhouse, Robert M; Weaver, Daniel B; Whitfield, Charles W; Wu, Yuanqing; Zdobnov, Evgeny M; Zhang, Lan; Zhu, Dianhui; Gibbs, Richard A

2014-01-30

The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

Finding the missing honey bee genes: lessons learned from a genome upgrade

PubMed Central

2014-01-01

Background The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. Results Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. Conclusions Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination. PMID:24479613

Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.

PubMed

Chin, Chen-Shan; Alexander, David H; Marks, Patrick; Klammer, Aaron A; Drake, James; Heiner, Cheryl; Clum, Alicia; Copeland, Alex; Huddleston, John; Eichler, Evan E; Turner, Stephen W; Korlach, Jonas

2013-06-01

We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

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.

SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing

PubMed Central

Bankevich, Anton; Nurk, Sergey; Antipov, Dmitry; Gurevich, Alexey A.; Dvorkin, Mikhail; Kulikov, Alexander S.; Lesin, Valery M.; Nikolenko, Sergey I.; Pham, Son; Prjibelski, Andrey D.; Pyshkin, Alexey V.; Sirotkin, Alexander V.; Vyahhi, Nikolay; Tesler, Glenn; Pevzner, Pavel A.

2012-01-01

Abstract The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V−SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online (http://bioinf.spbau.ru/spades). It is distributed as open source software. PMID:22506599

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.

Multiple hybrid de novo genome assembly of finger millet, an orphan allotetraploid crop.

PubMed

Hatakeyama, Masaomi; Aluri, Sirisha; Balachadran, Mathi Thumilan; Sivarajan, Sajeevan Radha; Patrignani, Andrea; Grüter, Simon; Poveda, Lucy; Shimizu-Inatsugi, Rie; Baeten, John; Francoijs, Kees-Jan; Nataraja, Karaba N; Reddy, Yellodu A Nanja; Phadnis, Shamprasad; Ravikumar, Ramapura L; Schlapbach, Ralph; Sreeman, Sheshshayee M; Shimizu, Kentaro K

2017-09-05

Finger millet (Eleusine coracana (L.) Gaertn) is an important crop for food security because of its tolerance to drought, which is expected to be exacerbated by global climate changes. Nevertheless, it is often classified as an orphan/underutilized crop because of the paucity of scientific attention. Among several small millets, finger millet is considered as an excellent source of essential nutrient elements, such as iron and zinc; hence, it has potential as an alternate coarse cereal. However, high-quality genome sequence data of finger millet are currently not available. One of the major problems encountered in the genome assembly of this species was its polyploidy, which hampers genome assembly compared with a diploid genome. To overcome this problem, we sequenced its genome using diverse technologies with sufficient coverage and assembled it via a novel multiple hybrid assembly workflow that combines next-generation with single-molecule sequencing, followed by whole-genome optical mapping using the Bionano Irys® system. The total number of scaffolds was 1,897 with an N50 length >2.6 Mb and detection of 96% of the universal single-copy orthologs. The majority of the homeologs were assembled separately. This indicates that the proposed workflow is applicable to the assembly of other allotetraploid genomes. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

A Large Maize (Zea mays L.) SNP Genotyping Array: Development and Germplasm Genotyping, and Genetic Mapping to Compare with the B73 Reference Genome

PubMed Central

Ganal, Martin W.; Durstewitz, Gregor; Polley, Andreas; Bérard, Aurélie; Buckler, Edward S.; Charcosset, Alain; Clarke, Joseph D.; Graner, Eva-Maria; Hansen, Mark; Joets, Johann; Le Paslier, Marie-Christine; McMullen, Michael D.; Montalent, Pierre; Rose, Mark; Schön, Chris-Carolin; Sun, Qi; Walter, Hildrun; Martin, Olivier C.; Falque, Matthieu

2011-01-01

SNP genotyping arrays have been useful for many applications that require a large number of molecular markers such as high-density genetic mapping, genome-wide association studies (GWAS), and genomic selection. We report the establishment of a large maize SNP array and its use for diversity analysis and high density linkage mapping. The markers, taken from more than 800,000 SNPs, were selected to be preferentially located in genes and evenly distributed across the genome. The array was tested with a set of maize germplasm including North American and European inbred lines, parent/F1 combinations, and distantly related teosinte material. A total of 49,585 markers, including 33,417 within 17,520 different genes and 16,168 outside genes, were of good quality for genotyping, with an average failure rate of 4% and rates up to 8% in specific germplasm. To demonstrate this array's use in genetic mapping and for the independent validation of the B73 sequence assembly, two intermated maize recombinant inbred line populations – IBM (B73×Mo17) and LHRF (F2×F252) – were genotyped to establish two high density linkage maps with 20,913 and 14,524 markers respectively. 172 mapped markers were absent in the current B73 assembly and their placement can be used for future improvements of the B73 reference sequence. Colinearity of the genetic and physical maps was mostly conserved with some exceptions that suggest errors in the B73 assembly. Five major regions containing non-colinearities were identified on chromosomes 2, 3, 6, 7 and 9, and are supported by both independent genetic maps. Four additional non-colinear regions were found on the LHRF map only; they may be due to a lower density of IBM markers in those regions or to true structural rearrangements between lines. Given the array's high quality, it will be a valuable resource for maize genetics and many aspects of maize breeding. PMID:22174790

Mechanics and Physics of Solids, Uncertainy, and the Archetype-Genome Exemplar

NASA Astrophysics Data System (ADS)

Greene, M. Steven

This dissertation argues that the mechanics and physics of solids rely on a fundamental exemplar: the apparent properties of a system depend on the building blocks that comprise it. Building blocks are referred to as archetypes and apparent system properties as the system genome. Three entities are of importance: the archetype properties, the conformation of archetypes, and the properties of interactions activated by that conformation. The combination of these entities into the system genome is called assembly. To show the utility of the archetype-genome exemplar, the dissertation presents the mathematical construction and computational implementation of a new theory for solid mechanics that is a continuum manifestation of the assembly process. The so-called archetype-blending continuum theory aligns the form of globally valid balance laws with physics evolving in a material's composite constitutive response so that, by rethinking conventional micromechanics, the theory accounts naturally for each piece of the genome assembly triplet: archetypes, interactions, and their conformation. With the pieces of the triplet isolated in the theory, materials genome design concepts that separately control microstructure and property may be gleaned from exploration of the constitutive parameter space. A suite of simulations that apply the new theory to polymer nanocomposite materials demonstrate the ability of the theory to predict a robust material genome that includes damping properties, modulus weakening, local strain amplification, and size effects. The dissertation also presents a theoretical assessment of the importance of uncertainty propagation in the archetype-genome exemplar. The findings from a set of computational experiments on instances of a general class of microstructured materials suggest that when overlap occurs between the size of the system geometry and the features of the conformation, material genomes become less certain. Increasing nonuniformity of boundary conditions and the size of random field correlation lengths exacerbate this conclusion. These criteria are combined into a scalar metric used to assess the impact of archetype-level uncertainties on the material genome for general scenarios in solid mechanics. Exemplary benchmark problems include bending in elastoplasticity and instability-induced pattern transition in porous elastomer. The contributions of this dissertation are threefold: (1) the mathematical construction of a new continuum theory for mechanics and physics of solids, (2) implementation of the theory, and (3) theoretical assessment of the scenarios in which material genomes deviate from determinism.

A new chicken genome assembly provides insight into avian genome structure

USDA-ARS?s Scientific Manuscript database

The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3) built from combined long single molecule sequencing t...

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

PubMed

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

2015-01-01

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

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

PubMed Central

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

2013-01-01

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

Combinatorial pooling enables selective sequencing of the barley gene space.

PubMed

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J

2013-04-01

For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

Genome Annotation Generator: a simple tool for generating and correcting WGS annotation tables for NCBI submission.

