SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information

PubMed Central

2014-01-01

Background The recent introduction of the Pacific Biosciences RS single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. The long read sequence information is promised to enhance the quality of incomplete and inaccurate draft assemblies constructed from Next Generation Sequencing (NGS) data. Results Here we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using PacBio RS long read information as a backbone. On a test set comprising six bacterial draft genomes, assembled using either a single Illumina MiSeq or Roche 454 library, we show that even a 50× coverage of uncorrected PacBio RS long reads is sufficient to drastically reduce the number of contigs. Comparisons to the AHA scaffolder indicate our strategy is better capable of producing (nearly) complete bacterial genomes. Conclusions The current work describes our SSPACE-LongRead software which is designed to upgrade incomplete draft genomes using single molecule sequences. We conclude that the recent advances of the PacBio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner. PMID:24950923

CAR: contig assembly of prokaryotic draft genomes using rearrangements.

PubMed

Lu, Chin Lung; Chen, Kun-Tze; Huang, Shih-Yuan; Chiu, Hsien-Tai

2014-11-28

Next generation sequencing technology has allowed efficient production of draft genomes for many organisms of interest. However, most draft genomes are just collections of independent contigs, whose relative positions and orientations along the genome being sequenced are unknown. Although several tools have been developed to order and orient the contigs of draft genomes, more accurate tools are still needed. In this study, we present a novel reference-based contig assembly (or scaffolding) tool, named as CAR, that can efficiently and more accurately order and orient the contigs of a prokaryotic draft genome based on a reference genome of a related organism. Given a set of contigs in multi-FASTA format and a reference genome in FASTA format, CAR can output a list of scaffolds, each of which is a set of ordered and oriented contigs. For validation, we have tested CAR on a real dataset composed of several prokaryotic genomes and also compared its performance with several other reference-based contig assembly tools. Consequently, our experimental results have shown that CAR indeed performs better than all these other reference-based contig assembly tools in terms of sensitivity, precision and genome coverage. CAR serves as an efficient tool that can more accurately order and orient the contigs of a prokaryotic draft genome based on a reference genome. The web server of CAR is freely available at http://genome.cs.nthu.edu.tw/CAR/ and its stand-alone program can also be downloaded from the same website.

TCGA-assembler 2: software pipeline for retrieval and processing of TCGA/CPTAC data.

PubMed

Wei, Lin; Jin, Zhilin; Yang, Shengjie; Xu, Yanxun; Zhu, Yitan; Ji, Yuan

2018-05-01

The Cancer Genome Atlas (TCGA) program has produced huge amounts of cancer genomics data providing unprecedented opportunities for research. In 2014, we developed TCGA-Assembler, a software pipeline for retrieval and processing of public TCGA data. In 2016, TCGA data were transferred from the TCGA data portal to the Genomic Data Commons (GDCs), which is supported by a different set of data storage and retrieval mechanisms. In addition, new proteomics data of TCGA samples have been generated by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) program, which were not available for downloading through TCGA-Assembler. It is desirable to acquire and integrate data from both GDC and CPTAC. We develop TCGA-assembler 2 (TA2) to automatically download and integrate data from GDC and CPTAC. We make substantial improvement on the functionality of TA2 to enhance user experience and software performance. TA2 together with its previous version have helped more than 2000 researchers from 64 countries to access and utilize TCGA and CPTAC data in their research. Availability of TA2 will continue to allow existing and new users to conduct reproducible research based on TCGA and CPTAC data. http://www.compgenome.org/TCGA-Assembler/ or https://github.com/compgenome365/TCGA-Assembler-2. zhuyitan@gmail.com or koaeraser@gmail.com. Supplementary data are available at Bioinformatics online.

MetaCAA: A clustering-aided methodology for efficient assembly of metagenomic datasets.

PubMed

Reddy, Rachamalla Maheedhar; Mohammed, Monzoorul Haque; Mande, Sharmila S

2014-01-01

A key challenge in analyzing metagenomics data pertains to assembly of sequenced DNA fragments (i.e. reads) originating from various microbes in a given environmental sample. Several existing methodologies can assemble reads originating from a single genome. However, these methodologies cannot be applied for efficient assembly of metagenomic sequence datasets. In this study, we present MetaCAA - a clustering-aided methodology which helps in improving the quality of metagenomic sequence assembly. MetaCAA initially groups sequences constituting a given metagenome into smaller clusters. Subsequently, sequences in each cluster are independently assembled using CAP3, an existing single genome assembly program. Contigs formed in each of the clusters along with the unassembled reads are then subjected to another round of assembly for generating the final set of contigs. Validation using simulated and real-world metagenomic datasets indicates that MetaCAA aids in improving the overall quality of assembly. A software implementation of MetaCAA is available at https://metagenomics.atc.tcs.com/MetaCAA. Copyright © 2014 Elsevier Inc. All rights reserved.

Development and validation of microsatellite markers for Brachiaria ruziziensis obtained by partial genome assembly of Illumina single-end reads

PubMed Central

2013-01-01

Background Brachiaria ruziziensis is one of the most important forage species planted in the tropics. The application of genomic tools to aid the selection of superior genotypes can provide support to B. ruziziensis breeding programs. However, there is a complete lack of information about the B. ruziziensis genome. Also, the availability of genomic tools, such as molecular markers, to support B. ruziziensis breeding programs is rather limited. Recently, next-generation sequencing technologies have been applied to generate sequence data for the identification of microsatellite regions and primer design. In this study, we present a first validated set of SSR markers for Brachiaria ruziziensis, selected from a de novo partial genome assembly of single-end Illumina reads. Results A total of 85,567 perfect microsatellite loci were detected in contigs with a minimum 10X coverage. We selected a set of 500 microsatellite loci identified in contigs with minimum 100X coverage for primer design and synthesis, and tested a subset of 269 primer pairs, 198 of which were polymorphic on 11 representative B. ruziziensis accessions. Descriptive statistics for these primer pairs are presented, as well as estimates of marker transferability to other relevant brachiaria species. Finally, a set of 11 multiplex panels containing the 30 most informative markers was validated and proposed for B. ruziziensis genetic analysis. Conclusions We show that the detection and development of microsatellite markers from genome assembled Illumina single-end DNA sequences is highly efficient. The developed markers are readily suitable for genetic analysis and marker assisted selection of Brachiaria ruziziensis. The use of this approach for microsatellite marker development is promising for species with limited genomic information, whose breeding programs would benefit from the use of genomic tools. To our knowledge, this is the first set of microsatellite markers developed for this important species. PMID:23324172

Fast scaffolding with small independent mixed integer programs

PubMed Central

Salmela, Leena; Mäkinen, Veli; Välimäki, Niko; Ylinen, Johannes; Ukkonen, Esko

2011-01-01

Motivation: Assembling genomes from short read data has become increasingly popular, but the problem remains computationally challenging especially for larger genomes. We study the scaffolding phase of sequence assembly where preassembled contigs are ordered based on mate pair data. Results: We present MIP Scaffolder that divides the scaffolding problem into smaller subproblems and solves these with mixed integer programming. The scaffolding problem can be represented as a graph and the biconnected components of this graph can be solved independently. We present a technique for restricting the size of these subproblems so that they can be solved accurately with mixed integer programming. We compare MIP Scaffolder to two state of the art methods, SOPRA and SSPACE. MIP Scaffolder is fast and produces better or as good scaffolds as its competitors on large genomes. Availability: The source code of MIP Scaffolder is freely available at http://www.cs.helsinki.fi/u/lmsalmel/mip-scaffolder/. Contact: leena.salmela@cs.helsinki.fi PMID:21998153

Critical Assessment of Metagenome Interpretation – a benchmark of computational metagenomics software

PubMed Central

Sczyrba, Alexander; Hofmann, Peter; Belmann, Peter; Koslicki, David; Janssen, Stefan; Dröge, Johannes; Gregor, Ivan; Majda, Stephan; Fiedler, Jessika; Dahms, Eik; Bremges, Andreas; Fritz, Adrian; Garrido-Oter, Ruben; Jørgensen, Tue Sparholt; Shapiro, Nicole; Blood, Philip D.; Gurevich, Alexey; Bai, Yang; Turaev, Dmitrij; DeMaere, Matthew Z.; Chikhi, Rayan; Nagarajan, Niranjan; Quince, Christopher; Meyer, Fernando; Balvočiūtė, Monika; Hansen, Lars Hestbjerg; Sørensen, Søren J.; Chia, Burton K. H.; Denis, Bertrand; Froula, Jeff L.; Wang, Zhong; Egan, Robert; Kang, Dongwan Don; Cook, Jeffrey J.; Deltel, Charles; Beckstette, Michael; Lemaitre, Claire; Peterlongo, Pierre; Rizk, Guillaume; Lavenier, Dominique; Wu, Yu-Wei; Singer, Steven W.; Jain, Chirag; Strous, Marc; Klingenberg, Heiner; Meinicke, Peter; Barton, Michael; Lingner, Thomas; Lin, Hsin-Hung; Liao, Yu-Chieh; Silva, Genivaldo Gueiros Z.; Cuevas, Daniel A.; Edwards, Robert A.; Saha, Surya; Piro, Vitor C.; Renard, Bernhard Y.; Pop, Mihai; Klenk, Hans-Peter; Göker, Markus; Kyrpides, Nikos C.; Woyke, Tanja; Vorholt, Julia A.; Schulze-Lefert, Paul; Rubin, Edward M.; Darling, Aaron E.; Rattei, Thomas; McHardy, Alice C.

2018-01-01

In metagenome analysis, computational methods for assembly, taxonomic profiling and binning are key components facilitating downstream biological data interpretation. However, a lack of consensus about benchmarking datasets and evaluation metrics complicates proper performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on datasets of unprecedented complexity and realism. Benchmark metagenomes were generated from ~700 newly sequenced microorganisms and ~600 novel viruses and plasmids, including genomes with varying degrees of relatedness to each other and to publicly available ones and representing common experimental setups. Across all datasets, assembly and genome binning programs performed well for species represented by individual genomes, while performance was substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below the family level. Parameter settings substantially impacted performances, underscoring the importance of program reproducibility. While highlighting current challenges in computational metagenomics, the CAMI results provide a roadmap for software selection to answer specific research questions. PMID:28967888

RNA-programmed genome editing in human cells

PubMed Central

Jinek, Martin; East, Alexandra; Cheng, Aaron; Lin, Steven; Ma, Enbo; Doudna, Jennifer

2013-01-01

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells. DOI: http://dx.doi.org/10.7554/eLife.00471.001 PMID:23386978

Improved annotation with de novo transcriptome assembly in four social amoeba species.

PubMed

Singh, Reema; Lawal, Hajara M; Schilde, Christina; Glöckner, Gernot; Barton, Geoffrey J; Schaap, Pauline; Cole, Christian

2017-01-31

Annotation of gene models and transcripts is a fundamental step in genome sequencing projects. Often this is performed with automated prediction pipelines, which can miss complex and atypical genes or transcripts. RNA sequencing (RNA-seq) data can aid the annotation with empirical data. Here we present de novo transcriptome assemblies generated from RNA-seq data in four Dictyostelid species: D. discoideum, P. pallidum, D. fasciculatum and D. lacteum. The assemblies were incorporated with existing gene models to determine corrections and improvement on a whole-genome scale. This is the first time this has been performed in these eukaryotic species. An initial de novo transcriptome assembly was generated by Trinity for each species and then refined with Program to Assemble Spliced Alignments (PASA). The completeness and quality were assessed with the Benchmarking Universal Single-Copy Orthologs (BUSCO) and Transrate tools at each stage of the assemblies. The final datasets of 11,315-12,849 transcripts contained 5,610-7,712 updates and corrections to >50% of existing gene models including changes to hundreds or thousands of protein products. Putative novel genes are also identified and alternative splice isoforms were observed for the first time in P. pallidum, D. lacteum and D. fasciculatum. In taking a whole transcriptome approach to genome annotation with empirical data we have been able to enrich the annotations of four existing genome sequencing projects. In doing so we have identified updates to the majority of the gene annotations across all four species under study and found putative novel genes and transcripts which could be worthy for follow-up. The new transcriptome data we present here will be a valuable resource for genome curators in the Dictyostelia and we propose this effective methodology for use in other genome annotation projects.

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.

Virome Assembly and Annotation: A Surprise in the Namib Desert

PubMed Central

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

2017-01-01

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

The genome sequence of the colonial chordate, Botryllus schlosseri

PubMed Central

Voskoboynik, Ayelet; Neff, Norma F; Sahoo, Debashis; Newman, Aaron M; Pushkarev, Dmitry; Koh, Winston; Passarelli, Benedetto; Fan, H Christina; Mantalas, Gary L; Palmeri, Karla J; Ishizuka, Katherine J; Gissi, Carmela; Griggio, Francesca; Ben-Shlomo, Rachel; Corey, Daniel M; Penland, Lolita; White, Richard A; Weissman, Irving L; Quake, Stephen R

2013-01-01

Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI: http://dx.doi.org/10.7554/eLife.00569.001 PMID:23840927

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

PubMed

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

2014-01-01

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

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

USGS Publications Warehouse

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

2014-01-01

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

GapBlaster-A Graphical Gap Filler for Prokaryote Genomes.

PubMed

de Sá, Pablo H C G; Miranda, Fábio; Veras, Adonney; de Melo, Diego Magalhães; Soares, Siomar; Pinheiro, Kenny; Guimarães, Luis; Azevedo, Vasco; Silva, Artur; Ramos, Rommel T J

2016-01-01

The advent of NGS (Next Generation Sequencing) technologies has resulted in an exponential increase in the number of complete genomes available in biological databases. This advance has allowed the development of several computational tools enabling analyses of large amounts of data in each of the various steps, from processing and quality filtering to gap filling and manual curation. The tools developed for gap closure are very useful as they result in more complete genomes, which will influence downstream analyses of genomic plasticity and comparative genomics. However, the gap filling step remains a challenge for genome assembly, often requiring manual intervention. Here, we present GapBlaster, a graphical application to evaluate and close gaps. GapBlaster was developed via Java programming language. The software uses contigs obtained in the assembly of the genome to perform an alignment against a draft of the genome/scaffold, using BLAST or Mummer to close gaps. Then, all identified alignments of contigs that extend through the gaps in the draft sequence are presented to the user for further evaluation via the GapBlaster graphical interface. GapBlaster presents significant results compared to other similar software and has the advantage of offering a graphical interface for manual curation of the gaps. GapBlaster program, the user guide and the test datasets are freely available at https://sourceforge.net/projects/gapblaster2015/. It requires Sun JDK 8 and Blast or Mummer.

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

PubMed Central

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

2014-01-01

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

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

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.

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.

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

Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments

PubMed Central

Haas, Brian J; Salzberg, Steven L; Zhu, Wei; Pertea, Mihaela; Allen, Jonathan E; Orvis, Joshua; White, Owen; Buell, C Robin; Wortman, Jennifer R

2008-01-01

EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation. PMID:18190707

Enhancing genome assemblies by integrating non-sequence based data

PubMed Central

2011-01-01

Introduction Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. Methods The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Results Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. Conclusions We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses. PMID:21554765

Enhancing genome assemblies by integrating non-sequence based data.

PubMed

Heider, Thomas N; Lindsay, James; Wang, Chenwei; O'Neill, Rachel J; Pask, Andrew J

2011-05-28

Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses.

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.

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.

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.

Hal: an automated pipeline for phylogenetic analyses of genomic data.

PubMed

Robbertse, Barbara; Yoder, Ryan J; Boyd, Alex; Reeves, John; Spatafora, Joseph W

2011-02-07

The rapid increase in genomic and genome-scale data is resulting in unprecedented levels of discrete sequence data available for phylogenetic analyses. Major analytical impasses exist, however, prior to analyzing these data with existing phylogenetic software. Obstacles include the management of large data sets without standardized naming conventions, identification and filtering of orthologous clusters of proteins or genes, and the assembly of alignments of orthologous sequence data into individual and concatenated super alignments. Here we report the production of an automated pipeline, Hal that produces multiple alignments and trees from genomic data. These alignments can be produced by a choice of four alignment programs and analyzed by a variety of phylogenetic programs. In short, the Hal pipeline connects the programs BLASTP, MCL, user specified alignment programs, GBlocks, ProtTest and user specified phylogenetic programs to produce species trees. The script is available at sourceforge (http://sourceforge.net/projects/bio-hal/). The results from an example analysis of Kingdom Fungi are briefly discussed.

Mojo Hand, a TALEN design tool for genome editing applications.

PubMed

Neff, Kevin L; Argue, David P; Ma, Alvin C; Lee, Han B; Clark, Karl J; Ekker, Stephen C

2013-01-16

Recent studies of transcription activator-like (TAL) effector domains fused to nucleases (TALENs) demonstrate enormous potential for genome editing. Effective design of TALENs requires a combination of selecting appropriate genetic features, finding pairs of binding sites based on a consensus sequence, and, in some cases, identifying endogenous restriction sites for downstream molecular genetic applications. We present the web-based program Mojo Hand for designing TAL and TALEN constructs for genome editing applications (http://www.talendesign.org). We describe the algorithm and its implementation. The features of Mojo Hand include (1) automatic download of genomic data from the National Center for Biotechnology Information, (2) analysis of any DNA sequence to reveal pairs of binding sites based on a user-defined template, (3) selection of restriction-enzyme recognition sites in the spacer between the TAL monomer binding sites including options for the selection of restriction enzyme suppliers, and (4) output files designed for subsequent TALEN construction using the Golden Gate assembly method. Mojo Hand enables the rapid identification of TAL binding sites for use in TALEN design. The assembly of TALEN constructs, is also simplified by using the TAL-site prediction program in conjunction with a spreadsheet management aid of reagent concentrations and TALEN formulation. Mojo Hand enables scientists to more rapidly deploy TALENs for genome editing applications.

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.

Origins of the Human Genome Project.

PubMed

Watson, J D; Cook-Deegan, R M

1991-01-01

The Human Genome Project has become a reality. Building on a debate that dates back to 1985, several genome projects are now in full stride around the world, and more are likely to form in the next several years. Italy began its genome program in 1987, and the United Kingdom and U.S.S.R. in 1988. The European communities mounted several genome projects on yeast, bacteria, Drosophila, and Arabidospis thaliana (a rapidly growing plant with a small genome) in 1988, and in 1990 commenced a new 2-year program on the human genome. In the United States, we have completed the first year of operation of the National Center for Human Genome Research at the National Institutes of Health (NIH), now the largest single funding source for genome research in the world. There have been dedicated budgets focused on genome-scale research at NIH, the U.S. Department of Energy, and the Howard Hughes Medical Institute for several years, and results are beginning to accumulate. There were three annual meetings on genome mapping and sequencing at Cold Spring Harbor, New York, in the spring of 1988, 1989, and 1990; the talks have shifted from a discussion about how to approach problems to presenting results from experiments already performed. We have finally begun to work rather than merely talk. The purpose of genome projects is to assemble data on the structure of DNA in human chromosomes and those of other organisms. A second goal is to develop new technologies to perform mapping and sequencing. There have been impressive technical advances in the past 5 years since the debate about the human genome project began. We are on the verge of beginning pilot projects to test several approaches to sequencing long stretches of DNA, using both automation and manual methods. Ordered sets of yeast artificial chromosome and cosmid clones have been assembled to span more than 2 million base pairs of several human chromosomes, and a region of 10 million base pairs has been assembled for Caenorhabditis elegans by a collaboration between Washington University and the Medical Research Council laboratory in Cambridge, U.K. This project is now turning to sequencing C. elegans DNA as a logical extension of this work. These are but the first fruits of the genome project. There is much more to come.

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.

A Chromosome-Scale Assembly of the Bactrocera cucurbitae Genome Provides Insight to the Genetic Basis of white pupae

PubMed Central

Sim, Sheina B.; Geib, Scott M.

2017-01-01

Genetic sexing strains (GSS) used in sterile insect technique (SIT) programs are textbook examples of how classical Mendelian genetics can be directly implemented in the management of agricultural insect pests. Although the foundation of traditionally developed GSS are single locus, autosomal recessive traits, their genetic basis are largely unknown. With the advent of modern genomic techniques, the genetic basis of sexing traits in GSS can now be further investigated. This study is the first of its kind to integrate traditional genetic techniques with emerging genomics to characterize a GSS using the tephritid fruit fly pest Bactrocera cucurbitae as a model. These techniques include whole-genome sequencing, the development of a mapping population and linkage map, and quantitative trait analysis. The experiment designed to map the genetic sexing trait in B. cucurbitae, white pupae (wp), also enabled the generation of a chromosome-scale genome assembly by integrating the linkage map with the assembly. Quantitative trait loci analysis revealed SNP loci near position 42 MB on chromosome 3 to be tightly linked to wp. Gene annotation and synteny analysis show a near perfect relationship between chromosomes in B. cucurbitae and Muller elements A–E in Drosophila melanogaster. This chromosome-scale genome assembly is complete, has high contiguity, was generated using a minimal input DNA, and will be used to further characterize the genetic mechanisms underlying wp. Knowledge of the genetic basis of genetic sexing traits can be used to improve SIT in this species and expand it to other economically important Diptera. PMID:28450369

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.

Human Contamination in Public Genome Assemblies.

PubMed

Kryukov, Kirill; Imanishi, Tadashi

2016-01-01

Contamination in genome assembly can lead to wrong or confusing results when using such genome as reference in sequence comparison. Although bacterial contamination is well known, the problem of human-originated contamination received little attention. In this study we surveyed 45,735 available genome assemblies for evidence of human contamination. We used lineage specificity to distinguish between contamination and conservation. We found that 154 genome assemblies contain fragments that with high confidence originate as contamination from human DNA. Majority of contaminating human sequences were present in the reference human genome assembly for over a decade. We recommend that existing contaminated genomes should be revised to remove contaminated sequence, and that new assemblies should be thoroughly checked for presence of human DNA before submitting them to public databases.

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.

Improving draft genome contiguity with reference-derived in silico mate-pair libraries.

PubMed

Grau, José Horacio; Hackl, Thomas; Koepfli, Klaus-Peter; Hofreiter, Michael

2018-05-01

Contiguous genome assemblies are a highly valued biological resource because of the higher number of completely annotated genes and genomic elements that are usable compared to fragmented draft genomes. Nonetheless, contiguity is difficult to obtain if only low coverage data and/or only distantly related reference genome assemblies are available. In order to improve genome contiguity, we have developed Cross-Species Scaffolding-a new pipeline that imports long-range distance information directly into the de novo assembly process by constructing mate-pair libraries in silico. We show how genome assembly metrics and gene prediction dramatically improve with our pipeline by assembling two primate genomes solely based on ∼30x coverage of shotgun sequencing data.

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.

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.

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.

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

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.

De novo assembly of a haplotype-resolved human genome.

PubMed

Cao, Hongzhi; Wu, Honglong; Luo, Ruibang; Huang, Shujia; Sun, Yuhui; Tong, Xin; Xie, Yinlong; Liu, Binghang; Yang, Hailong; Zheng, Hancheng; Li, Jian; Li, Bo; Wang, Yu; Yang, Fang; Sun, Peng; Liu, Siyang; Gao, Peng; Huang, Haodong; Sun, Jing; Chen, Dan; He, Guangzhu; Huang, Weihua; Huang, Zheng; Li, Yue; Tellier, Laurent C A M; Liu, Xiao; Feng, Qiang; Xu, Xun; Zhang, Xiuqing; Bolund, Lars; Krogh, Anders; Kristiansen, Karsten; Drmanac, Radoje; Drmanac, Snezana; Nielsen, Rasmus; Li, Songgang; Wang, Jian; Yang, Huanming; Li, Yingrui; Wong, Gane Ka-Shu; Wang, Jun

2015-06-01

The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.

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

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

Catfish Genome Consortium; Wang, Shaolin; Peatman, Eric

2010-03-23

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

Katome: de novo DNA assembler implemented in rust

NASA Astrophysics Data System (ADS)

Neumann, Łukasz; Nowak, Robert M.; Kuśmirek, Wiktor

2017-08-01

Katome is a new de novo sequence assembler written in the Rust programming language, designed with respect to future parallelization of the algorithms, run time and memory usage optimization. The application uses new algorithms for the correct assembly of repetitive sequences. Performance and quality tests were performed on various data, comparing the new application to `dnaasm', `ABySS' and `Velvet' genome assemblers. Quality tests indicate that the new assembler creates more contigs than well-established solutions, but the contigs have better quality with regard to mismatches per 100kbp and indels per 100kbp. Additionally, benchmarks indicate that the Rust-based implementation outperforms `dnaasm', `ABySS' and `Velvet' assemblers, written in C++, in terms of assembly time. Lower memory usage in comparison to `dnaasm' is observed.

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.

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.

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.

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

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

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

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

Rapid hybrid de novo assembly of a microbial genome using only short reads: Corynebacterium pseudotuberculosis I19 as a case study.

PubMed

Cerdeira, Louise Teixeira; Carneiro, Adriana Ribeiro; Ramos, Rommel Thiago Jucá; de Almeida, Sintia Silva; D'Afonseca, Vivian; Schneider, Maria Paula Cruz; Baumbach, Jan; Tauch, Andreas; McCulloch, John Anthony; Azevedo, Vasco Ariston Carvalho; Silva, Artur

2011-08-01

Due to the advent of the so-called Next-Generation Sequencing (NGS) technologies the amount of monetary and temporal resources for whole-genome sequencing has been reduced by several orders of magnitude. Sequence reads can be assembled either by anchoring them directly onto an available reference genome (classical reference assembly), or can be concatenated by overlap (de novo assembly). The latter strategy is preferable because it tends to maintain the architecture of the genome sequence the however, depending on the NGS platform used, the shortness of read lengths cause tremendous problems the in the subsequent genome assembly phase, impeding closing of the entire genome sequence. To address the problem, we developed a multi-pronged hybrid de novo strategy combining De Bruijn graph and Overlap-Layout-Consensus methods, which was used to assemble from short reads the entire genome of Corynebacterium pseudotuberculosis strain I19, a bacterium with immense importance in veterinary medicine that causes Caseous Lymphadenitis in ruminants, principally ovines and caprines. Briefly, contigs were assembled de novo from the short reads and were only oriented using a reference genome by anchoring. Remaining gaps were closed using iterative anchoring of short reads by craning to gap flanks. Finally, we compare the genome sequence assembled using our hybrid strategy to a classical reference assembly using the same data as input and show that with the availability of a reference genome, it pays off to use the hybrid de novo strategy, rather than a classical reference assembly, because more genome sequences are preserved using the former. Copyright © 2011 Elsevier B.V. All rights reserved.

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 .

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

Genovo: De Novo Assembly for Metagenomes

NASA Astrophysics Data System (ADS)

Laserson, Jonathan; Jojic, Vladimir; Koller, Daphne

Next-generation sequencing technologies produce a large number of noisy reads from the DNA in a sample. Metagenomics and population sequencing aim to recover the genomic sequences of the species in the sample, which could be of high diversity. Methods geared towards single sequence reconstruction are not sensitive enough when applied in this setting. We introduce a generative probabilistic model of read generation from environmental samples and present Genovo, a novel de novo sequence assembler that discovers likely sequence reconstructions under the model. A Chinese restaurant process prior accounts for the unknown number of genomes in the sample. Inference is made by applying a series of hill-climbing steps iteratively until convergence. We compare the performance of Genovo to three other short read assembly programs across one synthetic dataset and eight metagenomic datasets created using the 454 platform, the largest of which has 311k reads. Genovo's reconstructions cover more bases and recover more genes than the other methods, and yield a higher assembly score.

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

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.

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

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

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.

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.

Bringing the fathead minnow (Pimephales promelas) into the ...

EPA Pesticide Factsheets

The fathead minnow (Pimephales promelas) is a well-established ecotoxicological model organism that has been widely used for regulatory ecotoxicity testing and research for over a half century. Throughout this time, a lot of knowledge has been gained about the fathead minnow’s biological responses to various xenobiotics. However, despite its importance as a model organism, the fathead minnow still has few publicly available gene sequences. Recently, Burns et al. (2015; Environ. Toxicol. Chem. 35:212) described the sequencing and de-novo assembly of the fathead minnow genome. Two draft genome assemblies are now publicly available on the GenBank database. However, on their own the draft assemblies remain of limited use to researchers who are primarily interested in the functional units of the genome, i.e. the genes. In the present study, an annotation pipeline, consisting of gene prediction, evidence alignment, and data synthesis, was applied to the fathead minnow SOAPdenovo assembly. Ab initio gene prediction was performed using AUGUSTUS, which provided a starting point of 43,345 gene predictions. Fathead minnow Expressed Sequence Tags (ESTs) and zebrafish protein-coding sequences (CDSs) were then aligned to the assembly using the corresponding spliced alignment methods of the program Exonerate. Of the over 240,000 EST alignments, 73% were successfully aligned with 90% or greater sequence identity and query coverage. Similarly, 39% of nearly 45,000 zebrafish co

Progress toward a low budget reference grade genome assembly

USDA-ARS?s Scientific Manuscript database

Reference quality de novo genome assemblies were once solely the domain of large, well-funded genome projects. While next-generation short read technology removed some of the cost barriers, accurate chromosome-scale assembly remains a real challenge. Here we present efforts to de novo assemble the...

Reference-guided assembly of four diverse Arabidopsis thaliana genomes

PubMed Central

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

2011-01-01

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

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.

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...

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.

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

FANTOM5 CAGE profiles of human and mouse reprocessed for GRCh38 and GRCm38 genome assemblies.

PubMed

Abugessaisa, Imad; Noguchi, Shuhei; Hasegawa, Akira; Harshbarger, Jayson; Kondo, Atsushi; Lizio, Marina; Severin, Jessica; Carninci, Piero; Kawaji, Hideya; Kasukawa, Takeya

2017-08-29

The FANTOM5 consortium described the promoter-level expression atlas of human and mouse by using CAGE (Cap Analysis of Gene Expression) with single molecule sequencing. In the original publications, GRCh37/hg19 and NCBI37/mm9 assemblies were used as the reference genomes of human and mouse respectively; later, the Genome Reference Consortium released newer genome assemblies GRCh38/hg38 and GRCm38/mm10. To increase the utility of the atlas in forthcoming researches, we reprocessed the data to make them available on the recent genome assemblies. The data include observed frequencies of transcription starting sites (TSSs) based on the realignment of CAGE reads, and TSS peaks that are converted from those based on the previous reference. Annotations of the peak names were also updated based on the latest public databases. The reprocessed results enable us to examine frequencies of transcription initiations on the recent genome assemblies and to refer promoters with updated information across the genome assemblies consistently.

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.

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

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

USDA-ARS?s Scientific Manuscript database

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

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.

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

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.

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

PubMed

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

2014-12-01

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

Single haplotype assembly of the human genome from a hydatidiform mole

PubMed Central

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

2014-01-01

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

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 genome resource to address mechanisms of developmental programming: determination of the fetal sheep heart transcriptome.

PubMed

Cox, Laura A; Glenn, Jeremy P; Spradling, Kimberly D; Nijland, Mark J; Garcia, Roy; Nathanielsz, Peter W; Ford, Stephen P

2012-06-15

The pregnant sheep has provided seminal insights into reproduction related to animal and human development (ovarian function, fertility, implantation, fetal growth, parturition and lactation). Fetal sheep physiology has been extensively studied since 1950, contributing significantly to the basis for our understanding of many aspects of fetal development and behaviour that remain in use in clinical practice today. Understanding mechanisms requires the combination of systems approaches uniquely available in fetal sheep with the power of genomic studies. Absence of the full range of sheep genomic resources has limited the full realization of the power of this model, impeding progress in emerging areas of pregnancy biology such as developmental programming. We have examined the expressed fetal sheep heart transcriptome using high-throughput sequencing technologies. In so doing we identified 36,737 novel transcripts and describe genes, gene variants and pathways relevant to fundamental developmental mechanisms. Genes with the highest expression levels and with novel exons in the fetal heart transcriptome are known to play central roles in muscle development. We show that high-throughput sequencing methods can generate extensive transcriptome information in the absence of an assembled and annotated genome for that species. The gene sequence data obtained provide a unique genomic resource for sheep specific genetic technology development and, combined with the polymorphism data, augment annotation and assembly of the sheep genome. In addition, identification and pathway analysis of novel fetal sheep heart transcriptome splice variants is a first step towards revealing mechanisms of genetic variation and gene environment interactions during fetal heart development.

A genome resource to address mechanisms of developmental programming: determination of the fetal sheep heart transcriptome

PubMed Central

Cox, Laura A; Glenn, Jeremy P; Spradling, Kimberly D; Nijland, Mark J; Garcia, Roy; Nathanielsz, Peter W; Ford, Stephen P

2012-01-01

The pregnant sheep has provided seminal insights into reproduction related to animal and human development (ovarian function, fertility, implantation, fetal growth, parturition and lactation). Fetal sheep physiology has been extensively studied since 1950, contributing significantly to the basis for our understanding of many aspects of fetal development and behaviour that remain in use in clinical practice today. Understanding mechanisms requires the combination of systems approaches uniquely available in fetal sheep with the power of genomic studies. Absence of the full range of sheep genomic resources has limited the full realization of the power of this model, impeding progress in emerging areas of pregnancy biology such as developmental programming. We have examined the expressed fetal sheep heart transcriptome using high-throughput sequencing technologies. In so doing we identified 36,737 novel transcripts and describe genes, gene variants and pathways relevant to fundamental developmental mechanisms. Genes with the highest expression levels and with novel exons in the fetal heart transcriptome are known to play central roles in muscle development. We show that high-throughput sequencing methods can generate extensive transcriptome information in the absence of an assembled and annotated genome for that species. The gene sequence data obtained provide a unique genomic resource for sheep specific genetic technology development and, combined with the polymorphism data, augment annotation and assembly of the sheep genome. In addition, identification and pathway analysis of novel fetal sheep heart transcriptome splice variants is a first step towards revealing mechanisms of genetic variation and gene environment interactions during fetal heart development. PMID:22508961

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.

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...

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

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.

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

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.

An ethnically relevant consensus Korean reference genome is a step towards personal reference genomes

PubMed Central

Cho, Yun Sung; Kim, Hyunho; Kim, Hak-Min; Jho, Sungwoong; Jun, JeHoon; Lee, Yong Joo; Chae, Kyun Shik; Kim, Chang Geun; Kim, Sangsoo; Eriksson, Anders; Edwards, Jeremy S.; Lee, Semin; Kim, Byung Chul; Manica, Andrea; Oh, Tae-Kwang; Church, George M.; Bhak, Jong

2016-01-01

Human genomes are routinely compared against a universal reference. However, this strategy could miss population-specific and personal genomic variations, which may be detected more efficiently using an ethnically relevant or personal reference. Here we report a hybrid assembly of a Korean reference genome (KOREF) for constructing personal and ethnic references by combining sequencing and mapping methods. We also build its consensus variome reference, providing information on millions of variants from 40 additional ethnically homogeneous genomes from the Korean Personal Genome Project. We find that the ethnically relevant consensus reference can be beneficial for efficient variant detection. Systematic comparison of human assemblies shows the importance of assembly quality, suggesting the necessity of new technologies to comprehensively map ethnic and personal genomic structure variations. In the era of large-scale population genome projects, the leveraging of ethnicity-specific genome assemblies as well as the human reference genome will accelerate mapping all human genome diversity. PMID:27882922

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.

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

A first generation BAC-based physical map of the rainbow trout genome

PubMed Central

Palti, Yniv; Luo, Ming-Cheng; Hu, Yuqin; Genet, Carine; You, Frank M; Vallejo, Roger L; Thorgaard, Gary H; Wheeler, Paul A; Rexroad, Caird E

2009-01-01

Background Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. A bacterial artificial chromosome (BAC) physical map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) for improving rainbow trout aquaculture production. This resource will also facilitate efforts to obtain and assemble a whole-genome reference sequence for this species. Results The physical map was constructed from DNA fingerprinting of 192,096 BAC clones using the 4-color high-information content fingerprinting (HICF) method. The clones were assembled into physical map contigs using the finger-printing contig (FPC) program. The map is composed of 4,173 contigs and 9,379 singletons. The total number of unique fingerprinting fragments (consensus bands) in contigs is 1,185,157, which corresponds to an estimated physical length of 2.0 Gb. The map assembly was validated by 1) comparison with probe hybridization results and agarose gel fingerprinting contigs; and 2) anchoring large contigs to the microsatellite-based genetic linkage map. Conclusion The production and validation of the first BAC physical map of the rainbow trout genome is described in this paper. We are currently integrating this map with the NCCCWA genetic map using more than 200 microsatellites isolated from BAC end sequences and by identifying BACs that harbor more than 300 previously mapped markers. The availability of an integrated physical and genetic map will enable detailed comparative genome analyses, fine mapping of QTL, positional cloning, selection of positional candidate genes for economically important traits and the incorporation of MAS into rainbow trout breeding programs. PMID:19814815

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.

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-...

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

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.

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.

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

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.

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.

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.

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

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

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.

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.

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

PubMed Central

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

2007-01-01

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

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...

Stewardship of the Maize B73 feference genome assembly

USDA-ARS?s Scientific Manuscript database

The release of version 4 of the B73 reference genome assembly is imminent. However, continued improvement of the assembly is likely to fall to the maize research community. Toward this end, and recognizing the importance of an accurate and well-curated reference genome, MaizeGDB, Gramene, and the Ge...

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).

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.

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

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).

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

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/.

Best practices for evaluating single nucleotide variant calling methods for microbial genomics

PubMed Central

Olson, Nathan D.; Lund, Steven P.; Colman, Rebecca E.; Foster, Jeffrey T.; Sahl, Jason W.; Schupp, James M.; Keim, Paul; Morrow, Jayne B.; Salit, Marc L.; Zook, Justin M.

2015-01-01

Innovations in sequencing technologies have allowed biologists to make incredible advances in understanding biological systems. As experience grows, researchers increasingly recognize that analyzing the wealth of data provided by these new sequencing platforms requires careful attention to detail for robust results. Thus far, much of the scientific Communit’s focus for use in bacterial genomics has been on evaluating genome assembly algorithms and rigorously validating assembly program performance. Missing, however, is a focus on critical evaluation of variant callers for these genomes. Variant calling is essential for comparative genomics as it yields insights into nucleotide-level organismal differences. Variant calling is a multistep process with a host of potential error sources that may lead to incorrect variant calls. Identifying and resolving these incorrect calls is critical for bacterial genomics to advance. The goal of this review is to provide guidance on validating algorithms and pipelines used in variant calling for bacterial genomics. First, we will provide an overview of the variant calling procedures and the potential sources of error associated with the methods. We will then identify appropriate datasets for use in evaluating algorithms and describe statistical methods for evaluating algorithm performance. As variant calling moves from basic research to the applied setting, standardized methods for performance evaluation and reporting are required; it is our hope that this review provides the groundwork for the development of these standards. PMID:26217378

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.

Assembling networks of microbial genomes using linear programming.

PubMed

Holloway, Catherine; Beiko, Robert G

2010-11-20

Microbial genomes exhibit complex sets of genetic affinities due to lateral genetic transfer. Assessing the relative contributions of parent-to-offspring inheritance and gene sharing is a vital step in understanding the evolutionary origins and modern-day function of an organism, but recovering and showing these relationships is a challenging problem. We have developed a new approach that uses linear programming to find between-genome relationships, by treating tables of genetic affinities (here, represented by transformed BLAST e-values) as an optimization problem. Validation trials on simulated data demonstrate the effectiveness of the approach in recovering and representing vertical and lateral relationships among genomes. Application of the technique to a set comprising Aquifex aeolicus and 75 other thermophiles showed an important role for large genomes as 'hubs' in the gene sharing network, and suggested that genes are preferentially shared between organisms with similar optimal growth temperatures. We were also able to discover distinct and common genetic contributors to each sequenced representative of genus Pseudomonas. The linear programming approach we have developed can serve as an effective inference tool in its own right, and can be an efficient first step in a more-intensive phylogenomic analysis.

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

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 .

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.

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.

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/.

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

PubMed

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

2016-08-17

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

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.

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

DOGMA: domain-based transcriptome and proteome quality assessment.

PubMed

Dohmen, Elias; Kremer, Lukas P M; Bornberg-Bauer, Erich; Kemena, Carsten

2016-09-01

Genome studies have become cheaper and easier than ever before, due to the decreased costs of high-throughput sequencing and the free availability of analysis software. However, the quality of genome or transcriptome assemblies can vary a lot. Therefore, quality assessment of assemblies and annotations are crucial aspects of genome analysis pipelines. We developed DOGMA, a program for fast and easy quality assessment of transcriptome and proteome data based on conserved protein domains. DOGMA measures the completeness of a given transcriptome or proteome and provides information about domain content for further analysis. DOGMA provides a very fast way to do quality assessment within seconds. DOGMA is implemented in Python and published under GNU GPL v.3 license. The source code is available on https://ebbgit.uni-muenster.de/domainWorld/DOGMA/ CONTACTS: e.dohmen@wwu.de or c.kemena@wwu.de 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.

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.

ILP-based maximum likelihood genome scaffolding

PubMed Central

2014-01-01

Background Interest in de novo genome assembly has been renewed in the past decade due to rapid advances in high-throughput sequencing (HTS) technologies which generate relatively short reads resulting in highly fragmented assemblies consisting of contigs. Additional long-range linkage information is typically used to orient, order, and link contigs into larger structures referred to as scaffolds. Due to library preparation artifacts and erroneous mapping of reads originating from repeats, scaffolding remains a challenging problem. In this paper, we provide a scalable scaffolding algorithm (SILP2) employing a maximum likelihood model capturing read mapping uncertainty and/or non-uniformity of contig coverage which is solved using integer linear programming. A Non-Serial Dynamic Programming (NSDP) paradigm is applied to render our algorithm useful in the processing of larger mammalian genomes. To compare scaffolding tools, we employ novel quantitative metrics in addition to the extant metrics in the field. We have also expanded the set of experiments to include scaffolding of low-complexity metagenomic samples. Results SILP2 achieves better scalability throughg a more efficient NSDP algorithm than previous release of SILP. The results show that SILP2 compares favorably to previous methods OPERA and MIP in both scalability and accuracy for scaffolding single genomes of up to human size, and significantly outperforms them on scaffolding low-complexity metagenomic samples. Conclusions Equipped with NSDP, SILP2 is able to scaffold large mammalian genomes, resulting in the longest and most accurate scaffolds. The ILP formulation for the maximum likelihood model is shown to be flexible enough to handle metagenomic samples. PMID:25253180

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

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

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...

Genome sequence of the olive tree, Olea europaea.

PubMed

Cruz, Fernando; Julca, Irene; Gómez-Garrido, Jèssica; Loska, Damian; Marcet-Houben, Marina; Cano, Emilio; Galán, Beatriz; Frias, Leonor; Ribeca, Paolo; Derdak, Sophia; Gut, Marta; Sánchez-Fernández, Manuel; García, Jose Luis; Gut, Ivo G; Vargas, Pablo; Alioto, Tyler S; Gabaldón, Toni

2016-06-27

The Mediterranean olive tree (Olea europaea subsp. europaea) was one of the first trees to be domesticated and is currently of major agricultural importance in the Mediterranean region as the source of olive oil. The molecular bases underlying the phenotypic differences among domesticated cultivars, or between domesticated olive trees and their wild relatives, remain poorly understood. Both wild and cultivated olive trees have 46 chromosomes (2n). A total of 543 Gb of raw DNA sequence from whole genome shotgun sequencing, and a fosmid library containing 155,000 clones from a 1,000+ year-old olive tree (cv. Farga) were generated by Illumina sequencing using different combinations of mate-pair and pair-end libraries. Assembly gave a final genome with a scaffold N50 of 443 kb, and a total length of 1.31 Gb, which represents 95 % of the estimated genome length (1.38 Gb). In addition, the associated fungus Aureobasidium pullulans was partially sequenced. Genome annotation, assisted by RNA sequencing from leaf, root, and fruit tissues at various stages, resulted in 56,349 unique protein coding genes, suggesting recent genomic expansion. Genome completeness, as estimated using the CEGMA pipeline, reached 98.79 %. The assembled draft genome of O. europaea will provide a valuable resource for the study of the evolution and domestication processes of this important tree, and allow determination of the genetic bases of key phenotypic traits. Moreover, it will enhance breeding programs and the formation of new varieties.

GenomeHubs: simple containerized setup of a custom Ensembl database and web server for any species

PubMed Central

Kumar, Sujai; Stevens, Lewis; Blaxter, Mark

2017-01-01

Abstract As the generation and use of genomic datasets is becoming increasingly common in all areas of biology, the need for resources to collate, analyse and present data from one or more genome projects is becoming more pressing. The Ensembl platform is a powerful tool to make genome data and cross-species analyses easily accessible through a web interface and a comprehensive application programming interface. Here we introduce GenomeHubs, which provide a containerized environment to facilitate the setup and hosting of custom Ensembl genome browsers. This simplifies mirroring of existing content and import of new genomic data into the Ensembl database schema. GenomeHubs also provide a set of analysis containers to decorate imported genomes with results of standard analyses and functional annotations and support export to flat files, including EMBL format for submission of assemblies and annotations to International Nucleotide Sequence Database Collaboration. Database URL: http://GenomeHubs.org PMID:28605774

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.

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

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.

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

High molecular weight DNA assembly in vivo for synthetic biology applications.

PubMed

Juhas, Mario; Ajioka, James W

2017-05-01

DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.

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.

AutoAssemblyD: a graphical user interface system for several genome assemblers.

PubMed

Veras, Adonney Allan de Oliveira; de Sá, Pablo Henrique Caracciolo Gomes; Azevedo, Vasco; Silva, Artur; Ramos, Rommel Thiago Jucá

2013-01-01

Next-generation sequencing technologies have increased the amount of biological data generated. Thus, bioinformatics has become important because new methods and algorithms are necessary to manipulate and process such data. However, certain challenges have emerged, such as genome assembly using short reads and high-throughput platforms. In this context, several algorithms have been developed, such as Velvet, Abyss, Euler-SR, Mira, Edna, Maq, SHRiMP, Newbler, ALLPATHS, Bowtie and BWA. However, most such assemblers do not have a graphical interface, which makes their use difficult for users without computing experience given the complexity of the assembler syntax. Thus, to make the operation of such assemblers accessible to users without a computing background, we developed AutoAssemblyD, which is a graphical tool for genome assembly submission and remote management by multiple assemblers through XML templates. AssemblyD is freely available at https://sourceforge.net/projects/autoassemblyd. It requires Sun jdk 6 or higher.

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.

Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis

PubMed Central

Shao, Changwei; Niu, Yongchao; Rastas, Pasi; Liu, Yang; Xie, Zhiyuan; Li, Hengde; Wang, Lei; Jiang, Yong; Tai, Shuaishuai; Tian, Yongsheng; Sakamoto, Takashi; Chen, Songlin

2015-01-01

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1–8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species. PMID:25762582

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.

PubMed

Wang, Zhiwen; Hobson, Neil; Galindo, Leonardo; Zhu, Shilin; Shi, Daihu; McDill, Joshua; Yang, Linfeng; Hawkins, Simon; Neutelings, Godfrey; Datla, Raju; Lambert, Georgina; Galbraith, David W; Grassa, Christopher J; Geraldes, Armando; Cronk, Quentin C; Cullis, Christopher; Dash, Prasanta K; Kumar, Polumetla A; Cloutier, Sylvie; Sharpe, Andrew G; Wong, Gane K-S; Wang, Jun; Deyholos, Michael K

2012-11-01

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

PubMed Central

Swart, Estienne C.; Bracht, John R.; Magrini, Vincent; Minx, Patrick; Chen, Xiao; Zhou, Yi; Khurana, Jaspreet S.; Goldman, Aaron D.; Nowacki, Mariusz; Schotanus, Klaas; Jung, Seolkyoung; Fulton, Robert S.; Ly, Amy; McGrath, Sean; Haub, Kevin; Wiggins, Jessica L.; Storton, Donna; Matese, John C.; Parsons, Lance; Chang, Wei-Jen; Bowen, Michael S.; Stover, Nicholas A.; Jones, Thomas A.; Eddy, Sean R.; Herrick, Glenn A.; Doak, Thomas G.; Wilson, Richard K.; Mardis, Elaine R.; Landweber, Laura F.

2013-01-01

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease. PMID:23382650

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.

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

USDA-ARS?s Scientific Manuscript database

Genome evolution influences a parasite’s’s pathogenicity, host-pathogen interactions, environmental constraints, and invasion biology, while genome assemblies form the basis of comparative sequence analyses. Given that closely related organisms typically maintain appreciable synteny, the genome asse...

Necklace: combining reference and assembled transcriptomes for more comprehensive RNA-Seq analysis.

PubMed

Davidson, Nadia M; Oshlack, Alicia

2018-05-01

RNA sequencing (RNA-seq) analyses can benefit from performing a genome-guided and de novo assembly, in particular for species where the reference genome or the annotation is incomplete. However, tools for integrating an assembled transcriptome with reference annotation are lacking. Necklace is a software pipeline that runs genome-guided and de novo assembly and combines the resulting transcriptomes with reference genome annotations. Necklace constructs a compact but comprehensive superTranscriptome out of the assembled and reference data. Reads are subsequently aligned and counted in preparation for differential expression testing. Necklace allows a comprehensive transcriptome to be built from a combination of assembled and annotated transcripts, which results in a more comprehensive transcriptome for the majority of organisms. In addition RNA-seq data are mapped back to this newly created superTranscript reference to enable differential expression testing with standard methods.

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.

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea

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

Bowers, Robert M.; Kyrpides, Nikos C.; Stepanauskas, Ramunas

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequencemore » (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.« less

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea

DOE PAGES

Bowers, Robert M.; Kyrpides, Nikos C.; Stepanauskas, Ramunas; ...

2017-08-08

Here, we present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a MetagenomeAssembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Genemore » Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.« less

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea

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

Bowers, Robert M.; Kyrpides, Nikos C.; Stepanauskas, Ramunas

Here, we present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a MetagenomeAssembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Genemore » Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.« less

High-resolution linkage map and chromosome-scale genome assembly for cassava (Manihot esculenta Crantz) from 10 populations.

PubMed

2014-12-11

Cassava (Manihot esculenta Crantz) is a major staple crop in Africa, Asia, and South America, and its starchy roots provide nourishment for 800 million people worldwide. Although native to South America, cassava was brought to Africa 400-500 years ago and is now widely cultivated across sub-Saharan Africa, but it is subject to biotic and abiotic stresses. To assist in the rapid identification of markers for pathogen resistance and crop traits, and to accelerate breeding programs, we generated a framework map for M. esculenta Crantz from reduced representation sequencing [genotyping-by-sequencing (GBS)]. The composite 2412-cM map integrates 10 biparental maps (comprising 3480 meioses) and organizes 22,403 genetic markers on 18 chromosomes, in agreement with the observed karyotype. We used the map to anchor 71.9% of the draft genome assembly and 90.7% of the predicted protein-coding genes. The chromosome-anchored genome sequence will be useful for breeding improvement by assisting in the rapid identification of markers linked to important traits, and in providing a framework for genomic selection-enhanced breeding of this important crop. Copyright © 2015 International Cassava Genetic Map Consortium (ICGMC).