PubMed

Geib, Scott M; Hall, Brian; Derego, Theodore; Bremer, Forest T; Cannoles, Kyle; Sim, Sheina B

2018-04-01

One of the most overlooked, yet critical, components of a whole genome sequencing (WGS) project is the submission and curation of the data to a genomic repository, most commonly the National Center for Biotechnology Information (NCBI). While large genome centers or genome groups have developed software tools for post-annotation assembly filtering, annotation, and conversion into the NCBI's annotation table format, these tools typically require back-end setup and connection to an Structured Query Language (SQL) database and/or some knowledge of programming (Perl, Python) to implement. With WGS becoming commonplace, genome sequencing projects are moving away from the genome centers and into the ecology or biology lab, where fewer resources are present to support the process of genome assembly curation. To fill this gap, we developed software to assess, filter, and transfer annotation and convert a draft genome assembly and annotation set into the NCBI annotation table (.tbl) format, facilitating submission to the NCBI Genome Assembly database. This software has no dependencies, is compatible across platforms, and utilizes a simple command to perform a variety of simple and complex post-analysis, pre-NCBI submission WGS project tasks. The Genome Annotation Generator is a consistent and user-friendly bioinformatics tool that can be used to generate a .tbl file that is consistent with the NCBI submission pipeline. The Genome Annotation Generator achieves the goal of providing a publicly available tool that will facilitate the submission of annotated genome assemblies to the NCBI. It is useful for any individual researcher or research group that wishes to submit a genome assembly of their study system to the NCBI.

Genome Annotation Generator: a simple tool for generating and correcting WGS annotation tables for NCBI submission

PubMed Central

Hall, Brian; Derego, Theodore; Bremer, Forest T; Cannoles, Kyle

2018-01-01

Abstract Background One of the most overlooked, yet critical, components of a whole genome sequencing (WGS) project is the submission and curation of the data to a genomic repository, most commonly the National Center for Biotechnology Information (NCBI). While large genome centers or genome groups have developed software tools for post-annotation assembly filtering, annotation, and conversion into the NCBI’s annotation table format, these tools typically require back-end setup and connection to an Structured Query Language (SQL) database and/or some knowledge of programming (Perl, Python) to implement. With WGS becoming commonplace, genome sequencing projects are moving away from the genome centers and into the ecology or biology lab, where fewer resources are present to support the process of genome assembly curation. To fill this gap, we developed software to assess, filter, and transfer annotation and convert a draft genome assembly and annotation set into the NCBI annotation table (.tbl) format, facilitating submission to the NCBI Genome Assembly database. This software has no dependencies, is compatible across platforms, and utilizes a simple command to perform a variety of simple and complex post-analysis, pre-NCBI submission WGS project tasks. Findings The Genome Annotation Generator is a consistent and user-friendly bioinformatics tool that can be used to generate a .tbl file that is consistent with the NCBI submission pipeline Conclusions The Genome Annotation Generator achieves the goal of providing a publicly available tool that will facilitate the submission of annotated genome assemblies to the NCBI. It is useful for any individual researcher or research group that wishes to submit a genome assembly of their study system to the NCBI. PMID:29635297

Toward allotetraploid cotton genome assembly: integration of a high-density molecular genetic linkage map with DNA sequence information

PubMed Central

2012-01-01

Background Cotton is the world’s most important natural textile fiber and a significant oilseed crop. Decoding cotton genomes will provide the ultimate reference and resource for research and utilization of the species. Integration of high-density genetic maps with genomic sequence information will largely accelerate the process of whole-genome assembly in cotton. Results In this paper, we update a high-density interspecific genetic linkage map of allotetraploid cultivated cotton. An additional 1,167 marker loci have been added to our previously published map of 2,247 loci. Three new marker types, InDel (insertion-deletion) and SNP (single nucleotide polymorphism) developed from gene information, and REMAP (retrotransposon-microsatellite amplified polymorphism), were used to increase map density. The updated map consists of 3,414 loci in 26 linkage groups covering 3,667.62 cM with an average inter-locus distance of 1.08 cM. Furthermore, genome-wide sequence analysis was finished using 3,324 informative sequence-based markers and publicly-available Gossypium DNA sequence information. A total of 413,113 EST and 195 BAC sequences were physically anchored and clustered by 3,324 sequence-based markers. Of these, 14,243 ESTs and 188 BACs from different species of Gossypium were clustered and specifically anchored to the high-density genetic map. A total of 2,748 candidate unigenes from 2,111 ESTs clusters and 63 BACs were mined for functional annotation and classification. The 337 ESTs/genes related to fiber quality traits were integrated with 132 previously reported cotton fiber quality quantitative trait loci, which demonstrated the important roles in fiber quality of these genes. Higher-level sequence conservation between different cotton species and between the A- and D-subgenomes in tetraploid cotton was found, indicating a common evolutionary origin for orthologous and paralogous loci in Gossypium. Conclusion This study will serve as a valuable genomic resource for tetraploid cotton genome assembly, for cloning genes related to superior agronomic traits, and for further comparative genomic analyses in Gossypium. PMID:23046547

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.

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).

De novo assembly of the pepper transcriptome (Capsicum annuum): a benchmark for in silico discovery of SNPs, SSRs and candidate genes.

PubMed

Ashrafi, Hamid; Hill, Theresa; Stoffel, Kevin; Kozik, Alexander; Yao, Jiqiang; Chin-Wo, Sebastian Reyes; Van Deynze, Allen

2012-10-30

Molecular breeding of pepper (Capsicum spp.) can be accelerated by developing DNA markers associated with transcriptomes in breeding germplasm. Before the advent of next generation sequencing (NGS) technologies, the majority of sequencing data were generated by the Sanger sequencing method. By leveraging Sanger EST data, we have generated a wealth of genetic information for pepper including thousands of SNPs and Single Position Polymorphic (SPP) markers. To complement and enhance these resources, we applied NGS to three pepper genotypes: Maor, Early Jalapeño and Criollo de Morelos-334 (CM334) to identify SNPs and SSRs in the assembly of these three genotypes. Two pepper transcriptome assemblies were developed with different purposes. The first reference sequence, assembled by CAP3 software, comprises 31,196 contigs from >125,000 Sanger-EST sequences that were mainly derived from a Korean F1-hybrid line, Bukang. Overlapping probes were designed for 30,815 unigenes to construct a pepper Affymetrix GeneChip® microarray for whole genome analyses. In addition, custom Python scripts were used to identify 4,236 SNPs in contigs of the assembly. A total of 2,489 simple sequence repeats (SSRs) were identified from the assembly, and primers were designed for the SSRs. Annotation of contigs using Blast2GO software resulted in information for 60% of the unigenes in the assembly. The second transcriptome assembly was constructed from more than 200 million Illumina Genome Analyzer II reads (80-120 nt) using a combination of Velvet, CLC workbench and CAP3 software packages. BWA, SAMtools and in-house Perl scripts were used to identify SNPs among three pepper genotypes. The SNPs were filtered to be at least 50 bp from any intron-exon junctions as well as flanking SNPs. More than 22,000 high-quality putative SNPs were identified. Using the MISA software, 10,398 SSR markers were also identified within the Illumina transcriptome assembly and primers were designed for the identified markers. The assembly was annotated by Blast2GO and 14,740 (12%) of annotated contigs were associated with functional proteins. Before availability of pepper genome sequence, assembling transcriptomes of this economically important crop was required to generate thousands of high-quality molecular markers that could be used in breeding programs. In order to have a better understanding of the assembled sequences and to identify candidate genes underlying QTLs, we annotated the contigs of Sanger-EST and Illumina transcriptome assemblies. These and other information have been curated in a database that we have dedicated for pepper project.

When Whole-Genome Alignments Just Won't Work: kSNP v2 Software for Alignment-Free SNP Discovery and Phylogenetics of Hundreds of Microbial Genomes

PubMed Central

Gardner, Shea N.; Hall, Barry G.

2013-01-01

Effective use of rapid and inexpensive whole genome sequencing for microbes requires fast, memory efficient bioinformatics tools for sequence comparison. The kSNP v2 software finds single nucleotide polymorphisms (SNPs) in whole genome data. kSNP v2 has numerous improvements over kSNP v1 including SNP gene annotation; better scaling for draft genomes available as assembled contigs or raw, unassembled reads; a tool to identify the optimal value of k; distribution of packages of executables for Linux and Mac OS X for ease of installation and user-friendly use; and a detailed User Guide. SNP discovery is based on k-mer analysis, and requires no multiple sequence alignment or the selection of a single reference genome. Most target sets with hundreds of genomes complete in minutes to hours. SNP phylogenies are built by maximum likelihood, parsimony, and distance, based on all SNPs, only core SNPs, or SNPs present in some intermediate user-specified fraction of targets. The SNP-based trees that result are consistent with known taxonomy. kSNP v2 can handle many gigabases of sequence in a single run, and if one or more annotated genomes are included in the target set, SNPs are annotated with protein coding and other information (UTRs, etc.) from Genbank file(s). We demonstrate application of kSNP v2 on sets of viral and bacterial genomes, and discuss in detail analysis of a set of 68 finished E. coli and Shigella genomes and a set of the same genomes to which have been added 47 assemblies and four “raw read” genomes of H104:H4 strains from the recent European E. coli outbreak that resulted in both bloody diarrhea and hemolytic uremic syndrome (HUS), and caused at least 50 deaths. PMID:24349125

When whole-genome alignments just won't work: kSNP v2 software for alignment-free SNP discovery and phylogenetics of hundreds of microbial genomes.

PubMed

Gardner, Shea N; Hall, Barry G

2013-01-01

Effective use of rapid and inexpensive whole genome sequencing for microbes requires fast, memory efficient bioinformatics tools for sequence comparison. The kSNP v2 software finds single nucleotide polymorphisms (SNPs) in whole genome data. kSNP v2 has numerous improvements over kSNP v1 including SNP gene annotation; better scaling for draft genomes available as assembled contigs or raw, unassembled reads; a tool to identify the optimal value of k; distribution of packages of executables for Linux and Mac OS X for ease of installation and user-friendly use; and a detailed User Guide. SNP discovery is based on k-mer analysis, and requires no multiple sequence alignment or the selection of a single reference genome. Most target sets with hundreds of genomes complete in minutes to hours. SNP phylogenies are built by maximum likelihood, parsimony, and distance, based on all SNPs, only core SNPs, or SNPs present in some intermediate user-specified fraction of targets. The SNP-based trees that result are consistent with known taxonomy. kSNP v2 can handle many gigabases of sequence in a single run, and if one or more annotated genomes are included in the target set, SNPs are annotated with protein coding and other information (UTRs, etc.) from Genbank file(s). We demonstrate application of kSNP v2 on sets of viral and bacterial genomes, and discuss in detail analysis of a set of 68 finished E. coli and Shigella genomes and a set of the same genomes to which have been added 47 assemblies and four "raw read" genomes of H104:H4 strains from the recent European E. coli outbreak that resulted in both bloody diarrhea and hemolytic uremic syndrome (HUS), and caused at least 50 deaths.

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

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

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

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

A novel genome-wide microsatellite resource for species of Eucalyptus with linkage-to-physical correspondence on the reference genome sequence.

PubMed

Grattapaglia, Dario; Mamani, Eva M C; Silva-Junior, Orzenil B; Faria, Danielle A

2015-03-01

Keystone species in their native ranges, eucalypts, are ecologically and genetically very diverse, growing naturally along extensive latitudinal and altitudinal ranges and variable environments. Besides their ecological importance, eucalypts are also the most widely planted trees for sustainable forestry in the world. We report the development of a novel collection of 535 microsatellites for species of Eucalyptus, 494 designed from ESTs and 41 from genomic libraries. A selected subset of 223 was evaluated for individual identification, parentage testing, and ancestral information content in the two most extensively studied species, Eucalyptus grandis and Eucalyptus globulus. Microsatellites showed high transferability and overlapping allele size range, suggesting they have arisen still in their common ancestor and confirming the extensive genome conservation between these two species. A consensus linkage map with 437 microsatellites, the most comprehensive microsatellite-only genetic map for Eucalyptus, was built by assembling segregation data from three mapping populations and anchored to the Eucalyptus genome. An overall colinearity between recombination-based and physical positioning of 84% of the mapped microsatellites was observed, with some ordering discrepancies and sporadic locus duplications, consistent with the recently described whole genome duplication events in Eucalyptus. The linkage map covered 95.2% of the 605.8-Mbp assembled genome sequence, placing one microsatellite every 1.55 Mbp on average, and an overall estimate of physical to recombination distance of 618 kbp/cM. The genetic parameters estimates together with linkage and physical position data for this large set of microsatellites should assist marker choice for genome-wide population genetics and comparative mapping in Eucalyptus. © 2014 John Wiley & Sons Ltd.

Complete mitochondrial genome of Lutzomyia (Nyssomyia) umbratilis (Diptera: Psychodidae), the main vector of Leishmania guyanensis.

PubMed

Kocher, Arthur; Gantier, Jean-Charles; Holota, Hélène; Jeziorski, Céline; Coissac, Eric; Bañuls, Anne-Laure; Girod, Romain; Gaborit, Pascal; Murienne, Jérôme

2016-11-01

The nearly complete mitochondrial genome of Lutzomyia umbratilis Ward & Fraiha, 1977 (Psychodidae: Phlebotominae), considered as the main vector of Leishmania guyanensis, is presented. The sequencing has been performed on an Illumina Hiseq 2500 platform, with a genome skimming strategy. The full nuclear ribosomal RNA segment was also assembled. The mitogenome of L. umbratilis was determined to be at least 15,717 bp-long and presents an architecture found in many mitogenomes of insect (13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and one non-coding region also referred as the control region). The control region contains a large repeated element of c. 370 bp and a poly-AT region of unknown length. This is the first mitogenome of Psychodidae to be described.

Whole-Genome Sequences of DA and F344 Rats with Different Susceptibilities to Arthritis, Autoimmunity, Inflammation and Cancer

PubMed Central

Guo, Xiaosen; Brenner, Max; Zhang, Xuemei; Laragione, Teresina; Tai, Shuaishuai; Li, Yanhong; Bu, Junjie; Yin, Ye; Shah, Anish A.; Kwan, Kevin; Li, Yingrui; Jun, Wang; Gulko, Pércio S.

2013-01-01

DA (D-blood group of Palm and Agouti, also known as Dark Agouti) and F344 (Fischer) are two inbred rat strains with differences in several phenotypes, including susceptibility to autoimmune disease models and inflammatory responses. While these strains have been extensively studied, little information is available about the DA and F344 genomes, as only the Brown Norway (BN) and spontaneously hypertensive rat strains have been sequenced to date. Here we report the sequencing of the DA and F344 genomes using next-generation Illumina paired-end read technology and the first de novo assembly of a rat genome. DA and F344 were sequenced with an average depth of 32-fold, covered 98.9% of the BN reference genome, and included 97.97% of known rat ESTs. New sequences could be assigned to 59 million positions with previously unknown data in the BN reference genome. Differences between DA, F344, and BN included 19 million positions in novel scaffolds, 4.09 million single nucleotide polymorphisms (SNPs) (including 1.37 million new SNPs), 458,224 short insertions and deletions, and 58,174 structural variants. Genetic differences between DA, F344, and BN, including high-impact SNPs and short insertions and deletions affecting >2500 genes, are likely to account for most of the phenotypic variation between these strains. The new DA and F344 genome sequencing data should facilitate gene discovery efforts in rat models of human disease. PMID:23695301

Whole-genome sequences of DA and F344 rats with different susceptibilities to arthritis, autoimmunity, inflammation and cancer.