High-resolution linkage map and chromosome-scale genome assembly for cassava ( Manihot esculenta Crantz) from 10 populations

DOE PAGES

Lyons, Jessica

2014-12-11

Cassava Manihot esculenta Crantz) is a major staple crop in Africa, Asia, and South America, and its starchy roots provide nourishment for 800 million people worldwide. Although native to South America, cassava was brought to Africa 400–500 years ago and is now widely cultivated across sub-Saharan Africa, but it is subject to biotic and abiotic stresses. To assist in the rapid identification of markers for pathogen resistance and crop traits, and to accelerate breeding programs, we generated a framework map for M. esculent Crantz from reduced representation sequencing [genotyping-by-sequencing (GBS)]. The composite 2412-cM map integrates 10 biparental maps (comprising 3480more » meioses) and organizes 22,403 genetic markers on 18 chromosomes, in agreement with the observed karyotype. Here, we used the map to anchor 71.9% of the draft genome assembly and 90.7% of the predicted protein-coding genes. The chromosome-anchored genome sequence will be useful for breeding improvement by assisting in the rapid identification of markers linked to important traits, and in providing a framework for genomic selectionenhanced breeding of this important crop.« less

High-resolution linkage map and chromosome-scale genome assembly for cassava ( Manihot esculenta Crantz) from 10 populations

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

Lyons, Jessica

Cassava Manihot esculenta Crantz) is a major staple crop in Africa, Asia, and South America, and its starchy roots provide nourishment for 800 million people worldwide. Although native to South America, cassava was brought to Africa 400–500 years ago and is now widely cultivated across sub-Saharan Africa, but it is subject to biotic and abiotic stresses. To assist in the rapid identification of markers for pathogen resistance and crop traits, and to accelerate breeding programs, we generated a framework map for M. esculent Crantz from reduced representation sequencing [genotyping-by-sequencing (GBS)]. The composite 2412-cM map integrates 10 biparental maps (comprising 3480more » meioses) and organizes 22,403 genetic markers on 18 chromosomes, in agreement with the observed karyotype. Here, we used the map to anchor 71.9% of the draft genome assembly and 90.7% of the predicted protein-coding genes. The chromosome-anchored genome sequence will be useful for breeding improvement by assisting in the rapid identification of markers linked to important traits, and in providing a framework for genomic selectionenhanced breeding of this important crop.« less

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

The sequence and de novo assembly of the giant panda genome

PubMed Central

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

2013-01-01

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

Genome Sequencing and Assembly by Long Reads in Plants

PubMed Central

Li, Changsheng; Lin, Feng; An, Dong; Huang, Ruidong

2017-01-01

Plant genomes generated by Sanger and Next Generation Sequencing (NGS) have provided insight into species diversity and evolution. However, Sanger sequencing is limited in its applications due to high cost, labor intensity, and low throughput, while NGS reads are too short to resolve abundant repeats and polyploidy, leading to incomplete or ambiguous assemblies. The advent and improvement of long-read sequencing by Third Generation Sequencing (TGS) methods such as PacBio and Nanopore have shown promise in producing high-quality assemblies for complex genomes. Here, we review the development of sequencing, introducing the application as well as considerations of experimental design in TGS of plant genomes. We also introduce recent revolutionary scaffolding technologies including BioNano, Hi-C, and 10× Genomics. We expect that the informative guidance for genome sequencing and assembly by long reads will benefit the initiation of scientists’ projects. PMID:29283420

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

USDA-ARS?s Scientific Manuscript database

The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high-resolution genome maps saturated with ordered markers to assist in anchoring and orienting BAC contigs/ sequence scaffolds for whole genome sequence assembly. Radiation hybrid (RH) mapping has proven to be an e...

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.

Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

PubMed Central

Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D.; Malinsky, Sophie; Bétermier, Mireille

2011-01-01

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics. PMID:21533177

Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

PubMed

Kapusta, Aurélie; Matsuda, Atsushi; Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D; Malinsky, Sophie; Bétermier, Mireille

2011-04-01

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.

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

USDA-ARS?s Scientific Manuscript database

Over the course of 20 months, we assembled a sugar beet genome (700 - 800 Mb) into a close representation of the nine haploid chromosomes of beet. This result was obtained by sequentially assembling sequences >40 kb in length, orienting these assemblies via optical mapping, and scaffolding with in v...

Evaluation of nine popular de novo assemblers in microbial genome assembly.

PubMed

Forouzan, Esmaeil; Maleki, Masoumeh Sadat Mousavi; Karkhane, Ali Asghar; Yakhchali, Bagher

2017-12-01

Next generation sequencing (NGS) technologies are revolutionizing biology, with Illumina being the most popular NGS platform. Short read assembly is a critical part of most genome studies using NGS. Hence, in this study, the performance of nine well-known assemblers was evaluated in the assembly of seven different microbial genomes. Effect of different read coverage and k-mer parameters on the quality of the assembly were also evaluated on both simulated and actual read datasets. Our results show that the performance of assemblers on real and simulated datasets could be significantly different, mainly because of coverage bias. According to outputs on actual read datasets, for all studied read coverages (of 7×, 25× and 100×), SPAdes and IDBA-UD clearly outperformed other assemblers based on NGA50 and accuracy metrics. Velvet is the most conservative assembler with the lowest NGA50 and error rate. Copyright © 2017. Published by Elsevier B.V.

Meraculous: De Novo Genome Assembly with Short Paired-End Reads

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

Chapman, Jarrod A.; Ho, Isaac; Sunkara, Sirisha

2011-08-18

We describe a new algorithm, meraculous, for whole genome assembly of deep paired-end short reads, and apply it to the assembly of a dataset of paired 75-bp Illumina reads derived from the 15.4 megabase genome of the haploid yeast Pichia stipitis. More than 95% of the genome is recovered, with no errors; half the assembled sequence is in contigs longer than 101 kilobases and in scaffolds longer than 269 kilobases. Incorporating fosmid ends recovers entire chromosomes. Meraculous relies on an efficient and conservative traversal of the subgraph of the k-mer (deBruijn) graph of oligonucleotides with unique high quality extensions inmore » the dataset, avoiding an explicit error correction step as used in other short-read assemblers. A novel memory-efficient hashing scheme is introduced. The resulting contigs are ordered and oriented using paired reads separated by ~280 bp or ~3.2 kbp, and many gaps between contigs can be closed using paired-end placements. Practical issues with the dataset are described, and prospects for assembling larger genomes are discussed.« less

Comparison of de novo assembly statistics of Cucumis sativus L.

NASA Astrophysics Data System (ADS)

Wojcieszek, Michał; Kuśmirek, Wiktor; Pawełkowicz, Magdalena; PlÄ der, Wojciech; Nowak, Robert M.

2017-08-01

Genome sequencing is the core of genomic research. With the development of NGS and lowering the cost of procedure there is another tight gap - genome assembly. Developing the proper tool for this task is essential as quality of genome has important impact on further research. Here we present comparison of several de Bruijn assemblers tested on C. sativus genomic reads. The assessment shows that newly developed software - dnaasm provides better results in terms of quantity and quality. The number of generated sequences is lower by 5 - 33% with even two fold higher N50. Quality check showed reliable results were generated by dnaasm. This provides us with very strong base for future genomic analysis.

A physical map of the bovine genome

PubMed Central

Snelling, Warren M; Chiu, Readman; Schein, Jacqueline E; Hobbs, Matthew; Abbey, Colette A; Adelson, David L; Aerts, Jan; Bennett, Gary L; Bosdet, Ian E; Boussaha, Mekki; Brauning, Rudiger; Caetano, Alexandre R; Costa, Marcos M; Crawford, Allan M; Dalrymple, Brian P; Eggen, André; Everts-van der Wind, Annelie; Floriot, Sandrine; Gautier, Mathieu; Gill, Clare A; Green, Ronnie D; Holt, Robert; Jann, Oliver; Jones, Steven JM; Kappes, Steven M; Keele, John W; de Jong, Pieter J; Larkin, Denis M; Lewin, Harris A; McEwan, John C; McKay, Stephanie; Marra, Marco A; Mathewson, Carrie A; Matukumalli, Lakshmi K; Moore, Stephen S; Murdoch, Brenda; Nicholas, Frank W; Osoegawa, Kazutoyo; Roy, Alice; Salih, Hanni; Schibler, Laurent; Schnabel, Robert D; Silveri, Licia; Skow, Loren C; Smith, Timothy PL; Sonstegard, Tad S; Taylor, Jeremy F; Tellam, Ross; Van Tassell, Curtis P; Williams, John L; Womack, James E; Wye, Natasja H; Yang, George; Zhao, Shaying

2007-01-01

Background Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project. Results A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly. Conclusion Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans. PMID:17697342

Annotated Draft Genome Assemblies for the Northern Bobwhite (Colinus virginianus) and the Scaled Quail (Callipepla squamata) Reveal Disparate Estimates of Modern Genome Diversity and Historic Effective Population Size.

PubMed

Oldeschulte, David L; Halley, Yvette A; Wilson, Miranda L; Bhattarai, Eric K; Brashear, Wesley; Hill, Joshua; Metz, Richard P; Johnson, Charles D; Rollins, Dale; Peterson, Markus J; Bickhart, Derek M; Decker, Jared E; Sewell, John F; Seabury, Christopher M

2017-09-07

Northern bobwhite ( Colinus virginianus ; hereafter bobwhite) and scaled quail ( Callipepla squamata ) populations have suffered precipitous declines across most of their US ranges. Illumina-based first- (v1.0) and second- (v2.0) generation draft genome assemblies for the scaled quail and the bobwhite produced N50 scaffold sizes of 1.035 and 2.042 Mb, thereby producing a 45-fold improvement in contiguity over the existing bobwhite assembly, and ≥90% of the assembled genomes were captured within 1313 and 8990 scaffolds, respectively. The scaled quail assembly (v1.0 = 1.045 Gb) was ∼20% smaller than the bobwhite (v2.0 = 1.254 Gb), which was supported by kmer-based estimates of genome size. Nevertheless, estimates of GC content (41.72%; 42.66%), genome-wide repetitive content (10.40%; 10.43%), and MAKER-predicted protein coding genes (17,131; 17,165) were similar for the scaled quail (v1.0) and bobwhite (v2.0) assemblies, respectively. BUSCO analyses utilizing 3023 single-copy orthologs revealed a high level of assembly completeness for the scaled quail (v1.0; 84.8%) and the bobwhite (v2.0; 82.5%), as verified by comparison with well-established avian genomes. We also detected 273 putative segmental duplications in the scaled quail genome (v1.0), and 711 in the bobwhite genome (v2.0), including some that were shared among both species. Autosomal variant prediction revealed ∼2.48 and 4.17 heterozygous variants per kilobase within the scaled quail (v1.0) and bobwhite (v2.0) genomes, respectively, and estimates of historic effective population size were uniformly higher for the bobwhite across all time points in a coalescent model. However, large-scale declines were predicted for both species beginning ∼15-20 KYA. Copyright © 2017 Oldeschulte et al.

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.

Draft genome of the lined seahorse, Hippocampus erectus.

PubMed

Lin, Qiang; Qiu, Ying; Gu, Ruobo; Xu, Meng; Li, Jia; Bian, Chao; Zhang, Huixian; Qin, Geng; Zhang, Yanhong; Luo, Wei; Chen, Jieming; You, Xinxin; Fan, Mingjun; Sun, Min; Xu, Pao; Venkatesh, Byrappa; Xu, Junming; Fu, Hongtuo; Shi, Qiong

2017-06-01

The lined seahorse, Hippocampus erectus , is an Atlantic species and mainly inhabits shallow sea beds or coral reefs. It has become very popular in China for its wide use in traditional Chinese medicine. In order to improve the aquaculture yield of this valuable fish species, we are trying to develop genomic resources for assistant selection in genetic breeding. Here, we provide whole genome sequencing, assembly, and gene annotation of the lined seahorse, which can enrich genome resource and further application for its molecular breeding. A total of 174.6 Gb (Gigabase) raw DNA sequences were generated by the Illumina Hiseq2500 platform. The final assembly of the lined seahorse genome is around 458 Mb, representing 94% of the estimated genome size (489 Mb by k-mer analysis). The contig N50 and scaffold N50 reached 14.57 kb and 1.97 Mb, respectively. Quality of the assembled genome was assessed by BUSCO with prediction of 85% of the known vertebrate genes and evaluated using the de novo assembled RNA-seq transcripts to prove a high mapping ratio (more than 99% transcripts could be mapped to the assembly). Using homology-based, de novo and transcriptome-based prediction methods, we predicted 20 788 protein-coding genes in the generated assembly, which is less than our previously reported gene number (23 458) of the tiger tail seahorse ( H. comes ). We report a draft genome of the lined seahorse. These generated genomic data are going to enrich genome resource of this economically important fish, and also provide insights into the genetic mechanisms of its iconic morphology and male pregnancy behavior. © The Authors 2017. Published by Oxford University Press.

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

Draft genome of the lined seahorse, Hippocampus erectus

PubMed Central

Lin, Qiang; Qiu, Ying; Gu, Ruobo; Xu, Meng; Li, Jia; Bian, Chao; Zhang, Huixian; Qin, Geng; Zhang, Yanhong; Luo, Wei; Chen, Jieming; You, Xinxin; Fan, Mingjun; Sun, Min; Xu, Pao; Venkatesh, Byrappa

2017-01-01

Abstract Background: The lined seahorse, Hippocampus erectus, is an Atlantic species and mainly inhabits shallow sea beds or coral reefs. It has become very popular in China for its wide use in traditional Chinese medicine. In order to improve the aquaculture yield of this valuable fish species, we are trying to develop genomic resources for assistant selection in genetic breeding. Here, we provide whole genome sequencing, assembly, and gene annotation of the lined seahorse, which can enrich genome resource and further application for its molecular breeding. Findings: A total of 174.6 Gb (Gigabase) raw DNA sequences were generated by the Illumina Hiseq2500 platform. The final assembly of the lined seahorse genome is around 458 Mb, representing 94% of the estimated genome size (489 Mb by k-mer analysis). The contig N50 and scaffold N50 reached 14.57 kb and 1.97 Mb, respectively. Quality of the assembled genome was assessed by BUSCO with prediction of 85% of the known vertebrate genes and evaluated using the de novo assembled RNA-seq transcripts to prove a high mapping ratio (more than 99% transcripts could be mapped to the assembly). Using homology-based, de novo and transcriptome-based prediction methods, we predicted 20 788 protein-coding genes in the generated assembly, which is less than our previously reported gene number (23 458) of the tiger tail seahorse (H. comes). Conclusion: We report a draft genome of the lined seahorse. These generated genomic data are going to enrich genome resource of this economically important fish, and also provide insights into the genetic mechanisms of its iconic morphology and male pregnancy behavior. PMID:28444302

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.

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.

Genome resources for climate-resilient cowpea, an essential crop for food security.

PubMed

Muñoz-Amatriaín, María; Mirebrahim, Hamid; Xu, Pei; Wanamaker, Steve I; Luo, MingCheng; Alhakami, Hind; Alpert, Matthew; Atokple, Ibrahim; Batieno, Benoit J; Boukar, Ousmane; Bozdag, Serdar; Cisse, Ndiaga; Drabo, Issa; Ehlers, Jeffrey D; Farmer, Andrew; Fatokun, Christian; Gu, Yong Q; Guo, Yi-Ning; Huynh, Bao-Lam; Jackson, Scott A; Kusi, Francis; Lawley, Cynthia T; Lucas, Mitchell R; Ma, Yaqin; Timko, Michael P; Wu, Jiajie; You, Frank; Barkley, Noelle A; Roberts, Philip A; Lonardi, Stefano; Close, Timothy J

2017-03-01

Cowpea (Vigna unguiculata L. Walp.) is a legume crop that is resilient to hot and drought-prone climates, and a primary source of protein in sub-Saharan Africa and other parts of the developing world. However, genome resources for cowpea have lagged behind most other major crops. Here we describe foundational genome resources and their application to the analysis of germplasm currently in use in West African breeding programs. Resources developed from the African cultivar IT97K-499-35 include a whole-genome shotgun (WGS) assembly, a bacterial artificial chromosome (BAC) physical map, and assembled sequences from 4355 BACs. These resources and WGS sequences of an additional 36 diverse cowpea accessions supported the development of a genotyping assay for 51 128 SNPs, which was then applied to five bi-parental RIL populations to produce a consensus genetic map containing 37 372 SNPs. This genetic map enabled the anchoring of 100 Mb of WGS and 420 Mb of BAC sequences, an exploration of genetic diversity along each linkage group, and clarification of macrosynteny between cowpea and common bean. The SNP assay enabled a diversity analysis of materials from West African breeding programs. Two major subpopulations exist within those materials, one of which has significant parentage from South and East Africa and more diversity. There are genomic regions of high differentiation between subpopulations, one of which coincides with a cluster of nodulin genes. The new resources and knowledge help to define goals and accelerate the breeding of improved varieties to address food security issues related to limited-input small-holder farming and climate stress. © 2016 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

PubMed Central

Hamilton, Eileen P; Kapusta, Aurélie; Huvos, Piroska E; Bidwell, Shelby L; Zafar, Nikhat; Tang, Haibao; Hadjithomas, Michalis; Krishnakumar, Vivek; Badger, Jonathan H; Caler, Elisabet V; Russ, Carsten; Zeng, Qiandong; Fan, Lin; Levin, Joshua Z; Shea, Terrance; Young, Sarah K; Hegarty, Ryan; Daza, Riza; Gujja, Sharvari; Wortman, Jennifer R; Birren, Bruce W; Nusbaum, Chad; Thomas, Jainy; Carey, Clayton M; Pritham, Ellen J; Feschotte, Cédric; Noto, Tomoko; Mochizuki, Kazufumi; Papazyan, Romeo; Taverna, Sean D; Dear, Paul H; Cassidy-Hanley, Donna M; Xiong, Jie; Miao, Wei; Orias, Eduardo; Coyne, Robert S

2016-01-01

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena’s germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum. DOI: http://dx.doi.org/10.7554/eLife.19090.001 PMID:27892853

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.

Components of Adenovirus Genome Packaging

PubMed Central

Ahi, Yadvinder S.; Mittal, Suresh K.

2016-01-01

Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging. PMID:27721809

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

Lineage-Specific Biology Revealed by a Finished Genome Assembly of the Mouse

PubMed Central

Hillier, LaDeana W.; Zody, Michael C.; Goldstein, Steve; She, Xinwe; Bult, Carol J.; Agarwala, Richa; Cherry, Joshua L.; DiCuccio, Michael; Hlavina, Wratko; Kapustin, Yuri; Meric, Peter; Maglott, Donna; Birtle, Zoë; Marques, Ana C.; Graves, Tina; Zhou, Shiguo; Teague, Brian; Potamousis, Konstantinos; Churas, Christopher; Place, Michael; Herschleb, Jill; Runnheim, Ron; Forrest, Daniel; Amos-Landgraf, James; Schwartz, David C.; Cheng, Ze; Lindblad-Toh, Kerstin; Eichler, Evan E.; Ponting, Chris P.

2009-01-01

The mouse (Mus musculus) is the premier animal model for understanding human disease and development. Here we show that a comprehensive understanding of mouse biology is only possible with the availability of a finished, high-quality genome assembly. The finished clone-based assembly of the mouse strain C57BL/6J reported here has over 175,000 fewer gaps and over 139 Mb more of novel sequence, compared with the earlier MGSCv3 draft genome assembly. In a comprehensive analysis of this revised genome sequence, we are now able to define 20,210 protein-coding genes, over a thousand more than predicted in the human genome (19,042 genes). In addition, we identified 439 long, non–protein-coding RNAs with evidence for transcribed orthologs in human. We analyzed the complex and repetitive landscape of 267 Mb of sequence that was missing or misassembled in the previously published assembly, and we provide insights into the reasons for its resistance to sequencing and assembly by whole-genome shotgun approaches. Duplicated regions within newly assembled sequence tend to be of more recent ancestry than duplicates in the published draft, correcting our initial understanding of recent evolution on the mouse lineage. These duplicates appear to be largely composed of sequence regions containing transposable elements and duplicated protein-coding genes; of these, some may be fixed in the mouse population, but at least 40% of segmentally duplicated sequences are copy number variable even among laboratory mouse strains. Mouse lineage-specific regions contain 3,767 genes drawn mainly from rapidly-changing gene families associated with reproductive functions. The finished mouse genome assembly, therefore, greatly improves our understanding of rodent-specific biology and allows the delineation of ancestral biological functions that are shared with human from derived functions that are not. PMID:19468303

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.

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.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

PubMed

Oldfield, Lauren M; Grzesik, Peter; Voorhies, Alexander A; Alperovich, Nina; MacMath, Derek; Najera, Claudia D; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N; Montague, Michael G; Friedman, Robert M; Desai, Prashant J; Vashee, Sanjay

2017-10-17

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS YA , replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods

PubMed Central

Grzesik, Peter; Voorhies, Alexander A.; Alperovich, Nina; MacMath, Derek; Najera, Claudia D.; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N.; Montague, Michael G.; Friedman, Robert M.; Desai, Prashant J.

2017-01-01

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats. PMID:28928148

Comparative scaffolding and gap filling of ancient bacterial genomes applied to two ancient Yersinia pestis genomes

PubMed Central

Doerr, Daniel; Chauve, Cedric

2017-01-01

Yersinia pestis is the causative agent of the bubonic plague, a disease responsible for several dramatic historical pandemics. Progress in ancient DNA (aDNA) sequencing rendered possible the sequencing of whole genomes of important human pathogens, including the ancient Y. pestis strains responsible for outbreaks of the bubonic plague in London in the 14th century and in Marseille in the 18th century, among others. However, aDNA sequencing data are still characterized by short reads and non-uniform coverage, so assembling ancient pathogen genomes remains challenging and often prevents a detailed study of genome rearrangements. It has recently been shown that comparative scaffolding approaches can improve the assembly of ancient Y. pestis genomes at a chromosome level. In the present work, we address the last step of genome assembly, the gap-filling stage. We describe an optimization-based method AGapEs (ancestral gap estimation) to fill in inter-contig gaps using a combination of a template obtained from related extant genomes and aDNA reads. We show how this approach can be used to refine comparative scaffolding by selecting contig adjacencies supported by a mix of unassembled aDNA reads and comparative signal. We applied our method to two Y. pestis data sets from the London and Marseilles outbreaks, for which we obtained highly improved genome assemblies for both genomes, comprised of, respectively, five and six scaffolds with 95 % of the assemblies supported by ancient reads. We analysed the genome evolution between both ancient genomes in terms of genome rearrangements, and observed a high level of synteny conservation between these strains. PMID:29114402

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

PubMed

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

2016-06-13

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

A Multi-Platform Draft de novo Genome Assembly and Comparative Analysis for the Scarlet Macaw (Ara macao)

PubMed Central

Seabury, Christopher M.; Dowd, Scot E.; Seabury, Paul M.; Raudsepp, Terje; Brightsmith, Donald J.; Liboriussen, Poul; Halley, Yvette; Fisher, Colleen A.; Owens, Elaine; Viswanathan, Ganesh; Tizard, Ian R.

2013-01-01

Data deposition to NCBI Genomes This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMXX00000000 (SMACv1.0, unscaffolded genome assembly). The version described in this paper is the first version (AMXX01000000). The scaffolded assembly (SMACv1.1) has been deposited at DDBJ/EMBL/GenBank under the accession AOUJ00000000, and is also the first version (AOUJ01000000). Strong biological interest in traits such as the acquisition and utilization of speech, cognitive abilities, and longevity catalyzed the utilization of two next-generation sequencing platforms to provide the first-draft de novo genome assembly for the large, new world parrot Ara macao (Scarlet Macaw). Despite the challenges associated with genome assembly for an outbred avian species, including 951,507 high-quality putative single nucleotide polymorphisms, the final genome assembly (>1.035 Gb) includes more than 997 Mb of unambiguous sequence data (excluding N’s). Cytogenetic analyses including ZooFISH revealed complex rearrangements associated with two scarlet macaw macrochromosomes (AMA6, AMA7), which supports the hypothesis that translocations, fusions, and intragenomic rearrangements are key factors associated with karyotype evolution among parrots. In silico annotation of the scarlet macaw genome provided robust evidence for 14,405 nuclear gene annotation models, their predicted transcripts and proteins, and a complete mitochondrial genome. Comparative analyses involving the scarlet macaw, chicken, and zebra finch genomes revealed high levels of nucleotide-based conservation as well as evidence for overall genome stability among the three highly divergent species. Application of a new whole-genome analysis of divergence involving all three species yielded prioritized candidate genes and noncoding regions for parrot traits of interest (i.e., speech, intelligence, longevity) which were independently supported by the results of previous human GWAS studies. We also observed evidence for genes and noncoding loci that displayed extreme conservation across the three avian lineages, thereby reflecting their likely biological and developmental importance among birds. PMID:23667475

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.

Ten steps to get started in Genome Assembly and Annotation

PubMed Central

Dominguez Del Angel, Victoria; Hjerde, Erik; Sterck, Lieven; Capella-Gutierrez, Salvadors; Notredame, Cederic; Vinnere Pettersson, Olga; Amselem, Joelle; Bouri, Laurent; Bocs, Stephanie; Klopp, Christophe; Gibrat, Jean-Francois; Vlasova, Anna; Leskosek, Brane L.; Soler, Lucile; Binzer-Panchal, Mahesh; Lantz, Henrik

2018-01-01

As a part of the ELIXIR-EXCELERATE efforts in capacity building, we present here 10 steps to facilitate researchers getting started in genome assembly and genome annotation. The guidelines given are broadly applicable, intended to be stable over time, and cover all aspects from start to finish of a general assembly and annotation project. Intrinsic properties of genomes are discussed, as is the importance of using high quality DNA. Different sequencing technologies and generally applicable workflows for genome assembly are also detailed. We cover structural and functional annotation and encourage readers to also annotate transposable elements, something that is often omitted from annotation workflows. The importance of data management is stressed, and we give advice on where to submit data and how to make your results Findable, Accessible, Interoperable, and Reusable (FAIR). PMID:29568489

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 High-Resolution SNP Array-Based Linkage Map Anchors a New Domestic Cat Draft Genome Assembly and Provides Detailed Patterns of Recombination.