PubMed

Guo, Xiaosen; Brenner, Max; Zhang, Xuemei; Laragione, Teresina; Tai, Shuaishuai; Li, Yanhong; Bu, Junjie; Yin, Ye; Shah, Anish A; Kwan, Kevin; Li, Yingrui; Jun, Wang; Gulko, Pércio S

2013-08-01

DA (D-blood group of Palm and Agouti, also known as Dark Agouti) and F344 (Fischer) are two inbred rat strains with differences in several phenotypes, including susceptibility to autoimmune disease models and inflammatory responses. While these strains have been extensively studied, little information is available about the DA and F344 genomes, as only the Brown Norway (BN) and spontaneously hypertensive rat strains have been sequenced to date. Here we report the sequencing of the DA and F344 genomes using next-generation Illumina paired-end read technology and the first de novo assembly of a rat genome. DA and F344 were sequenced with an average depth of 32-fold, covered 98.9% of the BN reference genome, and included 97.97% of known rat ESTs. New sequences could be assigned to 59 million positions with previously unknown data in the BN reference genome. Differences between DA, F344, and BN included 19 million positions in novel scaffolds, 4.09 million single nucleotide polymorphisms (SNPs) (including 1.37 million new SNPs), 458,224 short insertions and deletions, and 58,174 structural variants. Genetic differences between DA, F344, and BN, including high-impact SNPs and short insertions and deletions affecting >2500 genes, are likely to account for most of the phenotypic variation between these strains. The new DA and F344 genome sequencing data should facilitate gene discovery efforts in rat models of human disease.

Comparative genomic data of the Avian Phylogenomics Project.

PubMed

Zhang, Guojie; Li, Bo; Li, Cai; Gilbert, M Thomas P; Jarvis, Erich D; Wang, Jun

2014-01-01

The evolutionary relationships of modern birds are among the most challenging to understand in systematic biology and have been debated for centuries. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders, and used the genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomics analyses (Jarvis et al. in press; Zhang et al. in press). Here we release assemblies and datasets associated with the comparative genome analyses, which include 38 newly sequenced avian genomes plus previously released or simultaneously released genomes of Chicken, Zebra finch, Turkey, Pigeon, Peregrine falcon, Duck, Budgerigar, Adelie penguin, Emperor penguin and the Medium Ground Finch. We hope that this resource will serve future efforts in phylogenomics and comparative genomics. The 38 bird genomes were sequenced using the Illumina HiSeq 2000 platform and assembled using a whole genome shotgun strategy. The 48 genomes were categorized into two groups according to the N50 scaffold size of the assemblies: a high depth group comprising 23 species sequenced at high coverage (>50X) with multiple insert size libraries resulting in N50 scaffold sizes greater than 1 Mb (except the White-throated Tinamou and Bald Eagle); and a low depth group comprising 25 species sequenced at a low coverage (~30X) with two insert size libraries resulting in an average N50 scaffold size of about 50 kb. Repetitive elements comprised 4%-22% of the bird genomes. The assembled scaffolds allowed the homology-based annotation of 13,000 ~ 17000 protein coding genes in each avian genome relative to chicken, zebra finch and human, as well as comparative and sequence conservation analyses. Here we release full genome assemblies of 38 newly sequenced avian species, link genome assembly downloads for the 7 of the remaining 10 species, and provide a guideline of genomic data that has been generated and used in our Avian Phylogenomics Project. To the best of our knowledge, the Avian Phylogenomics Project is the biggest vertebrate comparative genomics project to date. The genomic data presented here is expected to accelerate further analyses in many fields, including phylogenetics, comparative genomics, evolution, neurobiology, development biology, and other related areas.

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

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

Zhou, Xiaofan; Peris, David; Kominek, Jacek

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

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

DOE PAGES

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

2016-09-16

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

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

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

Haplotype assembly in polyploid genomes and identical by descent shared tracts.

PubMed

Aguiar, Derek; Istrail, Sorin

2013-07-01

Genome-wide haplotype reconstruction from sequence data, or haplotype assembly, is at the center of major challenges in molecular biology and life sciences. For complex eukaryotic organisms like humans, the genome is vast and the population samples are growing so rapidly that algorithms processing high-throughput sequencing data must scale favorably in terms of both accuracy and computational efficiency. Furthermore, current models and methodologies for haplotype assembly (i) do not consider individuals sharing haplotypes jointly, which reduces the size and accuracy of assembled haplotypes, and (ii) are unable to model genomes having more than two sets of homologous chromosomes (polyploidy). Polyploid organisms are increasingly becoming the target of many research groups interested in the genomics of disease, phylogenetics, botany and evolution but there is an absence of theory and methods for polyploid haplotype reconstruction. In this work, we present a number of results, extensions and generalizations of compass graphs and our HapCompass framework. We prove the theoretical complexity of two haplotype assembly optimizations, thereby motivating the use of heuristics. Furthermore, we present graph theory-based algorithms for the problem of haplotype assembly using our previously developed HapCompass framework for (i) novel implementations of haplotype assembly optimizations (minimum error correction), (ii) assembly of a pair of individuals sharing a haplotype tract identical by descent and (iii) assembly of polyploid genomes. We evaluate our methods on 1000 Genomes Project, Pacific Biosciences and simulated sequence data. HapCompass is available for download at http://www.brown.edu/Research/Istrail_Lab/. Supplementary data are available at Bioinformatics online.

Safety analysis of a Russian phage cocktail: From MetaGenomic analysis to oral application in healthy human subjects

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

McCallin, Shawna, E-mail: semccallin@yahoo.com; Alam Sarker, Shafiqul, E-mail: sasarker@icddrb.org; Barretto, Caroline, E-mail: Caroline.Barretto@rdls.nestle.com

Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb,more » including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure. - Highlights: • We analyzed the composition of a commercial Russian phage cocktail. • The cocktail consists of at least 10 different phage genera. • No undesired genes were detected. • No adverse effects were seen upon oral application in a small human clinical trial.« less

Assessing the Gene Content of the Megagenome: Sugar Pine (Pinus lambertiana)

PubMed Central

Gonzalez-Ibeas, Daniel; Martinez-Garcia, Pedro J.; Famula, Randi A.; Delfino-Mix, Annette; Stevens, Kristian A.; Loopstra, Carol A.; Langley, Charles H.; Neale, David B.; Wegrzyn, Jill L.

2016-01-01

Sugar pine (Pinus lambertiana Douglas) is within the subgenus Strobus with an estimated genome size of 31 Gbp. Transcriptomic resources are of particular interest in conifers due to the challenges presented in their megagenomes for gene identification. In this study, we present the first comprehensive survey of the P. lambertiana transcriptome through deep sequencing of a variety of tissue types to generate more than 2.5 billion short reads. Third generation, long reads generated through PacBio Iso-Seq have been included for the first time in conifers to combat the challenges associated with de novo transcriptome assembly. A technology comparison is provided here to contribute to the otherwise scarce comparisons of second and third generation transcriptome sequencing approaches in plant species. In addition, the transcriptome reference was essential for gene model identification and quality assessment in the parallel project responsible for sequencing and assembly of the entire genome. In this study, the transcriptomic data were also used to address questions surrounding lineage-specific Dicer-like proteins in conifers. These proteins play a role in the control of transposable element proliferation and the related genome expansion in conifers. PMID:27799338

Assembly and comparison of two closely related Brassica napus genomes.