PubMed

Li, Gang; Hillier, LaDeana W; Grahn, Robert A; Zimin, Aleksey V; David, Victor A; Menotti-Raymond, Marilyn; Middleton, Rondo; Hannah, Steven; Hendrickson, Sher; Makunin, Alex; O'Brien, Stephen J; Minx, Pat; Wilson, Richard K; Lyons, Leslie A; Warren, Wesley C; Murphy, William J

2016-06-01

High-resolution genetic and physical maps are invaluable tools for building accurate genome assemblies, and interpreting results of genome-wide association studies (GWAS). Previous genetic and physical maps anchored good quality draft assemblies of the domestic cat genome, enabling the discovery of numerous genes underlying hereditary disease and phenotypes of interest to the biomedical science and breeding communities. However, these maps lacked sufficient marker density to order thousands of shorter scaffolds in earlier assemblies, which instead relied heavily on comparative mapping with related species. A high-resolution map would aid in validating and ordering chromosome scaffolds from existing and new genome assemblies. Here, we describe a high-resolution genetic linkage map of the domestic cat genome based on genotyping 453 domestic cats from several multi-generational pedigrees on the Illumina 63K SNP array. The final maps include 58,055 SNP markers placed relative to 6637 markers with unique positions, distributed across all autosomes and the X chromosome. Our final sex-averaged maps span a total autosomal length of 4464 cM, the longest described linkage map for any mammal, confirming length estimates from a previous microsatellite-based map. The linkage map was used to order and orient the scaffolds from a substantially more contiguous domestic cat genome assembly (Felis catus v8.0), which incorporated ∼20 × coverage of Illumina fragment reads. The new genome assembly shows substantial improvements in contiguity, with a nearly fourfold increase in N50 scaffold size to 18 Mb. We use this map to report probable structural errors in previous maps and assemblies, and to describe features of the recombination landscape, including a massive (∼50 Mb) recombination desert (of virtually zero recombination) on the X chromosome that parallels a similar desert on the porcine X chromosome in both size and physical location. Copyright © 2016 Li et al.

A high-quality carrot genome assembly provides new insights into carotenoid accumulation and asterid genome evolution

USDA-ARS?s Scientific Manuscript database

We report a chromosome-scale assembly and analysis of the Daucus carota genome, an important source of provitamin A in the human diet and the first sequenced genome among members of the Euasterid II clade. We characterized two new polyploidization events, both occurring after the divergence of carro...

Illuminating the Black Box of Genome Sequence Assembly: A Free Online Tool to Introduce Students to Bioinformatics

ERIC Educational Resources Information Center

Taylor, D. Leland; Campbell, A. Malcolm; Heyer, Laurie J.

2013-01-01

Next-generation sequencing technologies have greatly reduced the cost of sequencing genomes. With the current sequencing technology, a genome is broken into fragments and sequenced, producing millions of "reads." A computer algorithm pieces these reads together in the genome assembly process. PHAST is a set of online modules…

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.

Simplifier: a web tool to eliminate redundant NGS contigs.

PubMed

Ramos, Rommel Thiago Jucá; Carneiro, Adriana Ribeiro; Azevedo, Vasco; Schneider, Maria Paula; Barh, Debmalya; Silva, Artur

2012-01-01

Modern genomic sequencing technologies produce a large amount of data with reduced cost per base; however, this data consists of short reads. This reduction in the size of the reads, compared to those obtained with previous methodologies, presents new challenges, including a need for efficient algorithms for the assembly of genomes from short reads and for resolving repetitions. Additionally after abinitio assembly, curation of the hundreds or thousands of contigs generated by assemblers demands considerable time and computational resources. We developed Simplifier, a stand-alone software that selectively eliminates redundant sequences from the collection of contigs generated by ab initio assembly of genomes. Application of Simplifier to data generated by assembly of the genome of Corynebacterium pseudotuberculosis strain 258 reduced the number of contigs generated by ab initio methods from 8,004 to 5,272, a reduction of 34.14%; in addition, N50 increased from 1 kb to 1.5 kb. Processing the contigs of Escherichia coli DH10B with Simplifier reduced the mate-paired library 17.47% and the fragment library 23.91%. Simplifier removed redundant sequences from datasets produced by assemblers, thereby reducing the effort required for finalization of genome assembly in tests with data from Prokaryotic organisms. Simplifier is available at http://www.genoma.ufpa.br/rramos/softwares/simplifier.xhtmlIt requires Sun jdk 6 or higher.

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

PubMed Central

Eastman, Alexander W.; Yuan, Ze-Chun

2015-01-01

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

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-01-01

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

Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes.

PubMed

Valdes Franco, José A; Wang, Yi; Huo, Naxin; Ponciano, Grisel; Colvin, Howard A; McMahan, Colleen M; Gu, Yong Q; Belknap, William R

2018-04-19

Guayule (Parthenium argentatum A. Gray) is a rubber-producing desert shrub native to Mexico and the United States. Guayule represents an alternative to Hevea brasiliensis as a source for commercial natural rubber. The efficient application of modern molecular/genetic tools to guayule improvement requires characterization of its genome. The 1.6 Gb guayule genome was sequenced, assembled and annotated. The final 1.5 Gb assembly, while fragmented (N 50  = 22 kb), maps > 95% of the shotgun reads and is essentially complete. Approximately 40,000 transcribed, protein encoding genes were annotated on the assembly. Further characterization of this genome revealed 15 families of small, microsatellite-associated, transposable elements (TEs) with unexpected chromosomal distribution profiles. These SaTar (Satellite Targeted) elements, which are non-autonomous Mu-like elements (MULEs), were frequently observed in multimeric linear arrays of unrelated individual elements within which no individual element is interrupted by another. This uniformly non-nested TE multimer architecture has not been previously described in either eukaryotic or prokaryotic genomes. Five families of similarly distributed non-autonomous MULEs (microsatellite associated, modularly assembled) were characterized in the rice genome. Families of TEs with similar structures and distribution profiles were identified in sorghum and citrus. The sequencing and assembly of the guayule genome provides a foundation for application of current crop improvement technologies to this plant. In addition, characterization of this genome revealed SaTar elements with distribution profiles unique among TEs. Satar targeting appears based on an alternative MULE recombination mechanism with the potential to impact gene evolution.

Illumina Synthetic Long Read Sequencing Allows Recovery of Missing Sequences even in the “Finished” C. elegans Genome

PubMed Central

Li, Runsheng; Hsieh, Chia-Ling; Young, Amanda; Zhang, Zhihong; Ren, Xiaoliang; Zhao, Zhongying

2015-01-01

Most next-generation sequencing platforms permit acquisition of high-throughput DNA sequences, but the relatively short read length limits their use in genome assembly or finishing. Illumina has recently released a technology called Synthetic Long-Read Sequencing that can produce reads of unusual length, i.e., predominately around 10 Kb. However, a systematic assessment of their use in genome finishing and assembly is still lacking. We evaluate the promise and deficiency of the long reads in these aspects using isogenic C. elegans genome with no gap. First, the reads are highly accurate and capable of recovering most types of repetitive sequences. However, the presence of tandem repetitive sequences prevents pre-assembly of long reads in the relevant genomic region. Second, the reads are able to reliably detect missing but not extra sequences in the C. elegans genome. Third, the reads of smaller size are more capable of recovering repetitive sequences than those of bigger size. Fourth, at least 40 Kbp missing genomic sequences are recovered in the C. elegans genome using the long reads. Finally, an N50 contig size of at least 86 Kbp can be achieved with 24×reads but with substantial mis-assembly errors, highlighting a need for novel assembly algorithm for the long reads. PMID:26039588

Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus): applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis.

PubMed

Shao, Changwei; Niu, Yongchao; Rastas, Pasi; Liu, Yang; Xie, Zhiyuan; Li, Hengde; Wang, Lei; Jiang, Yong; Tai, Shuaishuai; Tian, Yongsheng; Sakamoto, Takashi; Chen, Songlin

2015-04-01

High-resolution genetic maps are essential for fine mapping of complex traits, genome assembly, and comparative genomic analysis. Single-nucleotide polymorphisms (SNPs) are the primary molecular markers used for genetic map construction. In this study, we identified 13,362 SNPs evenly distributed across the Japanese flounder (Paralichthys olivaceus) genome. Of these SNPs, 12,712 high-confidence SNPs were subjected to high-throughput genotyping and assigned to 24 consensus linkage groups (LGs). The total length of the genetic linkage map was 3,497.29 cM with an average distance of 0.47 cM between loci, thereby representing the densest genetic map currently reported for Japanese flounder. Nine positive quantitative trait loci (QTLs) forming two main clusters for Vibrio anguillarum disease resistance were detected. All QTLs could explain 5.1-8.38% of the total phenotypic variation. Synteny analysis of the QTL regions on the genome assembly revealed 12 immune-related genes, among them 4 genes strongly associated with V. anguillarum disease resistance. In addition, 246 genome assembly scaffolds with an average size of 21.79 Mb were anchored onto the LGs; these scaffolds, comprising 522.99 Mb, represented 95.78% of assembled genomic sequences. The mapped assembly scaffolds in Japanese flounder were used for genome synteny analyses against zebrafish (Danio rerio) and medaka (Oryzias latipes). Flounder and medaka were found to possess almost one-to-one synteny, whereas flounder and zebrafish exhibited a multi-syntenic correspondence. The newly developed high-resolution genetic map, which will facilitate QTL mapping, scaffold assembly, and genome synteny analysis of Japanese flounder, marks a milestone in the ongoing genome project for this species. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Comparative primate genomics: emerging patterns of genome content and dynamics

PubMed Central

Rogers, Jeffrey; Gibbs, Richard A.

2014-01-01

Preface Advances in genome sequencing technologies have created new opportunities for comparative primate genomics. Genome assemblies have been published for several primates, with analyses of several others underway. Whole genome assemblies for the great apes provide remarkable new information about the evolutionary origins of the human genome and the processes involved. Genomic data for macaques and other nonhuman primates provide valuable insight into genetic similarities and differences among species used as models for disease-related research. This review summarizes current knowledge regarding primate genome content and dynamics and offers a series of goals for the near future. PMID:24709753

Comparative primate genomics: emerging patterns of genome content and dynamics.

PubMed

Rogers, Jeffrey; Gibbs, Richard A

2014-05-01

Advances in genome sequencing technologies have created new opportunities for comparative primate genomics. Genome assemblies have been published for various primate species, and analyses of several others are underway. Whole-genome assemblies for the great apes provide remarkable new information about the evolutionary origins of the human genome and the processes involved. Genomic data for macaques and other non-human primates offer valuable insights into genetic similarities and differences among species that are used as models for disease-related research. This Review summarizes current knowledge regarding primate genome content and dynamics, and proposes a series of goals for the near future.

Assembling large genomes: analysis of the stick insect (Clitarchus hookeri) genome reveals a high repeat content and sex-biased genes associated with reproduction.

PubMed

Wu, Chen; Twort, Victoria G; Crowhurst, Ross N; Newcomb, Richard D; Buckley, Thomas R

2017-11-16

Stick insects (Phasmatodea) have a high incidence of parthenogenesis and other alternative reproductive strategies, yet the genetic basis of reproduction is poorly understood. Phasmatodea includes nearly 3000 species, yet only the genome of Timema cristinae has been published to date. Clitarchus hookeri is a geographical parthenogenetic stick insect distributed across New Zealand. Sexual reproduction dominates in northern habitats but is replaced by parthenogenesis in the south. Here, we present a de novo genome assembly of a female C. hookeri and use it to detect candidate genes associated with gamete production and development in females and males. We also explore the factors underlying large genome size in stick insects. The C. hookeri genome assembly was 4.2 Gb, similar to the flow cytometry estimate, making it the second largest insect genome sequenced and assembled to date. Like the large genome of Locusta migratoria, the genome of C. hookeri is also highly repetitive and the predicted gene models are much longer than those from most other sequenced insect genomes, largely due to longer introns. Miniature inverted repeat transposable elements (MITEs), absent in the much smaller T. cristinae genome, is the most abundant repeat type in the C. hookeri genome assembly. Mapping RNA-Seq reads from female and male gonadal transcriptomes onto the genome assembly resulted in the identification of 39,940 gene loci, 15.8% and 37.6% of which showed female-biased and male-biased expression, respectively. The genes that were over-expressed in females were mostly associated with molecular transportation, developmental process, oocyte growth and reproductive process; whereas, the male-biased genes were enriched in rhythmic process, molecular transducer activity and synapse. Several genes involved in the juvenile hormone synthesis pathway were also identified. The evolution of large insect genomes such as L. migratoria and C. hookeri genomes is most likely due to the accumulation of repetitive regions and intron elongation. MITEs contributed significantly to the growth of C. hookeri genome size yet are surprisingly absent from the T. cristinae genome. Sex-biased genes identified from gonadal tissues, including genes involved in juvenile hormone synthesis, provide interesting candidates for the further study of flexible reproduction in stick insects.

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 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.

Gene expression atlas of fruit ripening and transcriptome assembly from RNA-seq data in octoploid strawberry (Fragaria × ananassa).

PubMed

Sánchez-Sevilla, José F; Vallarino, José G; Osorio, Sonia; Bombarely, Aureliano; Posé, David; Merchante, Catharina; Botella, Miguel A; Amaya, Iraida; Valpuesta, Victoriano

2017-10-23

RNA-seq has been used to perform global expression analysis of the achene and the receptacle at four stages of fruit ripening, and of the roots and leaves of strawberry (Fragaria × ananassa). About 967 million reads and 191 Gb of sequence were produced, using Illumina sequencing. Mapping the reads in the related genome of the wild diploid Fragaria vesca revealed differences between the achene and receptacle development program, and reinforced the role played by ethylene in the ripening receptacle. For the strawberry transcriptome assembly, a de novo strategy was followed, generating separate assemblies for each of the ten tissues and stages sampled. The Trinity program was used for these assemblies, resulting in over 1.4 M isoforms. Filtering by a threshold of 0.3 FPKM, and doing Blastx (E-value < 1 e-30) against the UniProt database of plants reduced the number to 472,476 isoforms. Their assembly with the MIRA program (90% homology) resulted in 26,087 contigs. From these, 91.34 percent showed high homology to Fragaria vesca genes and 87.30 percent Fragaria iinumae (BlastN E-value < 1 e-100). Mapping back the reads on the MIRA contigs identified polymorphisms at nucleotide level, using FREEBAYES, as well as estimate their relative abundance in each sample.

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

PubMed Central

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

2010-01-01

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

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

PubMed Central

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

2013-01-01

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

Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding

PubMed Central

Vij, Shubha; Kuhl, Heiner; Kuznetsova, Inna S.; Komissarov, Aleksey; Yurchenko, Andrey A.; Van Heusden, Peter; Singh, Siddharth; Thevasagayam, Natascha M.; Prakki, Sai Rama Sridatta; Purushothaman, Kathiresan; Saju, Jolly M.; Jiang, Junhui; Mbandi, Stanley Kimbung; Jonas, Mario; Hin Yan Tong, Amy; Mwangi, Sarah; Lau, Doreen; Ngoh, Si Yan; Liew, Woei Chang; Shen, Xueyan; Hon, Lawrence S.; Drake, James P.; Boitano, Matthew; Hall, Richard; Chin, Chen-Shan; Lachumanan, Ramkumar; Korlach, Jonas; Trifonov, Vladimir; Kabilov, Marsel; Tupikin, Alexey; Green, Darrell; Moxon, Simon; Garvin, Tyler; Sedlazeck, Fritz J.; Vurture, Gregory W.; Gopalapillai, Gopikrishna; Kumar Katneni, Vinaya; Noble, Tansyn H.; Scaria, Vinod; Sivasubbu, Sridhar; Jerry, Dean R.; O'Brien, Stephen J.; Schatz, Michael C.; Dalmay, Tamás; Turner, Stephen W.; Lok, Si; Christoffels, Alan; Orbán, László

2016-01-01

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species’ native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics. PMID:27082250

Whole-genome sequencing in bacteriology: state of the art

PubMed Central

Dark, Michael J

2013-01-01

Over the last ten years, genome sequencing capabilities have expanded exponentially. There have been tremendous advances in sequencing technology, DNA sample preparation, genome assembly, and data analysis. This has led to advances in a number of facets of bacterial genomics, including metagenomics, clinical medicine, bacterial archaeology, and bacterial evolution. This review examines the strengths and weaknesses of techniques in bacterial genome sequencing, upcoming technologies, and assembly techniques, as well as highlighting recent studies that highlight new applications for bacterial genomics. PMID:24143115

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.

Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

PubMed Central

Aslam, Luqman; Beal, Kathryn; Ann Blomberg, Le; Bouffard, Pascal; Burt, David W.; Crasta, Oswald; Crooijmans, Richard P. M. A.; Cooper, Kristal; Coulombe, Roger A.; De, Supriyo; Delany, Mary E.; Dodgson, Jerry B.; Dong, Jennifer J.; Evans, Clive; Frederickson, Karin M.; Flicek, Paul; Florea, Liliana; Folkerts, Otto; Groenen, Martien A. M.; Harkins, Tim T.; Herrero, Javier; Hoffmann, Steve; Megens, Hendrik-Jan; Jiang, Andrew; de Jong, Pieter; Kaiser, Pete; Kim, Heebal; Kim, Kyu-Won; Kim, Sungwon; Langenberger, David; Lee, Mi-Kyung; Lee, Taeheon; Mane, Shrinivasrao; Marcais, Guillaume; Marz, Manja; McElroy, Audrey P.; Modise, Thero; Nefedov, Mikhail; Notredame, Cédric; Paton, Ian R.; Payne, William S.; Pertea, Geo; Prickett, Dennis; Puiu, Daniela; Qioa, Dan; Raineri, Emanuele; Ruffier, Magali; Salzberg, Steven L.; Schatz, Michael C.; Scheuring, Chantel; Schmidt, Carl J.; Schroeder, Steven; Searle, Stephen M. J.; Smith, Edward J.; Smith, Jacqueline; Sonstegard, Tad S.; Stadler, Peter F.; Tafer, Hakim; Tu, Zhijian (Jake); Van Tassell, Curtis P.; Vilella, Albert J.; Williams, Kelly P.; Yorke, James A.; Zhang, Liqing; Zhang, Hong-Bin; Zhang, Xiaojun; Zhang, Yang; Reed, Kent M.

2010-01-01

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest. PMID:20838655

Large-scale parallel genome assembler over cloud computing environment.

PubMed

Das, Arghya Kusum; Koppa, Praveen Kumar; Goswami, Sayan; Platania, Richard; Park, Seung-Jong

2017-06-01

The size of high throughput DNA sequencing data has already reached the terabyte scale. To manage this huge volume of data, many downstream sequencing applications started using locality-based computing over different cloud infrastructures to take advantage of elastic (pay as you go) resources at a lower cost. However, the locality-based programming model (e.g. MapReduce) is relatively new. Consequently, developing scalable data-intensive bioinformatics applications using this model and understanding the hardware environment that these applications require for good performance, both require further research. In this paper, we present a de Bruijn graph oriented Parallel Giraph-based Genome Assembler (GiGA), as well as the hardware platform required for its optimal performance. GiGA uses the power of Hadoop (MapReduce) and Giraph (large-scale graph analysis) to achieve high scalability over hundreds of compute nodes by collocating the computation and data. GiGA achieves significantly higher scalability with competitive assembly quality compared to contemporary parallel assemblers (e.g. ABySS and Contrail) over traditional HPC cluster. Moreover, we show that the performance of GiGA is significantly improved by using an SSD-based private cloud infrastructure over traditional HPC cluster. We observe that the performance of GiGA on 256 cores of this SSD-based cloud infrastructure closely matches that of 512 cores of traditional HPC cluster.

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

PubMed

Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea; Burdick, Ryan C; Levine, Louis; Li, Kelvin; Rein, Alan; Pathak, Vinay K; Hu, Wei-Shau

2017-08-15

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication. Copyright © 2017 American Society for Microbiology.

The Effects of Signal Erosion and Core Genome Reduction on the Identification of Diagnostic Markers

DTIC Science & Technology

2016-09-20

31 diagnostics for the identification of bacterial pathogens. To do this effectively, 32 genomics databases must be comprehensive to identify the...diverse B. 118 pseudomallei/mallei strains were sequenced, assembled, and deposited in public 119 databases (Supplemental Table 1); these genomes were...combined with 160 B. 120 pseudomallei/mallei genome assemblies already in public databases . Most of the 121 genomes (n=779) in this study were

Re-Assembly and Analysis of an Ancient Variola Virus Genome.

PubMed

Smithson, Chad; Imbery, Jacob; Upton, Chris

2017-09-08

We report a major improvement to the assembly of published short read sequencing data from an ancient variola virus (VARV) genome by the removal of contig-capping sequencing tags and manual searches for gap-spanning reads. The new assembly, together with camelpox and taterapox genomes, permitted new dates to be calculated for the last common ancestor of all VARV genomes. The analysis of recently sequenced VARV-like cowpox virus genomes showed that single nucleotide polymorphisms (SNPs) and amino acid changes in the vaccinia virus (VACV)-Cop-O1L ortholog, predicted to be associated with VARV host specificity and virulence, were introduced into the lineage before the divergence of these viruses. A comparison of the ancient and modern VARV genome sequences also revealed a measurable drift towards adenine + thymine (A + T) richness.

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

Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence.

PubMed

Milles, Sigrid; Jensen, Malene Ringkjøbing; Communie, Guillaume; Maurin, Damien; Schoehn, Guy; Ruigrok, Rob W H; Blackledge, Martin

2016-08-01

Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo-proteins (N) that bind the entire genome. N-RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self-organization of N into NC-like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real-time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA-sequence, with the genomic 5' end and poly-Adenine sequences assembling efficiently, while sequences such as poly-Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis.

PubMed

Sato, Kengo; Kuroki, Yoko; Kumita, Wakako; Fujiyama, Asao; Toyoda, Atsushi; Kawai, Jun; Iriki, Atsushi; Sasaki, Erika; Okano, Hideyuki; Sakakibara, Yasubumi

2015-11-20

The first draft of the common marmoset (Callithrix jacchus) genome was published by the Marmoset Genome Sequencing and Analysis Consortium. The draft was based on whole-genome shotgun sequencing, and the current assembly version is Callithrix_jacches-3.2.1, but there still exist 187,214 undetermined gap regions and supercontigs and relatively short contigs that are unmapped to chromosomes in the draft genome. We performed resequencing and assembly of the genome of common marmoset by deep sequencing with high-throughput sequencing technology. Several different sequence runs using Illumina sequencing platforms were executed, and 181 Gbp of high-quality bases including mate-pairs with long insert lengths of 3, 8, 20, and 40 Kbp were obtained, that is, approximately 60× coverage. The resequencing significantly improved the MGSAC draft genome sequence. The N50 of the contigs, which is a statistical measure used to evaluate assembly quality, doubled. As a result, 51% of the contigs (total length: 299 Mbp) that were unmapped to chromosomes in the MGSAC draft were merged with chromosomal contigs, and the improved genome sequence helped to detect 5,288 new genes that are homologous to human cDNAs and the gaps in 5,187 transcripts of the Ensembl gene annotations were completely filled.

Assembly and Multiplex Genome Integration of Metabolic Pathways in Yeast Using CasEMBLR.

PubMed

Jakočiūnas, Tadas; Jensen, Emil D; Jensen, Michael K; Keasling, Jay D

2018-01-01

Genome integration is a vital step for implementing large biochemical pathways to build a stable microbial cell factory. Although traditional strain construction strategies are well established for the model organism Saccharomyces cerevisiae, recent advances in CRISPR/Cas9-mediated genome engineering allow much higher throughput and robustness in terms of strain construction. In this chapter, we describe CasEMBLR, a highly efficient and marker-free genome engineering method for one-step integration of in vivo assembled expression cassettes in multiple genomic sites simultaneously. CasEMBLR capitalizes on the CRISPR/Cas9 technology to generate double-strand breaks in genomic loci, thus prompting native homologous recombination (HR) machinery to integrate exogenously derived homology templates. As proof-of-principle for microbial cell factory development, CasEMBLR was used for one-step assembly and marker-free integration of the carotenoid pathway from 15 exogenously supplied DNA parts into three targeted genomic loci. As a second proof-of-principle, a total of ten DNA parts were assembled and integrated in two genomic loci to construct a tyrosine production strain, and at the same time knocking out two genes. This new method complements and improves the field of genome engineering in S. cerevisiae by providing a more flexible platform for rapid and precise strain building.

Improving the annotation of the Heterorhabditis bacteriophora genome.

PubMed

McLean, Florence; Berger, Duncan; Laetsch, Dominik R; Schwartz, Hillel T; Blaxter, Mark

2018-04-01

Genome assembly and annotation remain exacting tasks. As the tools available for these tasks improve, it is useful to return to data produced with earlier techniques to assess their credibility and correctness. The entomopathogenic nematode Heterorhabditis bacteriophora is widely used to control insect pests in horticulture. The genome sequence for this species was reported to encode an unusually high proportion of unique proteins and a paucity of secreted proteins compared to other related nematodes. We revisited the H. bacteriophora genome assembly and gene predictions to determine whether these unusual characteristics were biological or methodological in origin. We mapped an independent resequencing dataset to the genome and used the blobtools pipeline to identify potential contaminants. While present (0.2% of the genome span, 0.4% of predicted proteins), assembly contamination was not significant. Re-prediction of the gene set using BRAKER1 and published transcriptome data generated a predicted proteome that was very different from the published one. The new gene set had a much reduced complement of unique proteins, better completeness values that were in line with other related species' genomes, and an increased number of proteins predicted to be secreted. It is thus likely that methodological issues drove the apparent uniqueness of the initial H. bacteriophora genome annotation and that similar contamination and misannotation issues affect other published genome assemblies.