PubMed

Bayer, Philipp E; Hurgobin, Bhavna; Golicz, Agnieszka A; Chan, Chon-Kit Kenneth; Yuan, Yuxuan; Lee, HueyTyng; Renton, Michael; Meng, Jinling; Li, Ruiyuan; Long, Yan; Zou, Jun; Bancroft, Ian; Chalhoub, Boulos; King, Graham J; Batley, Jacqueline; Edwards, David

2017-12-01

As an increasing number of plant genome sequences become available, it is clear that gene content varies between individuals, and the challenge arises to predict the gene content of a species. However, genome comparison is often confounded by variation in assembly and annotation. Differentiating between true gene absence and variation in assembly or annotation is essential for the accurate identification of conserved and variable genes in a species. Here, we present the de novo assembly of the B. napus cultivar Tapidor and comparison with an improved assembly of the Brassica napus cultivar Darmor-bzh. Both cultivars were annotated using the same method to allow comparison of gene content. We identified genes unique to each cultivar and differentiate these from artefacts due to variation in the assembly and annotation. We demonstrate that using a common annotation pipeline can result in different gene predictions, even for closely related cultivars, and repeat regions which collapse during assembly impact whole genome comparison. After accounting for differences in assembly and annotation, we demonstrate that the genome of Darmor-bzh contains a greater number of genes than the genome of Tapidor. Our results are the first step towards comparison of the true differences between B. napus genomes and highlight the potential sources of error in future production of a B. napus pangenome. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Detection and correction of false segmental duplications caused by genome mis-assembly

PubMed Central

2010-01-01

Diploid genomes with divergent chromosomes present special problems for assembly software as two copies of especially polymorphic regions may be mistakenly constructed, creating the appearance of a recent segmental duplication. We developed a method for identifying such false duplications and applied it to four vertebrate genomes. For each genome, we corrected mis-assemblies, improved estimates of the amount of duplicated sequence, and recovered polymorphisms between the sequenced chromosomes. PMID:20219098

High Quality Maize Centromere 10 Sequence Reveals Evidence of Frequent Recombination Events

PubMed Central

Wolfgruber, Thomas K.; Nakashima, Megan M.; Schneider, Kevin L.; Sharma, Anupma; Xie, Zidian; Albert, Patrice S.; Xu, Ronghui; Bilinski, Paul; Dawe, R. Kelly; Ross-Ibarra, Jeffrey; Birchler, James A.; Presting, Gernot G.

2016-01-01

The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR) has presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here, we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 × 10−6 and 5 × 10−5 for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb from the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length CR from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB) repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. In many cases examined here, DSB repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to efficiently repair frequent DSBs in centromeres. PMID:27047500

RNA-Seq Analysis Using De Novo Transcriptome Assembly as a Reference for the Salmon Louse Caligus rogercresseyi

PubMed Central

Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Nuñez-Acuña, Gustavo

2014-01-01

Despite the economic and environmental impacts that sea lice infestations have on salmon farming worldwide, genomic data generated by high-throughput transcriptome sequencing for different developmental stages, sexes, and strains of sea lice is still limited or unknown. In this study, RNA-seq analysis was performed using de novo transcriptome assembly as a reference for evidenced transcriptional changes from six developmental stages of the salmon louse Caligus rogercresseyi. EST-datasets were generated from the nauplius I, nauplius II, copepodid and chalimus stages and from female and male adults using MiSeq Illumina sequencing. A total of 151,788,682 transcripts were yielded, which were assembled into 83,444 high quality contigs and subsequently annotated into roughly 24,000 genes based on known proteins. To identify differential transcription patterns among salmon louse stages, cluster analyses were performed using normalized gene expression values. Herein, four clusters were differentially expressed between nauplius I–II and copepodid stages (604 transcripts), five clusters between copepodid and chalimus stages (2,426 transcripts), and six clusters between female and male adults (2,478 transcripts). Gene ontology analysis revealed that the nauplius I–II, copepodid and chalimus stages are mainly annotated to aminoacid transfer/repair/breakdown, metabolism, molting cycle, and nervous system development. Additionally, genes showing differential transcription in female and male adults were highly related to cytoskeletal and contractile elements, reproduction, cell development, morphogenesis, and transcription-translation processes. The data presented in this study provides the most comprehensive transcriptome resource available for C. rogercresseyi, which should be used for future genomic studies linked to host-parasite interactions. PMID:24691066

RNA-Seq analysis using de novo transcriptome assembly as a reference for the salmon louse Caligus rogercresseyi.

PubMed

Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Nuñez-Acuña, Gustavo

2014-01-01

Despite the economic and environmental impacts that sea lice infestations have on salmon farming worldwide, genomic data generated by high-throughput transcriptome sequencing for different developmental stages, sexes, and strains of sea lice is still limited or unknown. In this study, RNA-seq analysis was performed using de novo transcriptome assembly as a reference for evidenced transcriptional changes from six developmental stages of the salmon louse Caligus rogercresseyi. EST-datasets were generated from the nauplius I, nauplius II, copepodid and chalimus stages and from female and male adults using MiSeq Illumina sequencing. A total of 151,788,682 transcripts were yielded, which were assembled into 83,444 high quality contigs and subsequently annotated into roughly 24,000 genes based on known proteins. To identify differential transcription patterns among salmon louse stages, cluster analyses were performed using normalized gene expression values. Herein, four clusters were differentially expressed between nauplius I-II and copepodid stages (604 transcripts), five clusters between copepodid and chalimus stages (2,426 transcripts), and six clusters between female and male adults (2,478 transcripts). Gene ontology analysis revealed that the nauplius I-II, copepodid and chalimus stages are mainly annotated to aminoacid transfer/repair/breakdown, metabolism, molting cycle, and nervous system development. Additionally, genes showing differential transcription in female and male adults were highly related to cytoskeletal and contractile elements, reproduction, cell development, morphogenesis, and transcription-translation processes. The data presented in this study provides the most comprehensive transcriptome resource available for C. rogercresseyi, which should be used for future genomic studies linked to host-parasite interactions.

De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.

PubMed

Haas, Brian J; Papanicolaou, Alexie; Yassour, Moran; Grabherr, Manfred; Blood, Philip D; Bowden, Joshua; Couger, Matthew Brian; Eccles, David; Li, Bo; Lieber, Matthias; MacManes, Matthew D; Ott, Michael; Orvis, Joshua; Pochet, Nathalie; Strozzi, Francesco; Weeks, Nathan; Westerman, Rick; William, Thomas; Dewey, Colin N; Henschel, Robert; LeDuc, Richard D; Friedman, Nir; Regev, Aviv

2013-08-01

De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.

Safety analysis of a Russian phage cocktail: from metagenomic analysis to oral application in healthy human subjects.

PubMed

McCallin, Shawna; Alam Sarker, Shafiqul; Barretto, Caroline; Sultana, Shamima; Berger, Bernard; Huq, Sayeda; Krause, Lutz; Bibiloni, Rodrigo; Schmitt, Bertrand; Reuteler, Gloria; Brüssow, Harald

2013-09-01

Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb, including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Evaluation and validation of de novo and hybrid assembly techniques to derive high quality genome sequences

DOE PAGES

Utturkar, Sagar M.; Klingeman, Dawn Marie; Land, Miriam L.; ...

2014-06-14

Our motivation with this work was to assess the potential of different types of sequence data combined with de novo and hybrid assembly approaches to improve existing draft genome sequences. Our results show Illumina, 454 and PacBio sequencing technologies were used to generate de novo and hybrid genome assemblies for four different bacteria, which were assessed for quality using summary statistics (e.g. number of contigs, N50) and in silico evaluation tools. Differences in predictions of multiple copies of rDNA operons for each respective bacterium were evaluated by PCR and Sanger sequencing, and then the validated results were applied as anmore » additional criterion to rank assemblies. In general, assemblies using longer PacBio reads were better able to resolve repetitive regions. In this study, the combination of Illumina and PacBio sequence data assembled through the ALLPATHS-LG algorithm gave the best summary statistics and most accurate rDNA operon number predictions. This study will aid others looking to improve existing draft genome assemblies. As to availability and implementation–all assembly tools except CLC Genomics Workbench are freely available under GNU General Public License.« less

The genome of black cottonwood, Populus trichocarpa (Torr. & Gray)