Evolution and Diversity of Transposable Elements in Vertebrate Genomes.

PubMed

Sotero-Caio, Cibele G; Platt, Roy N; Suh, Alexander; Ray, David A

2017-01-01

Transposable elements (TEs) are selfish genetic elements that mobilize in genomes via transposition or retrotransposition and often make up large fractions of vertebrate genomes. Here, we review the current understanding of vertebrate TE diversity and evolution in the context of recent advances in genome sequencing and assembly techniques. TEs make up 4-60% of assembled vertebrate genomes, and deeply branching lineages such as ray-finned fishes and amphibians generally exhibit a higher TE diversity than the more recent radiations of birds and mammals. Furthermore, the list of taxa with exceptional TE landscapes is growing. We emphasize that the current bottleneck in genome analyses lies in the proper annotation of TEs and provide examples where superficial analyses led to misleading conclusions about genome evolution. Finally, recent advances in long-read sequencing will soon permit access to TE-rich genomic regions that previously resisted assembly including the gigantic, TE-rich genomes of salamanders and lungfishes. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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

PubMed

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

2017-01-01

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

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.

Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo) genome assembly and analysis

USDA-ARS?s Scientific Manuscript database

Next-generation sequencing technologies were used to rapidly and efficiently sequence the genome of the domestic turkey (Meleagris gallopavo). The current genome assembly (~1.1 Gb) includes 917 Mb of sequence assigned to chromosomes. Innate heterozygosity of the sequenced bird allowed discovery of...

HopBase: a unified resource for Humulus genomics

PubMed Central

Hill, Steven T.; Sudarsanam, Ramcharan

2017-01-01

Abstract Hop (Humulus lupulus L. var lupulus) is a dioecious plant of worldwide significance, used primarily for bittering and flavoring in brewing beer. Studies on the medicinal properties of several unique compounds produced by hop have led to additional interest from pharmacy and healthcare industries as well as livestock production as a natural antibiotic. Genomic research in hop has resulted a published draft genome and transcriptome assemblies. As research into the genomics of hop has gained interest, there is a critical need for centralized online genomic resources. To support the growing research community, we report the development of an online resource "HopBase.org." In addition to providing a gene annotation to the existing Shinsuwase draft genome, HopBase makes available genome assemblies and annotations for both the cultivar “Teamaker” and male hop accession number USDA 21422M. These genome assemblies, gene annotations, along with other common data, coupled with a genome browser and BLAST database enable the hop community to enter the genomic age. The HopBase genomic resource is accessible at http://hopbase.org and http://hopbase.cgrb.oregonstate.edu. PMID:28415075

SINE_scan: an efficient tool to discover short interspersed nuclear elements (SINEs) in large-scale genomic datasets.

PubMed

Mao, Hongliang; Wang, Hao

2017-03-01

Short Interspersed Nuclear Elements (SINEs) are transposable elements (TEs) that amplify through a copy-and-paste mode via RNA intermediates. The computational identification of new SINEs are challenging because of their weak structural signals and rapid diversification in sequences. Here we report SINE_Scan, a highly efficient program to predict SINE elements in genomic DNA sequences. SINE_Scan integrates hallmark of SINE transposition, copy number and structural signals to identify a SINE element. SINE_Scan outperforms the previously published de novo SINE discovery program. It shows high sensitivity and specificity in 19 plant and animal genome assemblies, of which sizes vary from 120 Mb to 3.5 Gb. It identifies numerous new families and substantially increases the estimation of the abundance of SINEs in these genomes. The code of SINE_Scan is freely available at http://github.com/maohlzj/SINE_Scan , implemented in PERL and supported on Linux. wangh8@fudan.edu.cn. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

SINE_scan: an efficient tool to discover short interspersed nuclear elements (SINEs) in large-scale genomic datasets

PubMed Central

Mao, Hongliang

2017-01-01

Abstract Motivation: Short Interspersed Nuclear Elements (SINEs) are transposable elements (TEs) that amplify through a copy-and-paste mode via RNA intermediates. The computational identification of new SINEs are challenging because of their weak structural signals and rapid diversification in sequences. Results: Here we report SINE_Scan, a highly efficient program to predict SINE elements in genomic DNA sequences. SINE_Scan integrates hallmark of SINE transposition, copy number and structural signals to identify a SINE element. SINE_Scan outperforms the previously published de novo SINE discovery program. It shows high sensitivity and specificity in 19 plant and animal genome assemblies, of which sizes vary from 120 Mb to 3.5 Gb. It identifies numerous new families and substantially increases the estimation of the abundance of SINEs in these genomes. Availability and Implementation: The code of SINE_Scan is freely available at http://github.com/maohlzj/SINE_Scan, implemented in PERL and supported on Linux. Contact: wangh8@fudan.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online. PMID:28062442

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.

LightAssembler: fast and memory-efficient assembly algorithm for high-throughput sequencing reads.

PubMed

El-Metwally, Sara; Zakaria, Magdi; Hamza, Taher

2016-11-01

The deluge of current sequenced data has exceeded Moore's Law, more than doubling every 2 years since the next-generation sequencing (NGS) technologies were invented. Accordingly, we will able to generate more and more data with high speed at fixed cost, but lack the computational resources to store, process and analyze it. With error prone high throughput NGS reads and genomic repeats, the assembly graph contains massive amount of redundant nodes and branching edges. Most assembly pipelines require this large graph to reside in memory to start their workflows, which is intractable for mammalian genomes. Resource-efficient genome assemblers combine both the power of advanced computing techniques and innovative data structures to encode the assembly graph efficiently in a computer memory. LightAssembler is a lightweight assembly algorithm designed to be executed on a desktop machine. It uses a pair of cache oblivious Bloom filters, one holding a uniform sample of [Formula: see text]-spaced sequenced [Formula: see text]-mers and the other holding [Formula: see text]-mers classified as likely correct, using a simple statistical test. LightAssembler contains a light implementation of the graph traversal and simplification modules that achieves comparable assembly accuracy and contiguity to other competing tools. Our method reduces the memory usage by [Formula: see text] compared to the resource-efficient assemblers using benchmark datasets from GAGE and Assemblathon projects. While LightAssembler can be considered as a gap-based sequence assembler, different gap sizes result in an almost constant assembly size and genome coverage. https://github.com/SaraEl-Metwally/LightAssembler CONTACT: sarah_almetwally4@mans.edu.egSupplementary 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.

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.

Updated sesame genome assembly and fine mapping of plant height and seed coat color QTLs using a new high-density genetic map.

PubMed

Wang, Linhai; Xia, Qiuju; Zhang, Yanxin; Zhu, Xiaodong; Zhu, Xiaofeng; Li, Donghua; Ni, Xuemei; Gao, Yuan; Xiang, Haitao; Wei, Xin; Yu, Jingyin; Quan, Zhiwu; Zhang, Xiurong

2016-01-05

Sesame is an important high-quality oil seed crop. The sesame genome was de novo sequenced and assembled in 2014 (version 1.0); however, the number of anchored pseudomolecules was higher than the chromosome number (2n = 2x = 26) due to the lack of a high-density genetic map with 13 linkage groups. We resequenced a permanent population consisting of 430 recombinant inbred lines and constructed a genetic map to improve the sesame genome assembly. We successfully anchored 327 scaffolds onto 13 pseudomolecules. The new genome assembly (version 2.0) included 97.5 % of the scaffolds greater than 150 kb in size present in assembly version 1.0 and increased the total pseudomolecule length from 233.7 to 258.4 Mb with 94.3 % of the genome assembled and 97.2 % of the predicted gene models anchored. Based on the new genome assembly, a bin map including 1,522 bins spanning 1090.99 cM was generated and used to identified 41 quantitative trait loci (QTLs) for sesame plant height and 9 for seed coat color. The plant height-related QTLs explained 3-24 % the phenotypic variation (mean value, 8 %), and 29 of them were detected in at least two field trials. Two major loci (qPH-8.2 and qPH-3.3) that contributed 23 and 18 % of the plant height were located in 350 and 928-kb spaces on Chr8 and Chr3, respectively. qPH-3.3, is predicted to be responsible for the semi-dwarf sesame plant phenotype and contains 102 candidate genes. This is the first report of a sesame semi-dwarf locus and provides an interesting opportunity for a plant architecture study of the sesame. For the sesame seed coat color, the QTLs of the color spaces L*, a*, and b* were detected with contribution rates of 3-46 %. qSCb-4.1 contributed approximately 39 % of the b* value and was located on Chr4 in a 199.9-kb space. A list of 32 candidate genes for the locus, including a predicted black seed coat-related gene, was determined by screening the newly anchored genome. This study offers a high-density genetic map and an improved assembly of the sesame genome. The number of linkage groups and pseudomolecules in this assembly equals the number of sesame chromosomes for the first time. The map and updated genome assembly are expected to serve as a platform for future comparative genomics and genetic studies.

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.

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

EPA Pesticide Factsheets

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

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome through long-read (>11 kb), single molecule, real-time sequencing

PubMed Central

Vembar, Shruthi Sridhar; Seetin, Matthew; Lambert, Christine; Nattestad, Maria; Schatz, Michael C.; Baybayan, Primo; Scherf, Artur; Smith, Melissa Laird

2016-01-01

The application of next-generation sequencing to estimate genetic diversity of Plasmodium falciparum, the most lethal malaria parasite, has proved challenging due to the skewed AT-richness [∼80.6% (A + T)] of its genome and the lack of technology to assemble highly polymorphic subtelomeric regions that contain clonally variant, multigene virulence families (Ex: var and rifin). To address this, we performed amplification-free, single molecule, real-time sequencing of P. falciparum genomic DNA and generated reads of average length 12 kb, with 50% of the reads between 15.5 and 50 kb in length. Next, using the Hierarchical Genome Assembly Process, we assembled the P. falciparum genome de novo and successfully compiled all 14 nuclear chromosomes telomere-to-telomere. We also accurately resolved centromeres [∼90–99% (A + T)] and subtelomeric regions and identified large insertions and duplications that add extra var and rifin genes to the genome, along with smaller structural variants such as homopolymer tract expansions. Overall, we show that amplification-free, long-read sequencing combined with de novo assembly overcomes major challenges inherent to studying the P. falciparum genome. Indeed, this technology may not only identify the polymorphic and repetitive subtelomeric sequences of parasite populations from endemic areas but may also evaluate structural variation linked to virulence, drug resistance and disease transmission. PMID:27345719

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.

A continuous genome assembly of the corkwing wrasse (Symphodus melops).

PubMed

Mattingsdal, Morten; Jentoft, Sissel; Tørresen, Ole K; Knutsen, Halvor; Hansen, Michael M; Robalo, Joana I; Zagrodzka, Zuzanna; André, Carl; Gonzalez, Enrique Blanco

2018-04-14

The wrasses (Labridae) are one of the most successful and species-rich families of the Perciformes order of teleost fish. Its members display great morphological diversity, and occupy distinct trophic levels in coastal waters and coral reefs. The cleaning behaviour displayed by some wrasses, such as corkwing wrasse (Symphodus melops), is of particular interest for the salmon aquaculture industry to combat and control sea lice infestation as an alternative to chemicals and pharmaceuticals. There are still few genome assemblies available within this fish family for comparative and functional studies, despite the rapid increase in genome resources generated during the past years. Here, we present a highly continuous genome assembly of the corkwing wrasse using PacBio SMRT sequencing (x28.8) followed by error correction with paired-end Illumina data (x132.9). The present genome assembly consists of 5040 contigs (N50 = 461,652 bp) and a total size of 614 Mbp, of which 8.5% of the genome sequence encode known repeated elements. The genome assembly covers 94.21% of highly conserved genes across ray-finned fish species. We find evidence for increased copy numbers specific for corkwing wrasse possibly highlighting diversification and adaptive processes in gene families including N-linked glycosylation (ST8SIA6) and stress response kinases (HIPK1). By comparative analyses, we discover that de novo repeats, often not properly investigated during genome annotation, encode hundreds of immune-related genes. This new genomic resource, together with the ballan wrasse (Labrus bergylta), will allow for in-depth comparative genomics as well as population genetic analyses for the understudied wrasses. Copyright © 2018 Elsevier Inc. All rights reserved.

The Draft Assembly of the Radically Organized Stylonychia lemnae Macronuclear Genome

PubMed Central

Aeschlimann, Samuel H.; Jönsson, Franziska; Postberg, Jan; Stover, Nicholas A.; Petera, Robert L.; Lipps, Hans-Joachim; Nowacki, Mariusz; Swart, Estienne C.

2014-01-01

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia’s macronucleus has a very unusual architecture, comprised variably and highly amplified “nanochromosomes,” each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements. PMID:24951568

☆DNA assembly technique simplifies the construction of infectious clone of fowl adenovirus.

PubMed

Zou, Xiao-Hui; Bi, Zhi-Xiang; Guo, Xiao-Juan; Zhang, Zun; Zhao, Yang; Wang, Min; Zhu, Ya-Lu; Jie, Hong-Ying; Yu, Yang; Hung, Tao; Lu, Zhuo-Zhuang

2018-07-01

Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix. E. coli competent cells were transformed with the assembled product, and plasmids (pKFAV4) were extracted and confirmed to contain viral genome by restriction analysis and sequencing. Virus was successfully rescued from linear pKFAV4-transfected chicken LMH cells. This approach was further verified in cloning of human adenovirus 5 genome. Our results indicated that DNA assembly technique simplified the construction of infectious clone of adenovirus, suggesting its possible application in virus traditional or reverse genetics. Copyright © 2018 Elsevier B.V. All rights reserved.

LPmerge: an R package for merging genetic maps by linear programming.

PubMed

Endelman, Jeffrey B; Plomion, Christophe

2014-06-01

Consensus genetic maps constructed from multiple populations are an important resource for both basic and applied research, including genome-wide association analysis, genome sequence assembly and studies of evolution. The LPmerge software uses linear programming to efficiently minimize the mean absolute error between the consensus map and the linkage maps from each population. This minimization is performed subject to linear inequality constraints that ensure the ordering of the markers in the linkage maps is preserved. When marker order is inconsistent between linkage maps, a minimum set of ordinal constraints is deleted to resolve the conflicts. LPmerge is on CRAN at http://cran.r-project.org/web/packages/LPmerge. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

TUMOR HAPLOTYPE ASSEMBLY ALGORITHMS FOR CANCER GENOMICS

PubMed Central

AGUIAR, DEREK; WONG, WENDY S.W.; ISTRAIL, SORIN

2014-01-01

The growing availability of inexpensive high-throughput sequence data is enabling researchers to sequence tumor populations within a single individual at high coverage. But, cancer genome sequence evolution and mutational phenomena like driver mutations and gene fusions are difficult to investigate without first reconstructing tumor haplotype sequences. Haplotype assembly of single individual tumor populations is an exceedingly difficult task complicated by tumor haplotype heterogeneity, tumor or normal cell sequence contamination, polyploidy, and complex patterns of variation. While computational and experimental haplotype phasing of diploid genomes has seen much progress in recent years, haplotype assembly in cancer genomes remains uncharted territory. In this work, we describe HapCompass-Tumor a computational modeling and algorithmic framework for haplotype assembly of copy number variable cancer genomes containing haplotypes at different frequencies and complex variation. We extend our polyploid haplotype assembly model and present novel algorithms for (1) complex variations, including copy number changes, as varying numbers of disjoint paths in an associated graph, (2) variable haplotype frequencies and contamination, and (3) computation of tumor haplotypes using simple cycles of the compass graph which constrain the space of haplotype assembly solutions. The model and algorithm are implemented in the software package HapCompass-Tumor which is available for download from http://www.brown.edu/Research/Istrail_Lab/. PMID:24297529

Genome sequence of Bradyrhizobium sp. WSM1253; a microsymbiont of Ornithopus compressus from the Greek Island of Sifnos

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

Tiwari, Ravi; Howieson, John; Yates, Ron

Bradyrhizobium sp. WSM1253 is a novel N 2-fixing bacterium isolated from a root nodule of the herbaceous annual legume Ornithopus compressus that was growing on the Greek Island of Sifnos. WSM1253 emerged as a strain of interest in an Australian program that was selecting inoculant quality bradyrhizobial strains for inoculation of Mediterranean species of lupins ( Lupinus angustifolius, L. princei, L. atlanticus, L. pilosus ). In this report we describe, for the first time, the genome sequence information and annotation of this legume microsymbiont. The 8,719,808 bp genome has a G + C content of 63.09 % with 71 contigsmore » arranged into two scaffolds. The assembled genome contains 8,432 protein-coding genes, 66 RNA genes and a single rRNA operon. In conclusion, this improved-high-quality draft rhizobial genome is one of 20 sequenced through a DOE Joint Genome Institute 2010 Community Sequencing Project.« less

Genome sequence of Bradyrhizobium sp. WSM1253; a microsymbiont of Ornithopus compressus from the Greek Island of Sifnos

DOE PAGES

Tiwari, Ravi; Howieson, John; Yates, Ron; ...

2015-11-30

Bradyrhizobium sp. WSM1253 is a novel N 2-fixing bacterium isolated from a root nodule of the herbaceous annual legume Ornithopus compressus that was growing on the Greek Island of Sifnos. WSM1253 emerged as a strain of interest in an Australian program that was selecting inoculant quality bradyrhizobial strains for inoculation of Mediterranean species of lupins ( Lupinus angustifolius, L. princei, L. atlanticus, L. pilosus ). In this report we describe, for the first time, the genome sequence information and annotation of this legume microsymbiont. The 8,719,808 bp genome has a G + C content of 63.09 % with 71 contigsmore » arranged into two scaffolds. The assembled genome contains 8,432 protein-coding genes, 66 RNA genes and a single rRNA operon. In conclusion, this improved-high-quality draft rhizobial genome is one of 20 sequenced through a DOE Joint Genome Institute 2010 Community Sequencing Project.« less

Whole-genome random sequencing and assembly of Haemophilus influenzae Rd

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

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

1995-07-28

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

Draft genome of the Peruvian scallop Argopecten purpuratus.

PubMed

Li, Chao; Liu, Xiao; Liu, Bo; Ma, Bin; Liu, Fengqiao; Liu, Guilong; Shi, Qiong; Wang, Chunde

2018-04-01

The Peruvian scallop, Argopecten purpuratus, is mainly cultured in southern Chile and Peru was introduced into China in the last century. Unlike other Argopecten scallops, the Peruvian scallop normally has a long life span of up to 7 to 10 years. Therefore, researchers have been using it to develop hybrid vigor. Here, we performed whole genome sequencing, assembly, and gene annotation of the Peruvian scallop, with an important aim to develop genomic resources for genetic breeding in scallops. A total of 463.19-Gb raw DNA reads were sequenced. A draft genome assembly of 724.78 Mb was generated (accounting for 81.87% of the estimated genome size of 885.29 Mb), with a contig N50 size of 80.11 kb and a scaffold N50 size of 1.02 Mb. Repeat sequences were calculated to reach 33.74% of the whole genome, and 26,256 protein-coding genes and 3,057 noncoding RNAs were predicted from the assembly. We generated a high-quality draft genome assembly of the Peruvian scallop, which will provide a solid resource for further genetic breeding and for the analysis of the evolutionary history of this economically important scallop.

SOPRA: Scaffolding algorithm for paired reads via statistical optimization.

PubMed

Dayarian, Adel; Michael, Todd P; Sengupta, Anirvan M

2010-06-24

High throughput sequencing (HTS) platforms produce gigabases of short read (<100 bp) data per run. While these short reads are adequate for resequencing applications, de novo assembly of moderate size genomes from such reads remains a significant challenge. These limitations could be partially overcome by utilizing mate pair technology, which provides pairs of short reads separated by a known distance along the genome. We have developed SOPRA, a tool designed to exploit the mate pair/paired-end information for assembly of short reads. The main focus of the algorithm is selecting a sufficiently large subset of simultaneously satisfiable mate pair constraints to achieve a balance between the size and the quality of the output scaffolds. Scaffold assembly is presented as an optimization problem for variables associated with vertices and with edges of the contig connectivity graph. Vertices of this graph are individual contigs with edges drawn between contigs connected by mate pairs. Similar graph problems have been invoked in the context of shotgun sequencing and scaffold building for previous generation of sequencing projects. However, given the error-prone nature of HTS data and the fundamental limitations from the shortness of the reads, the ad hoc greedy algorithms used in the earlier studies are likely to lead to poor quality results in the current context. SOPRA circumvents this problem by treating all the constraints on equal footing for solving the optimization problem, the solution itself indicating the problematic constraints (chimeric/repetitive contigs, etc.) to be removed. The process of solving and removing of constraints is iterated till one reaches a core set of consistent constraints. For SOLiD sequencer data, SOPRA uses a dynamic programming approach to robustly translate the color-space assembly to base-space. For assessing the quality of an assembly, we report the no-match/mismatch error rate as well as the rates of various rearrangement errors. Applying SOPRA to real data from bacterial genomes, we were able to assemble contigs into scaffolds of significant length (N50 up to 200 Kb) with very few errors introduced in the process. In general, the methodology presented here will allow better scaffold assemblies of any type of mate pair sequencing data.

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...

The tomato genome sequence provides insight into fleshy fruit evolution

USDA-ARS?s Scientific Manuscript database

The genome of the inbred tomato cultivar ‘Heinz 1706’ was sequenced and assembled using a combination of Sanger and “next generation” technologies. The predicted genome size is ~900 Mb, consistent with prior estimates, of which 760 Mb were assembled in 91 scaffolds aligned to the 12 tomato chromosom...

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...

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

USDA-ARS?s Scientific Manuscript database

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 ...

The reconstruction of 2,631 draft metagenome-assembled genomes from the global oceans.

PubMed

Tully, Benjamin J; Graham, Elaina D; Heidelberg, John F

2018-01-16

Microorganisms play a crucial role in mediating global biogeochemical cycles in the marine environment. By reconstructing the genomes of environmental organisms through metagenomics, researchers are able to study the metabolic potential of Bacteria and Archaea that are resistant to isolation in the laboratory. Utilizing the large metagenomic dataset generated from 234 samples collected during the Tara Oceans circumnavigation expedition, we were able to assemble 102 billion paired-end reads into 562 million contigs, which in turn were co-assembled and consolidated in to 7.2 million contigs ≥2 kb in length. Approximately 1 million of these contigs were binned to reconstruct draft genomes. In total, 2,631 draft genomes with an estimated completion of ≥50% were generated (1,491 draft genomes >70% complete; 603 genomes >90% complete). A majority of the draft genomes were manually assigned phylogeny based on sets of concatenated phylogenetic marker genes and/or 16S rRNA gene sequences. The draft genomes are now publically available for the research community at-large.

Improved assemblies using a source-agnostic pipeline for MetaGenomic Assembly by Merging (MeGAMerge) of contigs

DOE PAGES

Scholz, Matthew; Lo, Chien -Chi; Chain, Patrick S. G.

2014-10-01

Assembly of metagenomic samples is a very complex process, with algorithms designed to address sequencing platform-specific issues, (read length, data volume, and/or community complexity), while also faced with genomes that differ greatly in nucleotide compositional biases and in abundance. To address these issues, we have developed a post-assembly process: MetaGenomic Assembly by Merging (MeGAMerge). We compare this process to the performance of several assemblers, using both real, and in-silico generated samples of different community composition and complexity. MeGAMerge consistently outperforms individual assembly methods, producing larger contigs with an increased number of predicted genes, without replication of data. MeGAMerge contigs aremore » supported by read mapping and contig alignment data, when using synthetically-derived and real metagenomic data, as well as by gene prediction analyses and similarity searches. Ultimately, MeGAMerge is a flexible method that generates improved metagenome assemblies, with the ability to accommodate upcoming sequencing platforms, as well as present and future assembly algorithms.« less

Whole genome sequencing of Chinese clearhead icefish, Protosalanx hyalocranius.

PubMed

Liu, Kai; Xu, Dongpo; Li, Jia; Bian, Chao; Duan, Jinrong; Zhou, Yanfeng; Zhang, Minying; You, Xinxin; You, Yang; Chen, Jieming; Yu, Hui; Xu, Gangchun; Fang, Di-An; Qiang, Jun; Jiang, Shulun; He, Jie; Xu, Junmin; Shi, Qiong; Zhang, Zhiyong; Xu, Pao

2017-04-01

Chinese clearhead icefish, Protosalanx hyalocranius , is a representative icefish species with economic importance and special appearance. Due to its great economic value in China, the fish was introduced into Lake Dianchi and several other lakes from the Lake Taihu half a century ago. Similar to the Sinocyclocheilus cavefish, the clearhead icefish has certain cavefish-like traits, such as transparent body and nearly scaleless skin. Here, we provide the whole genome sequence of this surface-dwelling fish and generated a draft genome assembly, aiming at exploring molecular mechanisms for the biological interests. A total of 252.1 Gb of raw reads were sequenced. Subsequently, a novel draft genome assembly was generated, with the scaffold N50 reaching 1.163 Mb. The genome completeness was estimated to be 98.39 % by using the CEGMA evaluation. Finally, we annotated 19 884 protein-coding genes and observed that repeat sequences account for 24.43 % of the genome assembly. We report the first draft genome of the Chinese clearhead icefish. The genome assembly will provide a solid foundation for further molecular breeding and germplasm resource protection in Chinese clearhead icefish, as well as other icefishes. It is also a valuable genetic resource for revealing the molecular mechanisms for the cavefish-like characters. © The Authors 2017. Published by Oxford University Press.

Rewriting nature's assembly manual for a ssRNA virus.