Treesearch

G.A. Tuskan; S. DiFazio; S. Jansson; J. Bohlmann; I. Grigoriev; U. Hellsten; N. Putnam; S. Ralph; S. Rombauts; A. Salamov; J. Schein; L. Sterck; A. Aerts; R.R. Bhalerao; R.P. Bhalerao; D. Blaudez; W. Boerjan; A. Brun; A. Brunner; V. Busov; M. Campbell; J. Carlson; M. Chalot; J. Chapman; G.-L. Chen; D. Cooper; P.M. Coutinho; J. Couturier; S. Covert; Q. Cronk; R. Cunningham; J. Davis; S. Degroeve; A. Dejardin; C. dePamphilis; J. Detter; B. Dirks; U. Dubchak; S. Duplessis; J. Ehlting; B. Ellis; K. Gendler; D. Goodstein; M. Gribskov; J. Grimwood; A. Groover; L. Gunter; B. Hamberger; B. Heinze; Y. Helariutta; B. Henrissat; D. Holligan; R. Holt; W. Huang; N. Islam-Faridi; S. Jones; M. Jones-Rhoades; R. Jorgensen; C. Joshi; J. Kangasjarvi; J. Karlsson; C. Kelleher; R. Kirkpatrick; M. Kirst; A. Kohler; U. Kalluri; F. Larimer; J. Leebens-Mack; J.-C. Leple; P. Locascio; Y. Lou; S. Lucas; F. Martin; B. Montanini; C. Napoli; D.R. Nelson; C. Nelson; K. Nieminen; O. Nilsson; V. Pereda; G. Peter; R. Philippe; G. Pilate; A. Poliakov; J. Razumovskaya; P. Richardson; C. Rinaldi; K. Ritland; P. Rouze; D. Ryaboy; J. Schumtz; J. Schrader; B. Segerman; H. Shin; A. Siddiqui; F. Sterky; A. Terry; C.-J. Tsai; E. Uberbacher; P. Unneberg; J. Vahala; K. Wall; S. Wessler; G. Yang; T. Yin; C. Douglas; M. Marra; G. Sandberg; Y. Van de Peer; D. Rokhsar

2006-01-01

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs...

Outbred genome sequencing and CRISPR/Cas9 gene editing in butterflies

PubMed Central

Li, Xueyan; Fan, Dingding; Zhang, Wei; Liu, Guichun; Zhang, Lu; Zhao, Li; Fang, Xiaodong; Chen, Lei; Dong, Yang; Chen, Yuan; Ding, Yun; Zhao, Ruoping; Feng, Mingji; Zhu, Yabing; Feng, Yue; Jiang, Xuanting; Zhu, Deying; Xiang, Hui; Feng, Xikan; Li, Shuaicheng; Wang, Jun; Zhang, Guojie; Kronforst, Marcus R.; Wang, Wen

2015-01-01

Butterflies are exceptionally diverse but their potential as an experimental system has been limited by the difficulty of deciphering heterozygous genomes and a lack of genetic manipulation technology. Here we use a hybrid assembly approach to construct high-quality reference genomes for Papilio xuthus (contig and scaffold N50: 492 kb, 3.4 Mb) and Papilio machaon (contig and scaffold N50: 81 kb, 1.15 Mb), highly heterozygous species that differ in host plant affiliations, and adult and larval colour patterns. Integrating comparative genomics and analyses of gene expression yields multiple insights into butterfly evolution, including potential roles of specific genes in recent diversification. To functionally test gene function, we develop an efficient (up to 92.5%) CRISPR/Cas9 gene editing method that yields obvious phenotypes with three genes, Abdominal-B, ebony and frizzled. Our results provide valuable genomic and technological resources for butterflies and unlock their potential as a genetic model system. PMID:26354079

Draft genome sequence of pigeonpea (Cajanus cajan), an orphan legume crop of resource-poor farmers.

PubMed

Varshney, Rajeev K; Chen, Wenbin; Li, Yupeng; Bharti, Arvind K; Saxena, Rachit K; Schlueter, Jessica A; Donoghue, Mark T A; Azam, Sarwar; Fan, Guangyi; Whaley, Adam M; Farmer, Andrew D; Sheridan, Jaime; Iwata, Aiko; Tuteja, Reetu; Penmetsa, R Varma; Wu, Wei; Upadhyaya, Hari D; Yang, Shiaw-Pyng; Shah, Trushar; Saxena, K B; Michael, Todd; McCombie, W Richard; Yang, Bicheng; Zhang, Gengyun; Yang, Huanming; Wang, Jun; Spillane, Charles; Cook, Douglas R; May, Gregory D; Xu, Xun; Jackson, Scott A

2011-11-06

Pigeonpea is an important legume food crop grown primarily by smallholder farmers in many semi-arid tropical regions of the world. We used the Illumina next-generation sequencing platform to generate 237.2 Gb of sequence, which along with Sanger-based bacterial artificial chromosome end sequences and a genetic map, we assembled into scaffolds representing 72.7% (605.78 Mb) of the 833.07 Mb pigeonpea genome. Genome analysis predicted 48,680 genes for pigeonpea and also showed the potential role that certain gene families, for example, drought tolerance-related genes, have played throughout the domestication of pigeonpea and the evolution of its ancestors. Although we found a few segmental duplication events, we did not observe the recent genome-wide duplication events observed in soybean. This reference genome sequence will facilitate the identification of the genetic basis of agronomically important traits, and accelerate the development of improved pigeonpea varieties that could improve food security in many developing countries.

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

PubMed

Wang, Wenqin; Messing, Joachim

2011-01-01

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

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

PubMed Central

Wang, Wenqin; Messing, Joachim

2011-01-01

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

Evaluation of the utility of the new rainbow trout genome assembly for analyzing RNA-seq data from stress response experiments

USDA-ARS?s Scientific Manuscript database

The newly released rainbow trout genome assembly in NCBI RefSeq has greatly expanded our abilities for analyzing rainbow trout sequencing data. In this poster, we evaluate the utility of this genome assembly for analyzing RNA sequencing (RNA-seq) data of rainbow trout responses to various stressors,...

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.

ARKS: chromosome-scale scaffolding of human genome drafts with linked read kmers.

PubMed

Coombe, Lauren; Zhang, Jessica; Vandervalk, Benjamin P; Chu, Justin; Jackman, Shaun D; Birol, Inanc; Warren, René L

2018-06-20

The long-range sequencing information captured by linked reads, such as those available from 10× Genomics (10xG), helps resolve genome sequence repeats, and yields accurate and contiguous draft genome assemblies. We introduce ARKS, an alignment-free linked read genome scaffolding methodology that uses linked reads to organize genome assemblies further into contiguous drafts. Our approach departs from other read alignment-dependent linked read scaffolders, including our own (ARCS), and uses a kmer-based mapping approach. The kmer mapping strategy has several advantages over read alignment methods, including better usability and faster processing, as it precludes the need for input sequence formatting and draft sequence assembly indexing. The reliance on kmers instead of read alignments for pairing sequences relaxes the workflow requirements, and drastically reduces the run time. Here, we show how linked reads, when used in conjunction with Hi-C data for scaffolding, improve a draft human genome assembly of PacBio long-read data five-fold (baseline vs. ARKS NG50 = 4.6 vs. 23.1 Mbp, respectively). We also demonstrate how the method provides further improvements of a megabase-scale Supernova human genome assembly (NG50 = 14.74 Mbp vs. 25.94 Mbp before and after ARKS), which itself exclusively uses linked read data for assembly, with an execution speed six to nine times faster than competitive linked read scaffolders (~ 10.5 h compared to 75.7 h, on average). Following ARKS scaffolding of a human genome 10xG Supernova assembly (of cell line NA12878), fewer than 9 scaffolds cover each chromosome, except the largest (chromosome 1, n = 13). ARKS uses a kmer mapping strategy instead of linked read alignments to record and associate the barcode information needed to order and orient draft assembly sequences. The simplified workflow, when compared to that of our initial implementation, ARCS, markedly improves run time performances on experimental human genome datasets. Furthermore, the novel distance estimator in ARKS utilizes barcoding information from linked reads to estimate gap sizes. It accomplishes this by modeling the relationship between known distances of a region within contigs and calculating associated Jaccard indices. ARKS has the potential to provide correct, chromosome-scale genome assemblies, promptly. We expect ARKS to have broad utility in helping refine draft genomes.