PubMed

Patel, Nikesh; Wroblewski, Emma; Leonov, German; Phillips, Simon E V; Tuma, Roman; Twarock, Reidun; Stockley, Peter G

2017-11-14

Satellite tobacco necrosis virus (STNV) is one of the smallest viruses known. Its genome encodes only its coat protein (CP) subunit, relying on the polymerase of its helper virus TNV for replication. The genome has been shown to contain a cryptic set of dispersed assembly signals in the form of stem-loops that each present a minimal CP-binding motif AXXA in the loops. The genomic fragment encompassing nucleotides 1-127 is predicted to contain five such packaging signals (PSs). We have used mutagenesis to determine the critical assembly features in this region. These include the CP-binding motif, the relative placement of PS stem-loops, their number, and their folding propensity. CP binding has an electrostatic contribution, but assembly nucleation is dominated by the recognition of the folded PSs in the RNA fragment. Mutation to remove all AXXA motifs in PSs throughout the genome yields an RNA that is unable to assemble efficiently. In contrast, when a synthetic 127-nt fragment encompassing improved PSs is swapped onto the RNA otherwise lacking CP recognition motifs, assembly is partially restored, although the virus-like particles created are incomplete, implying that PSs outside this region are required for correct assembly. Swapping this improved region into the wild-type STNV1 sequence results in a better assembly substrate than the viral RNA, producing complete capsids and outcompeting the wild-type genome in head-to-head competition. These data confirm details of the PS-mediated assembly mechanism for STNV and identify an efficient approach for production of stable virus-like particles encapsidating nonnative RNAs or other cargoes. Copyright © 2017 the Author(s). Published by PNAS.

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome

PubMed Central

2009-01-01

Background Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. Results We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. Conclusion We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes. PMID:19656416

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome.

PubMed

Hamberger, Björn; Hall, Dawn; Yuen, Mack; Oddy, Claire; Hamberger, Britta; Keeling, Christopher I; Ritland, Carol; Ritland, Kermit; Bohlmann, Jörg

2009-08-06

Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes.

Draft Genome Sequence of a “Candidatus Liberibacter europaeus” Strain Assembled from Broom Psyllids (Arytainilla spartiophila) from New Zealand

PubMed Central

Thompson, Sarah M.; Kalamorz, Falk; David, Charles; Addison, Shea M.; Smith, Grant R.

2018-01-01

ABSTRACT Here, we report the draft genome sequence of “Candidatus Liberibacter europaeus” ASNZ1, assembled from broom psyllids (Arytainilla spartiophila) from New Zealand. The assembly comprises 15 contigs, with a total length of 1.33 Mb and a G+C content of 33.5%. PMID:29773636

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.

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

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

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

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

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

DOE PAGES

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

2017-07-18

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

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

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

PubMed Central

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

2017-01-01

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

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

ALLPATHS: Assembling Large Genomes with Short Illumina Reads

ScienceCinema

Gnerre, Sante

2018-02-06

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

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.

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...

De-Novo Assembly and Analysis of the Heterozygous Triploid Genome of the Wine Spoilage Yeast Dekkera bruxellensis AWRI1499

PubMed Central

Chambers, Paul J.; Pretorius, Isak S.

2012-01-01

Despite its industrial importance, the yeast species Dekkera (Brettanomyces) bruxellensis has remained poorly understood at the genetic level. In this study we describe whole genome sequencing and analysis for a prevalent wine spoilage strain, AWRI1499. The 12.7 Mb assembly, consisting of 324 contigs in 99 scaffolds (super-contigs) at 26-fold coverage, exhibits a relatively high density of single nucleotide polymorphisms (SNPs). Haplotype sampling for 1.2% of open reading frames suggested that the D. bruxellensis AWRI1499 genome is comprised of a moderately heterozygous diploid genome, in combination with a divergent haploid genome. Gene content analysis revealed enrichment in membrane proteins, particularly transporters, along with oxidoreductase enzymes. Availability of this assembly and annotation provides a resource for further investigation of genomic organization in this species, and functional characterization of genes that may confer important phenotypic traits. PMID:22470482

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

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).

Enabling Graph Appliance for Genome Assembly

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

Singh, Rina; Graves, Jeffrey A; Lee, Sangkeun

2015-01-01

In recent years, there has been a huge growth in the amount of genomic data available as reads generated from various genome sequencers. The number of reads generated can be huge, ranging from hundreds to billions of nucleotide, each varying in size. Assembling such large amounts of data is one of the challenging computational problems for both biomedical and data scientists. Most of the genome assemblers developed have used de Bruijn graph techniques. A de Bruijn graph represents a collection of read sequences by billions of vertices and edges, which require large amounts of memory and computational power to storemore » and process. This is the major drawback to de Bruijn graph assembly. Massively parallel, multi-threaded, shared memory systems can be leveraged to overcome some of these issues. The objective of our research is to investigate the feasibility and scalability issues of de Bruijn graph assembly on Cray s Urika-GD system; Urika-GD is a high performance graph appliance with a large shared memory and massively multithreaded custom processor designed for executing SPARQL queries over large-scale RDF data sets. However, to the best of our knowledge, there is no research on representing a de Bruijn graph as an RDF graph or finding Eulerian paths in RDF graphs using SPARQL for potential genome discovery. In this paper, we address the issues involved in representing a de Bruin graphs as RDF graphs and propose an iterative querying approach for finding Eulerian paths in large RDF graphs. We evaluate the performance of our implementation on real world ebola genome datasets and illustrate how genome assembly can be accomplished with Urika-GD using iterative SPARQL queries.« less

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).

Evolution of bird genomes-a transposon's-eye view.

PubMed

Kapusta, Aurélie; Suh, Alexander

2017-02-01

Birds, the most species-rich monophyletic group of land vertebrates, have been subject to some of the most intense sequencing efforts to date, making them an ideal case study for recent developments in genomics research. Here, we review how our understanding of bird genomes has changed with the recent sequencing of more than 75 species from all major avian taxa. We illuminate avian genome evolution from a previously neglected perspective: their repetitive genomic parasites, transposable elements (TEs) and endogenous viral elements (EVEs). We show that (1) birds are unique among vertebrates in terms of their genome organization; (2) information about the diversity of avian TEs and EVEs is changing rapidly; (3) flying birds have smaller genomes yet more TEs than flightless birds; (4) current second-generation genome assemblies fail to capture the variation in avian chromosome number and genome size determined with cytogenetics; (5) the genomic microcosm of bird-TE "arms races" has yet to be explored; and (6) upcoming third-generation genome assemblies suggest that birds exhibit stability in gene-rich regions and instability in TE-rich regions. We emphasize that integration of cytogenetics and single-molecule technologies with repeat-resolved genome assemblies is essential for understanding the evolution of (bird) genomes. © 2016 New York Academy of Sciences.

A comparative evaluation of genome assembly reconciliation tools.

PubMed

Alhakami, Hind; Mirebrahim, Hamid; Lonardi, Stefano

2017-05-18

The majority of eukaryotic genomes are unfinished due to the algorithmic challenges of assembling them. A variety of assembly and scaffolding tools are available, but it is not always obvious which tool or parameters to use for a specific genome size and complexity. It is, therefore, common practice to produce multiple assemblies using different assemblers and parameters, then select the best one for public release. A more compelling approach would allow one to merge multiple assemblies with the intent of producing a higher quality consensus assembly, which is the objective of assembly reconciliation. Several assembly reconciliation tools have been proposed in the literature, but their strengths and weaknesses have never been compared on a common dataset. We fill this need with this work, in which we report on an extensive comparative evaluation of several tools. Specifically, we evaluate contiguity, correctness, coverage, and the duplication ratio of the merged assembly compared to the individual assemblies provided as input. None of the tools we tested consistently improved the quality of the input GAGE and synthetic assemblies. Our experiments show an increase in contiguity in the consensus assembly when the original assemblies already have high quality. In terms of correctness, the quality of the results depends on the specific tool, as well as on the quality and the ranking of the input assemblies. In general, the number of misassemblies ranges from being comparable to the best of the input assembly to being comparable to the worst of the input assembly.

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.

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

PubMed

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

2018-02-09

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

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.

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

First draft genome of an iconic clownfish species (Amphiprion frenatus).

PubMed

Marcionetti, Anna; Rossier, Victor; Bertrand, Joris A M; Litsios, Glenn; Salamin, Nicolas

2018-02-17

Clownfishes (or anemonefishes) form an iconic group of coral reef fishes, principally known for their mutualistic interaction with sea anemones. They are characterized by particular life history traits, such as a complex social structure and mating system involving sequential hermaphroditism, coupled with an exceptionally long lifespan. Additionally, clownfishes are considered to be one of the rare groups to have experienced an adaptive radiation in the marine environment. Here, we assembled and annotated the first genome of a clownfish species, the tomato clownfish (Amphiprion frenatus). We obtained 17,801 assembled scaffolds, containing a total of 26,917 genes. The completeness of the assembly and annotation was satisfying, with 96.5% of the Actinopterygii Benchmarking Universal Single-Copy Orthologs (BUSCOs) being retrieved in A. frenatus assembly. The quality of the resulting assembly is comparable to other bony fish assemblies. This resource is valuable for advancing studies of the particular life history traits of clownfishes, as well as being useful for population genetic studies and the development of new phylogenetic markers. It will also open the way to comparative genomics. Indeed, future genomic comparison among closely related fishes may provide means to identify genes related to the unique adaptations to different sea anemone hosts, as well as better characterize the genomic signatures of an adaptive radiation. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

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

De novo assembly of the pepper transcriptome (Capsicum annuum): a benchmark for in silico discovery of SNPs, SSRs and candidate genes

PubMed Central

2012-01-01

Background 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. Results 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. Conclusions 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. PMID:23110314

SOBA: sequence ontology bioinformatics analysis.

PubMed

Moore, Barry; Fan, Guozhen; Eilbeck, Karen

2010-07-01

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

De Novo Assembly of the Donkey White Blood Cell Transcriptome and a Comparative Analysis of Phenotype-Associated Genes between Donkeys and Horses

PubMed Central

Xie, Feng-Yun; Feng, Yu-Long; Wang, Hong-Hui; Ma, Yun-Feng; Yang, Yang; Wang, Yin-Chao; Shen, Wei; Pan, Qing-Jie; Yin, Shen; Sun, Yu-Jiang; Ma, Jun-Yu

2015-01-01

Prior to the mechanization of agriculture and labor-intensive tasks, humans used donkeys (Equus africanus asinus) for farm work and packing. However, as mechanization increased, donkeys have been increasingly raised for meat, milk, and fur in China. To maintain the development of the donkey industry, breeding programs should focus on traits related to these new uses. Compared to conventional marker-assisted breeding plans, genome- and transcriptome-based selection methods are more efficient and effective. To analyze the coding genes of the donkey genome, we assembled the transcriptome of donkey white blood cells de novo. Using transcriptomic deep-sequencing data, we identified 264,714 distinct donkey unigenes and predicted 38,949 protein fragments. We annotated the donkey unigenes by BLAST searches against the non-redundant (NR) protein database. We also compared the donkey protein sequences with those of the horse (E. caballus) and wild horse (E. przewalskii), and linked the donkey protein fragments with mammalian phenotypes. As the outer ear size of donkeys and horses are obviously different, we compared the outer ear size-associated proteins in donkeys and horses. We identified three ear size-associated proteins, HIC1, PRKRA, and KMT2A, with sequence differences among the donkey, horse, and wild horse loci. Since the donkey genome sequence has not been released, the de novo assembled donkey transcriptome is helpful for preliminary investigations of donkey cultivars and for genetic improvement. PMID:26208029

De Novo Assembly of the Donkey White Blood Cell Transcriptome and a Comparative Analysis of Phenotype-Associated Genes between Donkeys and Horses.

PubMed

Xie, Feng-Yun; Feng, Yu-Long; Wang, Hong-Hui; Ma, Yun-Feng; Yang, Yang; Wang, Yin-Chao; Shen, Wei; Pan, Qing-Jie; Yin, Shen; Sun, Yu-Jiang; Ma, Jun-Yu

2015-01-01

Prior to the mechanization of agriculture and labor-intensive tasks, humans used donkeys (Equus africanus asinus) for farm work and packing. However, as mechanization increased, donkeys have been increasingly raised for meat, milk, and fur in China. To maintain the development of the donkey industry, breeding programs should focus on traits related to these new uses. Compared to conventional marker-assisted breeding plans, genome- and transcriptome-based selection methods are more efficient and effective. To analyze the coding genes of the donkey genome, we assembled the transcriptome of donkey white blood cells de novo. Using transcriptomic deep-sequencing data, we identified 264,714 distinct donkey unigenes and predicted 38,949 protein fragments. We annotated the donkey unigenes by BLAST searches against the non-redundant (NR) protein database. We also compared the donkey protein sequences with those of the horse (E. caballus) and wild horse (E. przewalskii), and linked the donkey protein fragments with mammalian phenotypes. As the outer ear size of donkeys and horses are obviously different, we compared the outer ear size-associated proteins in donkeys and horses. We identified three ear size-associated proteins, HIC1, PRKRA, and KMT2A, with sequence differences among the donkey, horse, and wild horse loci. Since the donkey genome sequence has not been released, the de novo assembled donkey transcriptome is helpful for preliminary investigations of donkey cultivars and for genetic improvement.

Herbicide targets and detoxification proteins in sugarcane: from gene assembly to structure modelling.

PubMed

Lloyd Evans, Dyfed; Joshi, Shailesh Vinay

2017-07-01

In a genome context, sugarcane is a classic orphan crop, in that no genome and only very few genes have been assembled. We have devised a novel exome assembly methodology that has allowed us to assemble and characterize 49 genes that serve as herbicide targets, safener interacting proteins, and members of herbicide detoxification pathways within the sugarcane genome. We have structurally modelled the products of each of these genes, as well as determining allelic, genomic, and RNA-Seq based polymorphisms for each gene. This study provides the largest collection of sugarcane structures modelled to date. We demonstrate that sugarcane genes are highly polymorphic, revealing that each genotype is evolving both uniquely and independently. In addition, we present an exome assembly system for orphan crops that can be executed on commodity infrastructure, making exome assembly practical for any group. In terms of knowledge about herbicide modes of action and detoxification, we have advanced sugarcane from a crop where no information about any herbicide-associated gene was available to the situation where sugarcane is now a species with the single largest collection of known and annotated herbicide-associated genes.

Metagenome assembly through clustering of next-generation sequencing data using protein sequences.

PubMed

Sim, Mikang; Kim, Jaebum

2015-02-01

The study of environmental microbial communities, called metagenomics, has gained a lot of attention because of the recent advances in next-generation sequencing (NGS) technologies. Microbes play a critical role in changing their environments, and the mode of their effect can be solved by investigating metagenomes. However, the difficulty of metagenomes, such as the combination of multiple microbes and different species abundance, makes metagenome assembly tasks more challenging. In this paper, we developed a new metagenome assembly method by utilizing protein sequences, in addition to the NGS read sequences. Our method (i) builds read clusters by using mapping information against available protein sequences, and (ii) creates contig sequences by finding consensus sequences through probabilistic choices from the read clusters. By using simulated NGS read sequences from real microbial genome sequences, we evaluated our method in comparison with four existing assembly programs. We found that our method could generate relatively long and accurate metagenome assemblies, indicating that the idea of using protein sequences, as a guide for the assembly, is promising. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2014-01-01

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

SvABA: genome-wide detection of structural variants and indels by local assembly.

PubMed

Wala, Jeremiah A; Bandopadhayay, Pratiti; Greenwald, Noah F; O'Rourke, Ryan; Sharpe, Ted; Stewart, Chip; Schumacher, Steve; Li, Yilong; Weischenfeldt, Joachim; Yao, Xiaotong; Nusbaum, Chad; Campbell, Peter; Getz, Gad; Meyerson, Matthew; Zhang, Cheng-Zhong; Imielinski, Marcin; Beroukhim, Rameen

2018-04-01

Structural variants (SVs), including small insertion and deletion variants (indels), are challenging to detect through standard alignment-based variant calling methods. Sequence assembly offers a powerful approach to identifying SVs, but is difficult to apply at scale genome-wide for SV detection due to its computational complexity and the difficulty of extracting SVs from assembly contigs. We describe SvABA, an efficient and accurate method for detecting SVs from short-read sequencing data using genome-wide local assembly with low memory and computing requirements. We evaluated SvABA's performance on the NA12878 human genome and in simulated and real cancer genomes. SvABA demonstrates superior sensitivity and specificity across a large spectrum of SVs and substantially improves detection performance for variants in the 20-300 bp range, compared with existing methods. SvABA also identifies complex somatic rearrangements with chains of short (<1000 bp) templated-sequence insertions copied from distant genomic regions. We applied SvABA to 344 cancer genomes from 11 cancer types and found that short templated-sequence insertions occur in ∼4% of all somatic rearrangements. Finally, we demonstrate that SvABA can identify sites of viral integration and cancer driver alterations containing medium-sized (50-300 bp) SVs. © 2018 Wala et al.; Published by Cold Spring Harbor Laboratory Press.

Nanopore DNA Sequencing and Genome Assembly on the International Space Station.

PubMed

Castro-Wallace, Sarah L; Chiu, Charles Y; John, Kristen K; Stahl, Sarah E; Rubins, Kathleen H; McIntyre, Alexa B R; Dworkin, Jason P; Lupisella, Mark L; Smith, David J; Botkin, Douglas J; Stephenson, Timothy A; Juul, Sissel; Turner, Daniel J; Izquierdo, Fernando; Federman, Scot; Stryke, Doug; Somasekar, Sneha; Alexander, Noah; Yu, Guixia; Mason, Christopher E; Burton, Aaron S

2017-12-21

We evaluated the performance of the MinION DNA sequencer in-flight on the International Space Station (ISS), and benchmarked its performance off-Earth against the MinION, Illumina MiSeq, and PacBio RS II sequencing platforms in terrestrial laboratories. Samples contained equimolar mixtures of genomic DNA from lambda bacteriophage, Escherichia coli (strain K12, MG1655) and Mus musculus (female BALB/c mouse). Nine sequencing runs were performed aboard the ISS over a 6-month period, yielding a total of 276,882 reads with no apparent decrease in performance over time. From sequence data collected aboard the ISS, we constructed directed assemblies of the ~4.6 Mb E. coli genome, ~48.5 kb lambda genome, and a representative M. musculus sequence (the ~16.3 kb mitochondrial genome), at 100%, 100%, and 96.7% consensus pairwise identity, respectively; de novo assembly of the E. coli genome from raw reads yielded a single contig comprising 99.9% of the genome at 98.6% consensus pairwise identity. Simulated real-time analyses of in-flight sequence data using an automated bioinformatic pipeline and laptop-based genomic assembly demonstrated the feasibility of sequencing analysis and microbial identification aboard the ISS. These findings illustrate the potential for sequencing applications including disease diagnosis, environmental monitoring, and elucidating the molecular basis for how organisms respond to spaceflight.

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

PubMed

Narzisi, Giuseppe; Mishra, Bud

2011-04-29

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

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.

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.

Application of long sequence reads to improve genomes for Clostridium thermocellum AD2, Clostridium thermocellum LQRI, and Pelosinus fermentans R7

DOE PAGES

Utturkar, Sagar M.; Bayer, Edward A.; Borovok, Ilya; ...

2016-09-29

Here, we and others have shown the utility of long sequence reads to improve genome assembly quality. In this study, we generated PacBio DNA sequence data to improve the assemblies of draft genomes for Clostridium thermocellum AD2, Clostridium thermocellum LQRI, and Pelosinus fermentans R7.

Hybrid genome assembly and annotation of Paenibacillus pasadenensis strain R16 reveals insights on endophytic life style and antifungal activity

PubMed Central

Passera, Alessandro; Marcolungo, Luca; Brasca, Milena; Quaglino, Fabio; Cantaloni, Chiara; Delledonne, Massimo

2018-01-01

Bacteria of the Paenibacillus genus are becoming important in many fields of science, including agriculture, for their positive effects on the health of plants. However, there are little information available on this genus compared to other bacteria (such as Bacillus or Pseudomonas), especially when considering genomic information. Sequencing the genomes of plant-beneficial bacteria is a crucial step to identify the genetic elements underlying the adaptation to life inside a plant host and, in particular, which of these features determine the differences between a helpful microorganism and a pathogenic one. In this study, we have characterized the genome of Paenibacillus pasadenensis, strain R16, recently investigated for its antifungal activities and plant-associated features. An hybrid assembly approach was used integrating the very precise reads obtained by Illumina technology and long fragments acquired with Oxford Nanopore Technology (ONT) sequencing. De novo genome assembly based solely on Illumina reads generated a relatively fragmented assembly of 5.72 Mbp in 99 ungapped sequences with an N50 length of 544 Kbp; hybrid assembly, integrating Illumina and ONT reads, improved the assembly quality, generating a genome of 5.75 Mbp, organized in 6 contigs with an N50 length of 3.4 Mbp. Annotation of the latter genome identified 4987 coding sequences, of which 1610 are hypothetical proteins. Enrichment analysis identified pathways of particular interest for the endophyte biology, including the chitin-utilization pathway and the incomplete siderophore pathway which hints at siderophore parasitism. In addition the analysis led to the identification of genes for the production of terpenes, as for example farnesol, that was hypothesized as the main antifungal molecule produced by the strain. The functional analysis on the genome confirmed several plant-associated, plant-growth promotion, and biocontrol traits of strain R16, thus adding insights in the genetic bases of these complex features, and of the Paenibacillus genus in general. PMID:29351296

De novo assembling and primary analysis of genome and transcriptome of gray whale Eschrichtius robustus.

PubMed

Moskalev, Alexey А; Kudryavtseva, Anna V; Graphodatsky, Alexander S; Beklemisheva, Violetta R; Serdyukova, Natalya A; Krutovsky, Konstantin V; Sharov, Vadim V; Kulakovskiy, Ivan V; Lando, Andrey S; Kasianov, Artem S; Kuzmin, Dmitry A; Putintseva, Yuliya A; Feranchuk, Sergey I; Shaposhnikov, Mikhail V; Fraifeld, Vadim E; Toren, Dmitri; Snezhkina, Anastasia V; Sitnik, Vasily V

2017-12-28

Gray whale, Eschrichtius robustus (E. robustus), is a single member of the family Eschrichtiidae, which is considered to be the most primitive in the class Cetacea. Gray whale is often described as a "living fossil". It is adapted to extreme marine conditions and has a high life expectancy (77 years). The assembly of a gray whale genome and transcriptome will allow to carry out further studies of whale evolution, longevity, and resistance to extreme environment. In this work, we report the first de novo assembly and primary analysis of the E. robustus genome and transcriptome based on kidney and liver samples. The presented draft genome assembly is complete by 55% in terms of a total genome length, but only by 24% in terms of the BUSCO complete gene groups, although 10,895 genes were identified. Transcriptome annotation and comparison with other whale species revealed robust expression of DNA repair and hypoxia-response genes, which is expected for whales. This preliminary study of the gray whale genome and transcriptome provides new data to better understand the whale evolution and the mechanisms of their adaptation to the hypoxic conditions.

The reconstruction of 2,631 draft metagenome-assembled genomes from the global oceans

PubMed Central

Tully, Benjamin J.; Graham, Elaina D.; Heidelberg, John F.

2018-01-01

Microorganisms play a crucial role in mediating global biogeochemical cycles in the marine environment. By reconstructing the genomes of environmental organisms through metagenomics, researchers are able to study the metabolic potential of Bacteria and Archaea that are resistant to isolation in the laboratory. Utilizing the large metagenomic dataset generated from 234 samples collected during the Tara Oceans circumnavigation expedition, we were able to assemble 102 billion paired-end reads into 562 million contigs, which in turn were co-assembled and consolidated in to 7.2 million contigs ≥2 kb in length. Approximately 1 million of these contigs were binned to reconstruct draft genomes. In total, 2,631 draft genomes with an estimated completion of ≥50% were generated (1,491 draft genomes >70% complete; 603 genomes >90% complete). A majority of the draft genomes were manually assigned phylogeny based on sets of concatenated phylogenetic marker genes and/or 16S rRNA gene sequences. The draft genomes are now publically available for the research community at-large. PMID:29337314

Phylogenic study of Lemnoideae (duckweeds) through complete chloroplast genomes for eight accessions.

PubMed

Ding, Yanqiang; Fang, Yang; Guo, Ling; Li, Zhidan; He, Kaize; Zhao, Yun; Zhao, Hai

2017-01-01

Phylogenetic relationship within different genera of Lemnoideae, a kind of small aquatic monocotyledonous plants, was not well resolved, using either morphological characters or traditional markers. Given that rich genetic information in chloroplast genome makes them particularly useful for phylogenetic studies, we used chloroplast genomes to clarify the phylogeny within Lemnoideae. DNAs were sequenced with next-generation sequencing. The duckweeds chloroplast genomes were indirectly filtered from the total DNA data, or directly obtained from chloroplast DNA data. To test the reliability of assembling the chloroplast genome based on the filtration of the total DNA, two methods were used to assemble the chloroplast genome of Landoltia punctata strain ZH0202. A phylogenetic tree was built on the basis of the whole chloroplast genome sequences using MrBayes v.3.2.6 and PhyML 3.0. Eight complete duckweeds chloroplast genomes were assembled, with lengths ranging from 165,775 bp to 171,152 bp, and each contains 80 protein-coding sequences, four rRNAs, 30 tRNAs and two pseudogenes. The identity of L. punctata strain ZH0202 chloroplast genomes assembled through two methods was 100%, and their sequences and lengths were completely identical. The chloroplast genome comparison demonstrated that the differences in chloroplast genome sizes among the Lemnoideae primarily resulted from variation in non-coding regions, especially from repeat sequence variation. The phylogenetic analysis demonstrated that the different genera of Lemnoideae are derived from each other in the following order: Spirodela , Landoltia , Lemna , Wolffiella , and Wolffia . This study demonstrates potential of whole chloroplast genome DNA as an effective option for phylogenetic studies of Lemnoideae. It also showed the possibility of using chloroplast DNA data to elucidate those phylogenies which were not yet solved well by traditional methods even in plants other than duckweeds.