Genomics and introgression: discovery and mapping of thousands of species-diagnostic SNPs using RAD sequencing

USGS Publications Warehouse

Hand, Brian K.; Hether, Tyler D; Kovach, Ryan P.; Muhlfeld, Clint C.; Amish, Stephen J.; Boyer, Matthew C.; O’Rourke, Sean M.; Miller, Michael R.; Lowe, Winsor H.; Hohenlohe, Paul A.; Luikart, Gordon

2015-01-01

Invasive hybridization and introgression pose a serious threat to the persistence of many native species. Understanding the effects of hybridization on native populations (e.g., fitness consequences) requires numerous species-diagnostic loci distributed genome-wide. Here we used RAD sequencing to discover thousands of single-nucleotide polymorphisms (SNPs) that are diagnostic between rainbow trout (RBT, Oncorhynchus mykiss), the world’s most widely introduced fish, and native westslope cutthroat trout (WCT, O. clarkii lewisi) in the northern Rocky Mountains, USA. We advanced previous work that identified 4,914 species-diagnostic loci by using longer sequence reads (100 bp vs. 60 bp) and a larger set of individuals (n = 84). We sequenced RAD libraries for individuals from diverse sampling sources, including native populations of WCT and hatchery broodstocks of WCT and RBT. We also took advantage of a newly released reference genome assembly for RBT to align our RAD loci. In total, we discovered 16,788 putatively diagnostic SNPs, 10,267 of which we mapped to anchored chromosome locations on the RBT genome. A small portion of previously discovered putative diagnostic loci (325 of 4,914) were no longer diagnostic (i.e., fixed between species) based on our wider survey of non-hybridized RBT and WCT individuals. Our study suggests that RAD loci mapped to a draft genome assembly could provide the marker density required to identify genes and chromosomal regions influencing selection in admixed populations of conservation concern and evolutionary interest.

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics1

PubMed Central

Weitemier, Kevin; Straub, Shannon C. K.; Cronn, Richard C.; Fishbein, Mark; Schmickl, Roswitha; McDonnell, Angela; Liston, Aaron

2014-01-01

• Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • Methods and Results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. • Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics. PMID:25225629

RNA-seq analysis of Rubus idaeus cv. Nova: transcriptome sequencing and de novo assembly for subsequent functional genomics approaches.

PubMed

Hyun, Tae Kyung; Lee, Sarah; Kumar, Dhinesh; Rim, Yeonggil; Kumar, Ritesh; Lee, Sang Yeol; Lee, Choong Hwan; Kim, Jae-Yean

2014-10-01

Using Illumina sequencing technology, we have generated the large-scale transcriptome sequencing data containing abundant information on genes involved in the metabolic pathways in R. idaeus cv. Nova fruits. Rubus idaeus (Red raspberry) is one of the important economical crops that possess numerous nutrients, micronutrients and phytochemicals with essential health benefits to human. The molecular mechanism underlying the ripening process and phytochemical biosynthesis in red raspberry is attributed to the changes in gene expression, but very limited transcriptomic and genomic information in public databases is available. To address this issue, we generated more than 51 million sequencing reads from R. idaeus cv. Nova fruit using Illumina RNA-Seq technology. After de novo assembly, we obtained 42,604 unigenes with an average length of 812 bp. At the protein level, Nova fruit transcriptome showed 77 and 68 % sequence similarities with Rubus coreanus and Fragaria versa, respectively, indicating the evolutionary relationship between them. In addition, 69 % of assembled unigenes were annotated using public databases including NCBI non-redundant, Cluster of Orthologous Groups and Gene ontology database, suggesting that our transcriptome dataset provides a valuable resource for investigating metabolic processes in red raspberry. To analyze the relationship between several novel transcripts and the amounts of metabolites such as γ-aminobutyric acid and anthocyanins, real-time PCR and target metabolite analysis were performed on two different ripening stages of Nova. This is the first attempt using Illumina sequencing platform for RNA sequencing and de novo assembly of Nova fruit without reference genome. Our data provide the most comprehensive transcriptome resource available for Rubus fruits, and will be useful for understanding the ripening process and for breeding R. idaeus cultivars with improved fruit quality.

Analysis of Illumina Microbial Assemblies

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

Clum, Alicia; Foster, Brian; Froula, Jeff

2010-05-28

Since the emerging of second generation sequencing technologies, the evaluation of different sequencing approaches and their assembly strategies for different types of genomes has become an important undertaken. Next generation sequencing technologies dramatically increase sequence throughput while decreasing cost, making them an attractive tool for whole genome shotgun sequencing. To compare different approaches for de-novo whole genome assembly, appropriate tools and a solid understanding of both quantity and quality of the underlying sequence data are crucial. Here, we performed an in-depth analysis of short-read Illumina sequence assembly strategies for bacterial and archaeal genomes. Different types of Illumina libraries as wellmore » as different trim parameters and assemblers were evaluated. Results of the comparative analysis and sequencing platforms will be presented. The goal of this analysis is to develop a cost-effective approach for the increased throughput of the generation of high quality microbial genomes.« less

The Power and Potential of Genomics in Weed Biology and Management.

PubMed

Ravet, Karl; Patterson, Eric L; Krähmer, Hansjörg; Hamouzová, Kateřina; Fan, Longjiang; Jasieniuk, Marie; Lawton-Rauh, Amy; Malone, Jenna M; Scott McElroy, J; Merotto, Aldo; Westra, Philip; Preston, Christopher; Vila-Aiub, Martin M; Busi, Roberto; Tranel, Patrick J; Reinhardt, Carl; Saski, Christopher; Beffa, Roland; Neve, Paul; Gaines, Todd A

2018-04-24

There have been previous calls for, and efforts focused on, realizing the power and potential of weed genomics for better understanding of weeds. Sustained advances in genome sequencing and assembly technologies now make it possible for individual research groups to generate reference genomes for multiple weed species at reasonable costs. Here, we present the outcomes from several meetings, discussions, and workshops focused on establishing an International Weed Genomics Consortium (IWGC) for a coordinated international effort in weed genomics. We review the 'state of the art' in genomics and weed genomics, including technologies, applications, and on-going weed genome projects. We also report the outcomes from a workshop and a global survey of the weed science community to identify priority species, key biological questions, and weed management applications that can be addressed through greater availability of, and access to, genomic resources. Major focus areas include the evolution of herbicide resistance and weedy traits, the development of molecular diagnostics, and the identification of novel targets and approaches for weed management. There is increasing interest in, and need for, weed genomics, and the establishment of the IWGC will provide the necessary global platform for communication and coordination of weed genomics research. This article is protected by copyright. All rights reserved.

Isoform Sequencing Provides a More Comprehensive View of the Panax ginseng Transcriptome.

PubMed

Jo, Ick-Hyun; Lee, Jinsu; Hong, Chi Eun; Lee, Dong Jin; Bae, Wonsil; Park, Sin-Gi; Ahn, Yong Ju; Kim, Young Chang; Kim, Jang Uk; Lee, Jung Woo; Hyun, Dong Yun; Rhee, Sung-Keun; Hong, Chang Pyo; Bang, Kyong Hwan; Ryu, Hojin

2017-09-15

Korean ginseng ( Panax ginseng C.A. Meyer) has been widely used for medicinal purposes and contains potent plant secondary metabolites, including ginsenosides. To obtain transcriptomic data that offers a more comprehensive view of functional genomics in P. ginseng , we generated genome-wide transcriptome data from four different P. ginseng tissues using PacBio isoform sequencing (Iso-Seq) technology. A total of 135,317 assembled transcripts were generated with an average length of 3.2 kb and high assembly completeness. Of those unigenes, 67.5% were predicted to be complete full-length (FL) open reading frames (ORFs) and exhibited a high gene annotation rate. Furthermore, we successfully identified unique full-length genes involved in triterpenoid saponin synthesis and plant hormonal signaling pathways, including auxin and cytokinin. Studies on the functional genomics of P. ginseng seedlings have confirmed the rapid upregulation of negative feed-back loops by auxin and cytokinin signaling cues. The conserved evolutionary mechanisms in the auxin and cytokinin canonical signaling pathways of P. ginseng are more complex than those in Arabidopsis thaliana . Our analysis also revealed a more detailed view of transcriptome-wide alternative isoforms for 88 genes. Finally, transposable elements (TEs) were also identified, suggesting transcriptional activity of TEs in P. ginseng . In conclusion, our results suggest that long-read, full-length or partial-unigene data with high-quality assemblies are invaluable resources as transcriptomic references in P. ginseng and can be used for comparative analyses in closely related medicinal plants.