Phylogenic study of Lemnoideae (duckweeds) through complete chloroplast genomes for eight accessions

PubMed Central

Ding, Yanqiang; Fang, Yang; Guo, Ling; Li, Zhidan; He, Kaize

2017-01-01

Background Phylogenetic relationship within different genera of Lemnoideae, a kind of small aquatic monocotyledonous plants, was not well resolved, using either morphological characters or traditional markers. Given that rich genetic information in chloroplast genome makes them particularly useful for phylogenetic studies, we used chloroplast genomes to clarify the phylogeny within Lemnoideae. Methods DNAs were sequenced with next-generation sequencing. The duckweeds chloroplast genomes were indirectly filtered from the total DNA data, or directly obtained from chloroplast DNA data. To test the reliability of assembling the chloroplast genome based on the filtration of the total DNA, two methods were used to assemble the chloroplast genome of Landoltia punctata strain ZH0202. A phylogenetic tree was built on the basis of the whole chloroplast genome sequences using MrBayes v.3.2.6 and PhyML 3.0. Results Eight complete duckweeds chloroplast genomes were assembled, with lengths ranging from 165,775 bp to 171,152 bp, and each contains 80 protein-coding sequences, four rRNAs, 30 tRNAs and two pseudogenes. The identity of L. punctata strain ZH0202 chloroplast genomes assembled through two methods was 100%, and their sequences and lengths were completely identical. The chloroplast genome comparison demonstrated that the differences in chloroplast genome sizes among the Lemnoideae primarily resulted from variation in non-coding regions, especially from repeat sequence variation. The phylogenetic analysis demonstrated that the different genera of Lemnoideae are derived from each other in the following order: Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia. Discussion This study demonstrates potential of whole chloroplast genome DNA as an effective option for phylogenetic studies of Lemnoideae. It also showed the possibility of using chloroplast DNA data to elucidate those phylogenies which were not yet solved well by traditional methods even in plants other than duckweeds. PMID:29302399

A ddRAD-based genetic map and its integration with the genome assembly of Japanese eel (Anguilla japonica) provides insights into genome evolution after the teleost-specific genome duplication

PubMed Central

2014-01-01

Background Recent advancements in next-generation sequencing technology have enabled cost-effective sequencing of whole or partial genomes, permitting the discovery and characterization of molecular polymorphisms. Double-digest restriction-site associated DNA sequencing (ddRAD-seq) is a powerful and inexpensive approach to developing numerous single nucleotide polymorphism (SNP) markers and constructing a high-density genetic map. To enrich genomic resources for Japanese eel (Anguilla japonica), we constructed a ddRAD-based genetic map using an Ion Torrent Personal Genome Machine and anchored scaffolds of the current genome assembly to 19 linkage groups of the Japanese eel. Furthermore, we compared the Japanese eel genome with genomes of model fishes to infer the history of genome evolution after the teleost-specific genome duplication. Results We generated the ddRAD-based linkage map of the Japanese eel, where the maps for female and male spanned 1748.8 cM and 1294.5 cM, respectively, and were arranged into 19 linkage groups. A total of 2,672 SNP markers and 115 Simple Sequence Repeat markers provide anchor points to 1,252 scaffolds covering 151 Mb (13%) of the current genome assembly of the Japanese eel. Comparisons among the Japanese eel, medaka, zebrafish and spotted gar genomes showed highly conserved synteny among teleosts and revealed part of the eight major chromosomal rearrangement events that occurred soon after the teleost-specific genome duplication. Conclusions The ddRAD-seq approach combined with the Ion Torrent Personal Genome Machine sequencing allowed us to conduct efficient and flexible SNP genotyping. The integration of the genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits and for investigating comparative genomics of the Japanese eel. PMID:24669946

A ddRAD-based genetic map and its integration with the genome assembly of Japanese eel (Anguilla japonica) provides insights into genome evolution after the teleost-specific genome duplication.

PubMed

Kai, Wataru; Nomura, Kazuharu; Fujiwara, Atushi; Nakamura, Yoji; Yasuike, Motoshige; Ojima, Nobuhiko; Masaoka, Tetsuji; Ozaki, Akiyuki; Kazeto, Yukinori; Gen, Koichiro; Nagao, Jiro; Tanaka, Hideki; Kobayashi, Takanori; Ototake, Mitsuru

2014-03-26

Recent advancements in next-generation sequencing technology have enabled cost-effective sequencing of whole or partial genomes, permitting the discovery and characterization of molecular polymorphisms. Double-digest restriction-site associated DNA sequencing (ddRAD-seq) is a powerful and inexpensive approach to developing numerous single nucleotide polymorphism (SNP) markers and constructing a high-density genetic map. To enrich genomic resources for Japanese eel (Anguilla japonica), we constructed a ddRAD-based genetic map using an Ion Torrent Personal Genome Machine and anchored scaffolds of the current genome assembly to 19 linkage groups of the Japanese eel. Furthermore, we compared the Japanese eel genome with genomes of model fishes to infer the history of genome evolution after the teleost-specific genome duplication. We generated the ddRAD-based linkage map of the Japanese eel, where the maps for female and male spanned 1748.8 cM and 1294.5 cM, respectively, and were arranged into 19 linkage groups. A total of 2,672 SNP markers and 115 Simple Sequence Repeat markers provide anchor points to 1,252 scaffolds covering 151 Mb (13%) of the current genome assembly of the Japanese eel. Comparisons among the Japanese eel, medaka, zebrafish and spotted gar genomes showed highly conserved synteny among teleosts and revealed part of the eight major chromosomal rearrangement events that occurred soon after the teleost-specific genome duplication. The ddRAD-seq approach combined with the Ion Torrent Personal Genome Machine sequencing allowed us to conduct efficient and flexible SNP genotyping. The integration of the genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits and for investigating comparative genomics of the Japanese eel.

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.

De novo genome assembly of the red silk cotton tree (Bombax ceiba).

PubMed

Gao, Yong; Wang, Haibo; Liu, Chao; Chu, Honglong; Dai, Dongqin; Song, Shengnan; Yu, Long; Han, Lihong; Fu, Yi; Tian, Bin; Tang, Lizhou

2018-05-01

Bombax ceiba L. (the red silk cotton tree) is a large deciduous tree that is distributed in tropical and sub-tropical Asia as well as northern Australia. It has great economic and ecological importance, with several applications in industry and traditional medicine in many Asian countries. To facilitate further utilization of this plant resource, we present here the draft genome sequence for B. ceiba. We assembled a relatively intact genome of B. ceiba by using PacBio single-molecule sequencing and BioNano optical mapping technologies. The final draft genome is approximately 895 Mb long, with contig and scaffold N50 sizes of 1.0 Mb and 2.06 Mb, respectively. The high-quality draft genome assembly of B. ceiba will be a valuable resource enabling further genetic improvement and more effective use of this tree species.

Metagenomic assembly through the lens of validation: recent advances in assessing and improving the quality of genomes assembled from metagenomes.

PubMed

Olson, Nathan D; Treangen, Todd J; Hill, Christopher M; Cepeda-Espinoza, Victoria; Ghurye, Jay; Koren, Sergey; Pop, Mihai

2017-08-07

Metagenomic samples are snapshots of complex ecosystems at work. They comprise hundreds of known and unknown species, contain multiple strain variants and vary greatly within and across environments. Many microbes found in microbial communities are not easily grown in culture making their DNA sequence our only clue into their evolutionary history and biological function. Metagenomic assembly is a computational process aimed at reconstructing genes and genomes from metagenomic mixtures. Current methods have made significant strides in reconstructing DNA segments comprising operons, tandem gene arrays and syntenic blocks. Shorter, higher-throughput sequencing technologies have become the de facto standard in the field. Sequencers are now able to generate billions of short reads in only a few days. Multiple metagenomic assembly strategies, pipelines and assemblers have appeared in recent years. Owing to the inherent complexity of metagenome assembly, regardless of the assembly algorithm and sequencing method, metagenome assemblies contain errors. Recent developments in assembly validation tools have played a pivotal role in improving metagenomics assemblers. Here, we survey recent progress in the field of metagenomic assembly, provide an overview of key approaches for genomic and metagenomic assembly validation and demonstrate the insights that can be derived from assemblies through the use of assembly validation strategies. We also discuss the potential for impact of long-read technologies in metagenomics. We conclude with a discussion of future challenges and opportunities in the field of metagenomic assembly and validation. © The Author 2017. Published by Oxford University Press.

Plastid genome sequence of an ornamental and editable fruit tree of Rosaceae, Prunus mume.

PubMed

Wang, Shuo; Gao, Cheng-Wen; Gao, Li-Zhi

2016-11-01

Here we assembled and analyzed the complete chloroplast genome of Prunus mume, a popular ornamental and editable fruit tree of Rosaceae. The cp genome exhibited a circular DNA molecule of 157 712 bp with a typical quadripartite structure consisted of two inverted repeat regions (IRa and IRb) of 26 394 bp separated by large (LSC) and small (SSC) single-copy regions of 85 861 and 19 063 bp, respectively. It encoded 112 unique genes, 19 of which were duplicated in the IR regions, giving a total of 131 genes. Eighteen of these genes harbored one or two introns. GC content was 38.9%, and coding regions accounted for 51.3% of the genome. Phylogenetic analysis showed that P. mume clustered with P. persica and P. kansuensis in the genus Punus. This newly determined chloroplast genome will enhance modern breeding programs for the purpose of genetic improvement of this valuable plant.

Nucleic Acid Binding by Mason-Pfizer Monkey Virus CA Promotes Virus Assembly and Genome Packaging

PubMed Central

Füzik, Tibor; Píchalová, Růžena; Schur, Florian K. M.; Strohalmová, Karolína; Křížová, Ivana; Hadravová, Romana; Rumlová, Michaela; Briggs, John A. G.

2016-01-01

ABSTRACT The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCE Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV. PMID:26912613

Final Report for LDRD Project 02-ERD-069: Discovering the Unknown Mechanism(s) of Virulence in a BW, Class A Select Agent

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

Chain, P; Garcia, E

2003-02-06

The goal of this proposed effort was to assess the difficulty in identifying and characterizing virulence candidate genes in an organism for which very limited data exists. This was accomplished by first addressing the finishing phase of draft-sequenced F. tularensis genomes and conducting comparative analyses to determine the coding potential of each genome; to discover the differences in genome structure and content, and to identify potential genes whose products may be involved in the F. tularensis virulence process. The project was divided into three parts: (1) Genome finishing: This part involves determining the order and orientation of the consensus sequencesmore » of contigs obtained from Phrap assemblies of random draft genomic sequences. This tedious process consists of linking contig ends using information embedded in each sequence file that relates the sequence to the original cloned insert. Since inserts are sequenced from both ends, we can establish a link between these paired-ends in different contigs and thus order and orient contigs. Since these genomes carry numerous copies of insertion sequences, these repeated elements ''confuse'' the Phrap assembly program. It is thus necessary to break these contigs apart at the repeated sequences and individually join the proper flanking regions using paired-end information, or using results of comparisons against a similar genome. Larger repeated elements such as the small subunit ribosomal RNA operon require verification with PCR. Tandem repeats require manual intervention and typically rely on single nucleotide polymorphisms to be resolved. Remaining gaps require PCR reactions and sequencing. Once the genomes have been ''closed'', low quality regions are addressed by resequencing reactions. (2) Genome analysis: The final consensus sequences are processed by combining the results of three gene modelers: Glimmer, Critica and Generation. The final gene models are submitted to a battery of homology searches and domain prediction programs in order to annotate them (e.g. BLAST, Pfam, TIGRfam, COG, KEGG, InterPro, TMhmm, SignalP). The genome structure is also assessed in terms of G+C content, GC bias (GC skew), and locations of repeated regions (e.g. IS elements) and phage-like genes. (3) Comparative genomics: The results of the various genome analyses are compared between the finished (or almost finished) genomes. Here, we have compared the F. tularensis genomes from the extremely lethal strain Schu4 (subsp. tularensis), the vaccine strain LVS (subsp. holartica), and strain UT01-4992 of the less virulent, opportunistic subsp. novicida. Regions present in the highly virulent strain that are absent from the other less virulent strains may provide insight into what factors are required for the high level of virulence.« less

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data.

PubMed

de Andrade, Roberto R S; Vaslin, Maite F S

2014-03-07

Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/.

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data

PubMed Central

2014-01-01

Background Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. Methods In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. Results The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. Conclusions SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. Availability and implementation SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/. PMID:24607237

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

PubMed

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

2016-01-01

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

New genomic resources for switchgrass: a BAC library and comparative analysis of homoeologous genomic regions harboring bioenergy traits

PubMed Central

2011-01-01

Background Switchgrass, a C4 species and a warm-season grass native to the prairies of North America, has been targeted for development into an herbaceous biomass fuel crop. Genetic improvement of switchgrass feedstock traits through marker-assisted breeding and biotechnology approaches calls for genomic tools development. Establishment of integrated physical and genetic maps for switchgrass will accelerate mapping of value added traits useful to breeding programs and to isolate important target genes using map based cloning. The reported polyploidy series in switchgrass ranges from diploid (2X = 18) to duodecaploid (12X = 108). Like in other large, repeat-rich plant genomes, this genomic complexity will hinder whole genome sequencing efforts. An extensive physical map providing enough information to resolve the homoeologous genomes would provide the necessary framework for accurate assembly of the switchgrass genome. Results A switchgrass BAC library constructed by partial digestion of nuclear DNA with EcoRI contains 147,456 clones covering the effective genome approximately 10 times based on a genome size of 3.2 Gigabases (~1.6 Gb effective). Restriction digestion and PFGE analysis of 234 randomly chosen BACs indicated that 95% of the clones contained inserts, ranging from 60 to 180 kb with an average of 120 kb. Comparative sequence analysis of two homoeologous genomic regions harboring orthologs of the rice OsBRI1 locus, a low-copy gene encoding a putative protein kinase and associated with biomass, revealed that orthologous clones from homoeologous chromosomes can be unambiguously distinguished from each other and correctly assembled to respective fingerprint contigs. Thus, the data obtained not only provide genomic resources for further analysis of switchgrass genome, but also improve efforts for an accurate genome sequencing strategy. Conclusions The construction of the first switchgrass BAC library and comparative analysis of homoeologous harboring OsBRI1 orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy. PMID:21767393

New genomic resources for switchgrass: a BAC library and comparative analysis of homoeologous genomic regions harboring bioenergy traits.

PubMed

Saski, Christopher A; Li, Zhigang; Feltus, Frank A; Luo, Hong

2011-07-18

Switchgrass, a C4 species and a warm-season grass native to the prairies of North America, has been targeted for development into an herbaceous biomass fuel crop. Genetic improvement of switchgrass feedstock traits through marker-assisted breeding and biotechnology approaches calls for genomic tools development. Establishment of integrated physical and genetic maps for switchgrass will accelerate mapping of value added traits useful to breeding programs and to isolate important target genes using map based cloning. The reported polyploidy series in switchgrass ranges from diploid (2X = 18) to duodecaploid (12X = 108). Like in other large, repeat-rich plant genomes, this genomic complexity will hinder whole genome sequencing efforts. An extensive physical map providing enough information to resolve the homoeologous genomes would provide the necessary framework for accurate assembly of the switchgrass genome. A switchgrass BAC library constructed by partial digestion of nuclear DNA with EcoRI contains 147,456 clones covering the effective genome approximately 10 times based on a genome size of 3.2 Gigabases (~1.6 Gb effective). Restriction digestion and PFGE analysis of 234 randomly chosen BACs indicated that 95% of the clones contained inserts, ranging from 60 to 180 kb with an average of 120 kb. Comparative sequence analysis of two homoeologous genomic regions harboring orthologs of the rice OsBRI1 locus, a low-copy gene encoding a putative protein kinase and associated with biomass, revealed that orthologous clones from homoeologous chromosomes can be unambiguously distinguished from each other and correctly assembled to respective fingerprint contigs. Thus, the data obtained not only provide genomic resources for further analysis of switchgrass genome, but also improve efforts for an accurate genome sequencing strategy. The construction of the first switchgrass BAC library and comparative analysis of homoeologous harboring OsBRI1 orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy.

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.

Draft genome sequence of ramie, Boehmeria nivea (L.) Gaudich.

PubMed

Luan, Ming-Bao; Jian, Jian-Bo; Chen, Ping; Chen, Jun-Hui; Chen, Jian-Hua; Gao, Qiang; Gao, Gang; Zhou, Ju-Hong; Chen, Kun-Mei; Guang, Xuan-Min; Chen, Ji-Kang; Zhang, Qian-Qian; Wang, Xiao-Fei; Fang, Long; Sun, Zhi-Min; Bai, Ming-Zhou; Fang, Xiao-Dong; Zhao, Shan-Cen; Xiong, He-Ping; Yu, Chun-Ming; Zhu, Ai-Guo

2018-05-01

Ramie, Boehmeria nivea (L.) Gaudich, family Urticaceae, is a plant native to eastern Asia, and one of the world's oldest fibre crops. It is also used as animal feed and for the phytoremediation of heavy metal-contaminated farmlands. Thus, the genome sequence of ramie was determined to explore the molecular basis of its fibre quality, protein content and phytoremediation. For further understanding ramie genome, different paired-end and mate-pair libraries were combined to generate 134.31 Gb of raw DNA sequences using the Illumina whole-genome shotgun sequencing approach. The highly heterozygous B. nivea genome was assembled using the Platanus Genome Assembler, which is an effective tool for the assembly of highly heterozygous genome sequences. The final length of the draft genome of this species was approximately 341.9 Mb (contig N50 = 22.62 kb, scaffold N50 = 1,126.36 kb). Based on ramie genome annotations, 30,237 protein-coding genes were predicted, and the repetitive element content was 46.3%. The completeness of the final assembly was evaluated by benchmarking universal single-copy orthologous genes (BUSCO); 90.5% of the 1,440 expected embryophytic genes were identified as complete, and 4.9% were identified as fragmented. Phylogenetic analysis based on single-copy gene families and one-to-one orthologous genes placed ramie with mulberry and cannabis, within the clade of urticalean rosids. Genome information of ramie will be a valuable resource for the conservation of endangered Boehmeria species and for future studies on the biogeography and characteristic evolution of members of Urticaceae. © 2018 John Wiley & Sons Ltd.

Estimating gene gain and loss rates in the presence of error in genome assembly and annotation using CAFE 3.

PubMed

Han, Mira V; Thomas, Gregg W C; Lugo-Martinez, Jose; Hahn, Matthew W

2013-08-01

Current sequencing methods produce large amounts of data, but genome assemblies constructed from these data are often fragmented and incomplete. Incomplete and error-filled assemblies result in many annotation errors, especially in the number of genes present in a genome. This means that methods attempting to estimate rates of gene duplication and loss often will be misled by such errors and that rates of gene family evolution will be consistently overestimated. Here, we present a method that takes these errors into account, allowing one to accurately infer rates of gene gain and loss among genomes even with low assembly and annotation quality. The method is implemented in the newest version of the software package CAFE, along with several other novel features. We demonstrate the accuracy of the method with extensive simulations and reanalyze several previously published data sets. Our results show that errors in genome annotation do lead to higher inferred rates of gene gain and loss but that CAFE 3 sufficiently accounts for these errors to provide accurate estimates of important evolutionary parameters.

Draft Genome Sequences of 12 Dry-Heat-Resistant Bacillus Strains Isolated from the Cleanrooms Where the Viking Spacecraft Were Assembled

PubMed Central

Seuylemezian, Arman; Cooper, Kerry; Schubert, Wayne

2018-01-01

ABSTRACT Spore-forming microorganisms are of concern for forward contamination because they can survive harsh interplanetary travel. Here, we report the draft genome sequences of 12 spore-forming strains isolated from the Manned Spacecraft Operations Building (MSOB) and the Vehicle Assembly Building (VAB) in Cape Canaveral, FL, where the Viking spacecraft were assembled. PMID:29567731

The draft genome sequence of forest musk deer (Moschus berezovskii).

PubMed

Fan, Zhenxin; Li, Wujiao; Jin, Jiazheng; Cui, Kai; Yan, Chaochao; Peng, Changjun; Jian, Zuoyi; Bu, Ping; Price, Megan; Zhang, Xiuyue; Shen, Yongmei; Li, Jing; Q, Wenhua; Yue, Bisong

2018-04-01

The forest musk deer, Moschus berezovskii, is one of seven musk deer (Moschus spp.) and is distributed in Southwest China. Akin to other musk deer, the forest musk deer has been traditionally and is currently hunted for its musk (i.e., global perfume industry). Considerable hunting pressure and habitat loss have caused significant population declines. Consequently, the Chinese government commenced captive breeding programs for musk harvesting in the 1950s. However, the prevalence of fatal diseases is considerably restricting population increases. Disease severity and extent are exacerbated by inbreeding and genetic diversity declines in captive musk deer populations. It is essential that knowledge of captive and wild forest musk deer populations' immune system and genome be gained in order to improve their physical and genetic health. We have thus sequenced the whole genome of the forest musk deer, completed the genomic assembly and annotation, and performed preliminary bioinformatic analyses. A total of 407 Gb raw reads from whole-genome sequencing were generated using the Illumina HiSeq 4000 platform. The final genome assembly is around 2.72 Gb, with a contig N50 length of 22.6 kb and a scaffold N50 length of 2.85 Mb. We identified 24,352 genes and found that 42.05% of the genome is composed of repetitive elements. We also detected 1,236 olfactory receptor genes. The genome-wide phylogenetic tree indicated that the forest musk deer was within the order Artiodactyla, and it appeared as the sister clade of four members of Bovidae. In total, 576 genes were under positive selection in the forest musk deer lineage. We provide the first genome sequence and gene annotation for the forest musk deer. The availability of these resources will be very useful for the conservation and captive breeding of this endangered and economically important species and for reconstructing the evolutionary history of the order Artiodactyla.

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.

The present and future of de novo whole-genome assembly.

PubMed

Sohn, Jang-Il; Nam, Jin-Wu

2018-01-01

As the advent of next-generation sequencing (NGS) technology, various de novo assembly algorithms based on the de Bruijn graph have been developed to construct chromosome-level sequences. However, numerous technical or computational challenges in de novo assembly still remain, although many bright ideas and heuristics have been suggested to tackle the challenges in both experimental and computational settings. In this review, we categorize de novo assemblers on the basis of the type of de Bruijn graphs (Hamiltonian and Eulerian) and discuss the challenges of de novo assembly for short NGS reads regarding computational complexity and assembly ambiguity. Then, we discuss how the limitations of the short reads can be overcome by using a single-molecule sequencing platform that generates long reads of up to several kilobases. In fact, the long read assembly has caused a paradigm shift in whole-genome assembly in terms of algorithms and supporting steps. We also summarize (i) hybrid assemblies using both short and long reads and (ii) overlap-based assemblies for long reads and discuss their challenges and future prospects. This review provides guidelines to determine the optimal approach for a given input data type, computational budget or genome. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Bonus Organisms in High-Throughput Eukaryotic Whole-Genome Shorgun Assembly

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

Pangilinan, Jasmyn; Shapiro, Harris; Tu, Hank

2006-02-06

The DOE Joint Genome Institute has sequenced over 50 eukaryotic genomes, ranging in size from 15 MB to 1.6 GB, over a wide range of organism types. In the course of doing so, it has become clear that a substantial fraction of these data sets contains bonus organisms, usually prokaryotes, in addition to the desired genome. While some of these additional organisms are extraneous contamination, they are sometimes symbionts, and so can be of biological interest. Therefore, it is desirable to assemble the bonus organisms along with the main genome. This transforms the problem into one of metagenomic assembly, whichmore » is considerably more challenging than traditional whole-genome shotgun (WGS) assembly. The different organisms will usually be present at different sequence depths, which is difficult to handle in most WGS assemblers. In addition, with multiple distinct genomes present, chimerism can produce cross-organism combinations. Finally, there is no guarantee that only a single bonus organism will be present. For example, one JGI project contained at least two different prokaryotic contaminants, plus a 145 KB plasmid of unknown origin. We have developed techniques to routinely identify and handle such bonus organisms in a high-throughput sequencing environment. Approaches include screening and partitioning the unassembled data, and iterative subassemblies. These methods are applicable not only to bonus organisms, but also to desired components such as organelles. These procedures have the additional benefit of identifying, and allowing for the removal of, cloning artifacts such as E.coli and spurious vector inclusions.« less

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

PubMed Central

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

2012-01-01

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

The Microbial Genomes Atlas (MiGA) webserver: taxonomic and gene diversity analysis of Archaea and Bacteria at the whole genome level.

PubMed

Rodriguez-R, Luis M; Gunturu, Santosh; Harvey, William T; Rosselló-Mora, Ramon; Tiedje, James M; Cole, James R; Konstantinidis, Konstantinos T

2018-06-14

The small subunit ribosomal RNA gene (16S rRNA) has been successfully used to catalogue and study the diversity of prokaryotic species and communities but it offers limited resolution at the species and finer levels, and cannot represent the whole-genome diversity and fluidity. To overcome these limitations, we introduced the Microbial Genomes Atlas (MiGA), a webserver that allows the classification of an unknown query genomic sequence, complete or partial, against all taxonomically classified taxa with available genome sequences, as well as comparisons to other related genomes including uncultivated ones, based on the genome-aggregate Average Nucleotide and Amino Acid Identity (ANI/AAI) concepts. MiGA integrates best practices in sequence quality trimming and assembly and allows input to be raw reads or assemblies from isolate genomes, single-cell sequences, and metagenome-assembled genomes (MAGs). Further, MiGA can take as input hundreds of closely related genomes of the same or closely related species (a so-called 'Clade Project') to assess their gene content diversity and evolutionary relationships, and calculate important clade properties such as the pangenome and core gene sets. Therefore, MiGA is expected to facilitate a range of genome-based taxonomic and diversity studies, and quality assessment across environmental and clinical settings. MiGA is available at http://microbial-genomes.org/.