GRAbB: Selective Assembly of Genomic Regions, a New Niche for Genomic Research

PubMed Central

Zhang, Hao; van Diepeningen, Anne D.; van der Lee, Theo A. J.; Waalwijk, Cees; de Hoog, G. Sybren

2016-01-01

GRAbB (Genomic Region Assembly by Baiting) is a new program that is dedicated to assemble specific genomic regions from NGS data. This approach is especially useful when dealing with multi copy regions, such as mitochondrial genome and the rDNA repeat region, parts of the genome that are often neglected or poorly assembled, although they contain interesting information from phylogenetic or epidemiologic perspectives, but also single copy regions can be assembled. The program is capable of targeting multiple regions within a single run. Furthermore, GRAbB can be used to extract specific loci from NGS data, based on homology, like sequences that are used for barcoding. To make the assembly specific, a known part of the region, such as the sequence of a PCR amplicon or a homologous sequence from a related species must be specified. By assembling only the region of interest, the assembly process is computationally much less demanding and may lead to assemblies of better quality. In this study the different applications and functionalities of the program are demonstrated such as: exhaustive assembly (rDNA region and mitochondrial genome), extracting homologous regions or genes (IGS, RPB1, RPB2 and TEF1a), as well as extracting multiple regions within a single run. The program is also compared with MITObim, which is meant for the exhaustive assembly of a single target based on a similar query sequence. GRAbB is shown to be more efficient than MITObim in terms of speed, memory and disk usage. The other functionalities (handling multiple targets simultaneously and extracting homologous regions) of the new program are not matched by other programs. The program is available with explanatory documentation at https://github.com/b-brankovics/grabb. GRAbB has been tested on Ubuntu (12.04 and 14.04), Fedora (23), CentOS (7.1.1503) and Mac OS X (10.7). Furthermore, GRAbB is available as a docker repository: brankovics/grabb (https://hub.docker.com/r/brankovics/grabb/). PMID:27308864

GRAbB: Selective Assembly of Genomic Regions, a New Niche for Genomic Research.

PubMed

Brankovics, Balázs; Zhang, Hao; van Diepeningen, Anne D; van der Lee, Theo A J; Waalwijk, Cees; de Hoog, G Sybren

2016-06-01

GRAbB (Genomic Region Assembly by Baiting) is a new program that is dedicated to assemble specific genomic regions from NGS data. This approach is especially useful when dealing with multi copy regions, such as mitochondrial genome and the rDNA repeat region, parts of the genome that are often neglected or poorly assembled, although they contain interesting information from phylogenetic or epidemiologic perspectives, but also single copy regions can be assembled. The program is capable of targeting multiple regions within a single run. Furthermore, GRAbB can be used to extract specific loci from NGS data, based on homology, like sequences that are used for barcoding. To make the assembly specific, a known part of the region, such as the sequence of a PCR amplicon or a homologous sequence from a related species must be specified. By assembling only the region of interest, the assembly process is computationally much less demanding and may lead to assemblies of better quality. In this study the different applications and functionalities of the program are demonstrated such as: exhaustive assembly (rDNA region and mitochondrial genome), extracting homologous regions or genes (IGS, RPB1, RPB2 and TEF1a), as well as extracting multiple regions within a single run. The program is also compared with MITObim, which is meant for the exhaustive assembly of a single target based on a similar query sequence. GRAbB is shown to be more efficient than MITObim in terms of speed, memory and disk usage. The other functionalities (handling multiple targets simultaneously and extracting homologous regions) of the new program are not matched by other programs. The program is available with explanatory documentation at https://github.com/b-brankovics/grabb. GRAbB has been tested on Ubuntu (12.04 and 14.04), Fedora (23), CentOS (7.1.1503) and Mac OS X (10.7). Furthermore, GRAbB is available as a docker repository: brankovics/grabb (https://hub.docker.com/r/brankovics/grabb/).

A draft fur seal genome provides insights into factors affecting SNP validation and how to mitigate them.

PubMed

Humble, E; Martinez-Barrio, A; Forcada, J; Trathan, P N; Thorne, M A S; Hoffmann, M; Wolf, J B W; Hoffman, J I

2016-07-01

Custom genotyping arrays provide a flexible and accurate means of genotyping single nucleotide polymorphisms (SNPs) in a large number of individuals of essentially any organism. However, validation rates, defined as the proportion of putative SNPs that are verified to be polymorphic in a population, are often very low. A number of potential causes of assay failure have been identified, but none have been explored systematically. In particular, as SNPs are often developed from transcriptomes, parameters relating to the genomic context are rarely taken into account. Here, we assembled a draft Antarctic fur seal (Arctocephalus gazella) genome (assembly size: 2.41 Gb; scaffold/contig N50 : 3.1 Mb/27.5 kb). We then used this resource to map the probe sequences of 144 putative SNPs genotyped in 480 individuals. The number of probe-to-genome mappings and alignment length together explained almost a third of the variation in validation success, indicating that sequence uniqueness and proximity to intron-exon boundaries play an important role. The same pattern was found after mapping the probe sequences to the Walrus and Weddell seal genomes, suggesting that the genomes of species divergent by as much as 23 million years can hold information relevant to SNP validation outcomes. Additionally, reanalysis of genotyping data from seven previous studies found the same two variables to be significantly associated with SNP validation success across a variety of taxa. Finally, our study reveals considerable scope for validation rates to be improved, either by simply filtering for SNPs whose flanking sequences align uniquely and completely to a reference genome, or through predictive modelling. © 2015 John Wiley & Sons Ltd.

Integrated physical map of bread wheat chromosome arm 7DS to facilitate gene cloning and comparative studies.

PubMed

Tulpová, Zuzana; Luo, Ming-Cheng; Toegelová, Helena; Visendi, Paul; Hayashi, Satomi; Vojta, Petr; Paux, Etienne; Kilian, Andrzej; Abrouk, Michaël; Bartoš, Jan; Hajdúch, Marián; Batley, Jacqueline; Edwards, David; Doležel, Jaroslav; Šimková, Hana

2018-03-08

Bread wheat (Triticum aestivum L.) is a staple food for a significant part of the world's population. The growing demand on its production can be satisfied by improving yield and resistance to biotic and abiotic stress. Knowledge of the genome sequence would aid in discovering genes and QTLs underlying these traits and provide a basis for genomics-assisted breeding. Physical maps and BAC clones associated with them have been valuable resources from which to generate a reference genome of bread wheat and to assist map-based gene cloning. As a part of a joint effort coordinated by the International Wheat Genome Sequencing Consortium, we have constructed a BAC-based physical map of bread wheat chromosome arm 7DS consisting of 895 contigs and covering 94% of its estimated length. By anchoring BAC contigs to one radiation hybrid map and three high resolution genetic maps, we assigned 73% of the assembly to a distinct genomic position. This map integration, interconnecting a total of 1713 markers with ordered and sequenced BAC clones from a minimal tiling path, provides a tool to speed up gene cloning in wheat. The process of physical map assembly included the integration of the 7DS physical map with a whole-genome physical map of Aegilops tauschii and a 7DS Bionano genome map, which together enabled efficient scaffolding of physical-map contigs, even in the non-recombining region of the genetic centromere. Moreover, this approach facilitated a comparison of bread wheat and its ancestor at BAC-contig level and revealed a reconstructed region in the 7DS pericentromere. Copyright © 2018. Published by Elsevier B.V.

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

PubMed

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

2012-09-11

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