Sequencing, De Novo Assembly, and Annotation of the Complete Genome of a New Thraustochytrid Species, Strain CCAP_4062/3

PubMed Central

Seddiki, Khawla; Godart, François; Aiese Cigliano, Riccardo; Sanseverino, Walter; Barakat, Mohamed; Ortet, Philippe; Rébeillé, Fabrice; Maréchal, Eric

2018-01-01

ABSTRACT Thraustochytrids are ecologically and biotechnologically relevant marine species. We report here the de novo assembly and annotation of the whole-genome sequence of a new thraustochytrid strain, CCAP_4062/3. The genome size was estimated at 38.7 Mb with 11,853 predicted coding sequences, and the GC content was scored at 57%. PMID:29545303

preAssemble: a tool for automatic sequencer trace data processing.

PubMed

Adzhubei, Alexei A; Laerdahl, Jon K; Vlasova, Anna V

2006-01-17

Trace or chromatogram files (raw data) are produced by automatic nucleic acid sequencing equipment or sequencers. Each file contains information which can be interpreted by specialised software to reveal the sequence (base calling). This is done by the sequencer proprietary software or publicly available programs. Depending on the size of a sequencing project the number of trace files can vary from just a few to thousands of files. Sequencing quality assessment on various criteria is important at the stage preceding clustering and contig assembly. Two major publicly available packages--Phred and Staden are used by preAssemble to perform sequence quality processing. The preAssemble pre-assembly sequence processing pipeline has been developed for small to large scale automatic processing of DNA sequencer chromatogram (trace) data. The Staden Package Pregap4 module and base-calling program Phred are utilized in the pipeline, which produces detailed and self-explanatory output that can be displayed with a web browser. preAssemble can be used successfully with very little previous experience, however options for parameter tuning are provided for advanced users. preAssemble runs under UNIX and LINUX operating systems. It is available for downloading and will run as stand-alone software. It can also be accessed on the Norwegian Salmon Genome Project web site where preAssemble jobs can be run on the project server. preAssemble is a tool allowing to perform quality assessment of sequences generated by automatic sequencing equipment. preAssemble is flexible since both interactive jobs on the preAssemble server and the stand alone downloadable version are available. Virtually no previous experience is necessary to run a default preAssemble job, on the other hand options for parameter tuning are provided. Consequently preAssemble can be used as efficiently for just several trace files as for large scale sequence processing.

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.

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.

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.

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.

Assembly of 913 microbial genomes from metagenomic sequencing of the cow rumen.

PubMed

Stewart, Robert D; Auffret, Marc D; Warr, Amanda; Wiser, Andrew H; Press, Maximilian O; Langford, Kyle W; Liachko, Ivan; Snelling, Timothy J; Dewhurst, Richard J; Walker, Alan W; Roehe, Rainer; Watson, Mick

2018-02-28

The cow rumen is adapted for the breakdown of plant material into energy and nutrients, a task largely performed by enzymes encoded by the rumen microbiome. Here we present 913 draft bacterial and archaeal genomes assembled from over 800 Gb of rumen metagenomic sequence data derived from 43 Scottish cattle, using both metagenomic binning and Hi-C-based proximity-guided assembly. Most of these genomes represent previously unsequenced strains and species. The draft genomes contain over 69,000 proteins predicted to be involved in carbohydrate metabolism, over 90% of which do not have a good match in public databases. Inclusion of the 913 genomes presented here improves metagenomic read classification by sevenfold against our own data, and by fivefold against other publicly available rumen datasets. Thus, our dataset substantially improves the coverage of rumen microbial genomes in the public databases and represents a valuable resource for biomass-degrading enzyme discovery and studies of the rumen microbiome.

Physical Maps for Genome Analysis of Serotype A and D Strains of the Fungal Pathogen Cryptococcus neoformans

PubMed Central

Schein, Jacqueline E.; Tangen, Kristin L.; Chiu, Readman; Shin, Heesun; Lengeler, Klaus B.; MacDonald, William Kim; Bosdet, Ian; Heitman, Joseph; Jones, Steven J.M.; Marra, Marco A.; Kronstad, James W.

2002-01-01

The basidiomycete fungus Cryptococcus neoformans is an important opportunistic pathogen of humans that poses a significant threat to immunocompromised individuals. Isolates of C. neoformans are classified into serotypes (A, B, C, D, and AD) based on antigenic differences in the polysaccharide capsule that surrounds the fungal cells. Genomic and EST sequencing projects are underway for the serotype D strain JEC21 and the serotype A strain H99. As part of a genomics program for C. neoformans, we have constructed fingerprinted bacterial artificial chromosome (BAC) clone physical maps for strains H99 and JEC21 to support the genomic sequencing efforts and to provide an initial comparison of the two genomes. The BAC clones represented an estimated 10-fold redundant coverage of the genomes of each serotype and allowed the assembly of 20 contigs each for H99 and JEC21. We found that the genomes of the two strains are sufficiently distinct to prevent coassembly of the two maps when combined fingerprint data are used to construct contigs. Hybridization experiments placed 82 markers on the JEC21 map and 102 markers on the H99 map, enabling contigs to be linked with specific chromosomes identified by electrophoretic karyotyping. These markers revealed both extensive similarity in gene order (conservation of synteny) between JEC21 and H99 as well as examples of chromosomal rearrangements including inversions and translocations. Sequencing reads were generated from the ends of the BAC clones to allow correlation of genomic shotgun sequence data with physical map contigs. The BAC maps therefore represent a valuable resource for the generation, assembly, and finishing of the genomic sequence of both JEC21 and H99. The physical maps also serve as a link between map-based and sequence-based data, providing a powerful resource for continued genomic studies. [This paper is dedicated to the memory of Michael Smith, Founding Director of the Biotechnology Laboratory and the BC Cancer Agency Genome Sciences Centre. Supplemental material is available online at http://www.genome.org.] PMID:12213782

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.

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.

Genome Sequence of the Freshwater Yangtze Finless Porpoise.

PubMed

Yuan, Yuan; Zhang, Peijun; Wang, Kun; Liu, Mingzhong; Li, Jing; Zheng, Jingsong; Wang, Ding; Xu, Wenjie; Lin, Mingli; Dong, Lijun; Zhu, Chenglong; Qiu, Qiang; Li, Songhai

2018-04-16

The Yangtze finless porpoise ( Neophocaena asiaeorientalis ssp. asiaeorientalis ) is a subspecies of the narrow-ridged finless porpoise ( N. asiaeorientalis ). In total, 714.28 gigabases (Gb) of raw reads were generated by whole-genome sequencing of the Yangtze finless porpoise, using an Illumina HiSeq 2000 platform. After filtering the low-quality and duplicated reads, we assembled a draft genome of 2.22 Gb, with contig N50 and scaffold N50 values of 46.69 kilobases (kb) and 1.71 megabases (Mb), respectively. We identified 887.63 Mb of repetitive sequences and predicted 18,479 protein-coding genes in the assembled genome. The phylogenetic tree showed a relationship between the Yangtze finless porpoise and the Yangtze River dolphin, which diverged approximately 20.84 million years ago. In comparisons with the genomes of 10 other mammals, we detected 44 species-specific gene families, 164 expanded gene families, and 313 positively selected genes in the Yangtze finless porpoise genome. The assembled genome sequence and underlying sequence data are available at the National Center for Biotechnology Information under BioProject accession number PRJNA433603.

Genome Sequence of the Freshwater Yangtze Finless Porpoise

PubMed Central

Yuan, Yuan; Zhang, Peijun; Wang, Kun; Liu, Mingzhong; Li, Jing; Zheng, Jinsong; Wang, Ding; Xu, Wenjie; Lin, Mingli; Dong, Lijun; Zhu, Chenglong; Qiu, Qiang

2018-01-01

The Yangtze finless porpoise (Neophocaena asiaeorientalis ssp. asiaeorientalis) is a subspecies of the narrow-ridged finless porpoise (N. asiaeorientalis). In total, 714.28 gigabases (Gb) of raw reads were generated by whole-genome sequencing of the Yangtze finless porpoise, using an Illumina HiSeq 2000 platform. After filtering the low-quality and duplicated reads, we assembled a draft genome of 2.22 Gb, with contig N50 and scaffold N50 values of 46.69 kilobases (kb) and 1.71 megabases (Mb), respectively. We identified 887.63 Mb of repetitive sequences and predicted 18,479 protein-coding genes in the assembled genome. The phylogenetic tree showed a relationship between the Yangtze finless porpoise and the Yangtze River dolphin, which diverged approximately 20.84 million years ago. In comparisons with the genomes of 10 other mammals, we detected 44 species-specific gene families, 164 expanded gene families, and 313 positively selected genes in the Yangtze finless porpoise genome. The assembled genome sequence and underlying sequence data are available at the National Center for Biotechnology Information under BioProject accession number PRJNA433603. PMID:29659530

Whole Genome Sequencing of the Braconid Parasitoid Wasp Fopius arisanus, an Important Biocontrol Agent of Pest Tepritid Fruit Flies

PubMed Central

Geib, Scott M.; Liang, Guang Hong; Murphy, Terence D.; Sim, Sheina B.

2017-01-01

The braconid wasp Fopius arisanus (Sonan) is an important biological control agent of tropical and subtropical pest fruit flies, including two important global pests, the Mediterranean fruit fly (Ceratitis capitata), and the oriental fruit fly (Bactrocera dorsalis). The goal of this study was to develop foundational genomic resources for this species to provide tools that can be used to answer questions exploring the multitrophic interactions between the host and parasitoid in this important research system. Here, we present a whole genome assembly of F. arisanus, derived from a pool of haploid offspring from a single unmated female. The genome is ∼154 Mb in size, with a N50 contig and scaffold size of 51,867 bp and 0.98 Mb, respectively. Utilizing existing RNA-Seq data for this species, as well as publicly available peptide sequences from related Hymenoptera, a high quality gene annotation set, which includes 10,991 protein coding genes, was generated. Prior to this assembly submission, no RefSeq proteins were present for this species. Parasitic wasps play an important role in a diverse ecosystem as well as a role in biological control of agricultural pests. This whole genome assembly and annotation data represents the first genome-scale assembly for this species or any closely related Opiine, and are publicly available in the National Center for Biotechnology Information Genome and RefSeq databases, providing a much needed genomic resource for this hymenopteran group. PMID:28584080

Draft Genome Sequences of 12 Dry-Heat-Resistant Bacillus Strains Isolated from the Cleanrooms Where the Viking Spacecraft Were Assembled.

PubMed

Seuylemezian, Arman; Cooper, Kerry; Schubert, Wayne; Vaishampayan, Parag

2018-03-22

Spore-forming microorganisms are of concern for forward contamination because they can survive harsh interplanetary travel. Here, we report the draft genome sequences of 12 spore-forming strains isolated from the Manned Spacecraft Operations Building (MSOB) and the Vehicle Assembly Building (VAB) in Cape Canaveral, FL, where the Viking spacecraft were assembled. Copyright © 2018 Seuylemezian et al.

The Genome Sequence of a Widespread Apex Predator, the Golden Eagle (Aquila chrysaetos)

PubMed Central

Doyle, Jacqueline M.; Katzner, Todd E.; Bloom, Peter H.; Ji, Yanzhu; Wijayawardena, Bhagya K.; DeWoody, J. Andrew

2014-01-01

Biologists routinely use molecular markers to identify conservation units, to quantify genetic connectivity, to estimate population sizes, and to identify targets of selection. Many imperiled eagle populations require such efforts and would benefit from enhanced genomic resources. We sequenced, assembled, and annotated the first eagle genome using DNA from a male golden eagle (Aquila chrysaetos) captured in western North America. We constructed genomic libraries that were sequenced using Illumina technology and assembled the high-quality data to a depth of ∼40x coverage. The genome assembly includes 2,552 scaffolds >10 Kb and 415 scaffolds >1.2 Mb. We annotated 16,571 genes that are involved in myriad biological processes, including such disparate traits as beak formation and color vision. We also identified repetitive regions spanning 92 Mb (∼6% of the assembly), including LINES, SINES, LTR-RTs and DNA transposons. The mitochondrial genome encompasses 17,332 bp and is ∼91% identical to the Mountain Hawk-Eagle (Nisaetus nipalensis). Finally, the data reveal that several anonymous microsatellites commonly used for population studies are embedded within protein-coding genes and thus may not have evolved in a neutral fashion. Because the genome sequence includes ∼800,000 novel polymorphisms, markers can now be chosen based on their proximity to functional genes involved in migration, carnivory, and other biological processes. PMID:24759626

Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation.

PubMed

Miller, Jason R; Koren, Sergey; Dilley, Kari A; Puri, Vinita; Brown, David M; Harkins, Derek M; Thibaud-Nissen, Françoise; Rosen, Benjamin; Chen, Xiao-Guang; Tu, Zhijian; Sharakhov, Igor V; Sharakhova, Maria V; Sebra, Robert; Stockwell, Timothy B; Bergman, Nicholas H; Sutton, Granger G; Phillippy, Adam M; Piermarini, Peter M; Shabman, Reed S

2018-03-01

The 50-year-old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome. The C6/36 genome assembly has the largest contig N50 (3.3 Mbp) of any mosquito assembly, presents the sequences of both haplotypes for most of the diploid genome, reveals independent null mutations in both alleles of the Dicer locus, and indicates a male-specific genome. Gene annotation was computed with publicly available mosquito transcript sequences. Gene expression data from cell line RNA sequence identified enrichment of growth-related pathways and conspicuous deficiency in aquaporins and inward rectifier K+ channels. As a test of utility, RNA sequence data from Zika-infected cells were mapped to the C6/36 genome and transcriptome assemblies. Host subtraction reduced the data set by 89%, enabling faster characterization of nonhost reads. The C6/36 genome sequence and annotation should enable additional uses of the cell line to study arbovirus vector interactions and interventions aimed at restricting the spread of human disease.

Why Assembling Plant Genome Sequences Is So Challenging

PubMed Central

Claros, Manuel Gonzalo; Bautista, Rocío; Guerrero-Fernández, Darío; Benzerki, Hicham; Seoane, Pedro; Fernández-Pozo, Noé

2012-01-01

In spite of the biological and economic importance of plants, relatively few plant species have been sequenced. Only the genome sequence of plants with relatively small genomes, most of them angiosperms, in particular eudicots, has been determined. The arrival of next-generation sequencing technologies has allowed the rapid and efficient development of new genomic resources for non-model or orphan plant species. But the sequencing pace of plants is far from that of animals and microorganisms. This review focuses on the typical challenges of plant genomes that can explain why plant genomics is less developed than animal genomics. Explanations about the impact of some confounding factors emerging from the nature of plant genomes are given. As a result of these challenges and confounding factors, the correct assembly and annotation of plant genomes is hindered, genome drafts are produced, and advances in plant genomics are delayed. PMID:24832233

Eimeria genomics: Where are we now and where are we going?

PubMed

Blake, Damer P

2015-08-15

The evolution of sequencing technologies, from Sanger to next generation (NGS) and now the emerging third generation, has prompted a radical frameshift moving genomics from the specialist to the mainstream. For parasitology, genomics has moved fastest for the protozoa with sequence assemblies becoming available for multiple genera including Babesia, Cryptosporidium, Eimeria, Giardia, Leishmania, Neospora, Plasmodium, Theileria, Toxoplasma and Trypanosoma. Progress has commonly been slower for parasites of animals which lack zoonotic potential, but the deficit is now being redressed with impact likely in the areas of drug and vaccine development, molecular diagnostics and population biology. Genomics studies with the apicomplexan Eimeria species clearly illustrate the approaches and opportunities available. Specifically, more than ten years after initiation of a genome sequencing project a sequence assembly was published for Eimeria tenella in 2014, complemented by assemblies for all other Eimeria species which infect the chicken and Eimeria falciformis, a parasite of the mouse. Public access to these and other coccidian genome assemblies through resources such as GeneDB and ToxoDB now promotes comparative analysis, encouraging better use of shared resources and enhancing opportunities for development of novel diagnostic and control strategies. In the short term genomics resources support development of targeted and genome-wide genetic markers such as single nucleotide polymorphisms (SNPs), with whole genome re-sequencing becoming viable in the near future. Experimental power will develop rapidly as additional species, strains and isolates are sampled with particular emphasis on population structure and allelic diversity. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

PubMed

Driscoll, Connor B; Otten, Timothy G; Brown, Nathan M; Dreher, Theo W

2017-01-01

Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality . Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hyphomonadaceae strain UKL13-1 (3,501,508 bp and 56.12% GC), incertae sedis Betaproteobacterium strain UKL13-2 (3,387,087 bp and 54.98% GC), and incertae sedis Bacteroidetes strain UKL13-3 (3,236,529 bp and 37.33% GC). Each genome consists of a single circular chromosome with no identified plasmids. When compared with binned Illumina assemblies of the same three genomes, there was ~7% discrepancy in total genome length. Gaps where Illumina assemblies broke were often due to repetitive elements. Within these missing sequences were essential genes and genes associated with a variety of functional categories. Annotated gene content reveals that both Proteobacteria are aerobic anoxygenic phototrophs, with Betaproteobacterium UKL13-2 potentially capable of phototrophic oxidation of sulfur compounds. Both proteobacterial genomes contain transporters suggesting they are scavenging fixed nitrogen from A. flos-aquae in the form of ammonium. Bacteroidetes UKL13-3 has few completely annotated biosynthetic pathways, and has a comparatively higher proportion of unannotated genes. The genomes were detected in only a few other freshwater metagenomes, suggesting that these bacteria are not ubiquitous in freshwater systems. Our results indicate that long-read sequencing is a viable method for sequencing dominant members from low-diversity microbial communities, and should be considered for environmental metagenomics when conditions meet these requirements.

Assembly and features of secondary metabolite biosynthetic gene clusters in Streptomyces ansochromogenes.

PubMed

Zhong, Xingyu; Tian, Yuqing; Niu, Guoqing; Tan, Huarong

2013-07-01

A draft genome sequence of Streptomyces ansochromogenes 7100 was generated using 454 sequencing technology. In combination with local BLAST searches and gap filling techniques, a comprehensive antiSMASH-based method was adopted to assemble the secondary metabolite biosynthetic gene clusters in the draft genome of S. ansochromogenes. A total of at least 35 putative gene clusters were identified and assembled. Transcriptional analysis showed that 20 of the 35 gene clusters were expressed in either or all of the three different media tested, whereas the other 15 gene clusters were silent in all three different media. This study provides a comprehensive method to identify and assemble secondary metabolite biosynthetic gene clusters in draft genomes of Streptomyces, and will significantly promote functional studies of these secondary metabolite biosynthetic gene clusters.

Modeling biological problems in computer science: a case study in genome assembly.

PubMed

Medvedev, Paul

2018-01-30

As computer scientists working in bioinformatics/computational biology, we often face the challenge of coming up with an algorithm to answer a biological question. This occurs in many areas, such as variant calling, alignment and assembly. In this tutorial, we use the example of the genome assembly problem to demonstrate how to go from a question in the biological realm to a solution in the computer science realm. We show the modeling process step-by-step, including all the intermediate failed attempts. Please note this is not an introduction to how genome assembly algorithms work and, if treated as such, would be incomplete and unnecessarily long-winded. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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...

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

GRIDSS: sensitive and specific genomic rearrangement detection using positional de Bruijn graph assembly

PubMed Central

Do, Hongdo; Molania, Ramyar

2017-01-01

The identification of genomic rearrangements with high sensitivity and specificity using massively parallel sequencing remains a major challenge, particularly in precision medicine and cancer research. Here, we describe a new method for detecting rearrangements, GRIDSS (Genome Rearrangement IDentification Software Suite). GRIDSS is a multithreaded structural variant (SV) caller that performs efficient genome-wide break-end assembly prior to variant calling using a novel positional de Bruijn graph-based assembler. By combining assembly, split read, and read pair evidence using a probabilistic scoring, GRIDSS achieves high sensitivity and specificity on simulated, cell line, and patient tumor data, recently winning SV subchallenge #5 of the ICGC-TCGA DREAM8.5 Somatic Mutation Calling Challenge. On human cell line data, GRIDSS halves the false discovery rate compared to other recent methods while matching or exceeding their sensitivity. GRIDSS identifies nontemplate sequence insertions, microhomologies, and large imperfect homologies, estimates a quality score for each breakpoint, stratifies calls into high or low confidence, and supports multisample analysis. PMID:29097403

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

A hybrid-hierarchical genome assembly strategy to sequence the invasive golden mussel, Limnoperna fortunei

PubMed Central

Uliano-Silva, Marcela; Dondero, Francesco; Dan Otto, Thomas; Costa, Igor; Lima, Nicholas Costa Barroso; Americo, Juliana Alves; Mazzoni, Camila Junqueira; Prosdocimi, Francisco; Rebelo, Mauro de Freitas

2018-01-01

Abstract Background For more than 25 years, the golden mussel, Limnoperna fortunei, has aggressively invaded South American freshwaters, having travelled more than 5000 km upstream across 5 countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it. Findings We assembled the 1.6-Gb genome into 20 548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60 717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. Conclusions We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina paired-end, mate-paired, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei’s genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control. PMID:29267857

A hybrid-hierarchical genome assembly strategy to sequence the invasive golden mussel, Limnoperna fortunei.

PubMed

Uliano-Silva, Marcela; Dondero, Francesco; Dan Otto, Thomas; Costa, Igor; Lima, Nicholas Costa Barroso; Americo, Juliana Alves; Mazzoni, Camila Junqueira; Prosdocimi, Francisco; Rebelo, Mauro de Freitas

2018-02-01

For more than 25 years, the golden mussel, Limnoperna fortunei, has aggressively invaded South American freshwaters, having travelled more than 5000 km upstream across 5 countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it. We assembled the 1.6-Gb genome into 20 548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60 717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina paired-end, mate-paired, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei's genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control.

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

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

PubMed Central

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

2017-01-01

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

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

PubMed

Dai, Xinghong; Li, Zhihai; Lai, Mason; Shu, Sara; Du, Yushen; Zhou, Z Hong; Sun, Ren

2017-01-05

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.

The cacao Criollo genome v2.0: an improved version of the genome for genetic and functional genomic studies.

PubMed

Argout, X; Martin, G; Droc, G; Fouet, O; Labadie, K; Rivals, E; Aury, J M; Lanaud, C

2017-09-15

Theobroma cacao L., native to the Amazonian basin of South America, is an economically important fruit tree crop for tropical countries as a source of chocolate. The first draft genome of the species, from a Criollo cultivar, was published in 2011. Although a useful resource, some improvements are possible, including identifying misassemblies, reducing the number of scaffolds and gaps, and anchoring un-anchored sequences to the 10 chromosomes. We used a NGS-based approach to significantly improve the assembly of the Belizian Criollo B97-61/B2 genome. We combined four Illumina large insert size mate paired libraries with 52x of Pacific Biosciences long reads to correct misassembled regions and reduced the number of scaffolds. We then used genotyping by sequencing (GBS) methods to increase the proportion of the assembly anchored to chromosomes. The scaffold number decreased from 4,792 in assembly V1 to 554 in V2 while the scaffold N50 size has increased from 0.47 Mb in V1 to 6.5 Mb in V2. A total of 96.7% of the assembly was anchored to the 10 chromosomes compared to 66.8% in the previous version. Unknown sites (Ns) were reduced from 10.8% to 5.7%. In addition, we updated the functional annotations and performed a new RefSeq structural annotation based on RNAseq evidence. Theobroma cacao Criollo genome version 2 will be a valuable resource for the investigation of complex traits at the genomic level and for future comparative genomics and genetics studies in cacao tree. New functional tools and annotations are available on the Cocoa Genome Hub ( http://cocoa-genome-hub.southgreen.fr ).

Meraculous2

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

2014-06-01

meraculous2 is a whole genome shotgun assembler for short-reads that is capable of assembling large, polymorphic genomes with modest computational requirements. Meraculous relies on an efficient and conservative traversal of the subgraph of the k-mer (deBruijn) graph of oligonucleotides with unique high quality extensions in the dataset, avoiding an explicit error correction step as used in other short-read assemblers. Additional features include (1) handling of allelic variation using "bubble" structures within the deBruijn graph, (2) gap closing of repetitive and low quality regions using localized assemblies, and (3) an improved scaffolding algorithm that produces more complete assemblies without compromising onmore » scaffolding accuracy« 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

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

The platypus genome unraveled.

PubMed

O'Brien, Stephen J

2008-06-13

The genome of the platypus has been sequenced, assembled, and annotated by an international genomics team. Like the animal itself the platypus genome contains an amalgam of mammal, reptile, and bird-like features.

A genome-wide screening of BEL-Pao like retrotransposons in Anopheles gambiae by the LTR_STRUC program.

PubMed

Marsano, Renè Massimiliano; Caizzi, Ruggiero

2005-09-12

The advanced status of assembly of the nematoceran Anopheles gambiae genomic sequence allowed us to perform a wide genome analysis to looking at the presence of Long Terminal Repeats (LTRs) in the range of 10 kb by means of the LTR_STRUC tool. More than three hundred sequences were retrieved and 210 were treated as putative complete retrotransposons that were individually analysed with respect to known retrotransposons of A. gambiae and D. melanogaster. The results show that the vast majority of the retrotransposons analysed belong to the Ty3/gypsy class and only 8% to the Ty1/copia class. In addition, phylogenetic analysis allowed us to characterize in more detail the relationship of a large BEL-Pao lineage in which a single family was shown to harbour an additional env gene.

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

Complete mitochondrial genome sequences of the northern spotted owl (Strix occidentalis caurina) and the barred owl (Strix varia; Aves: Strigiformes: Strigidae) confirm the presence of a duplicated control region

PubMed Central

Henderson, James B.; Sellas, Anna B.; Fuchs, Jérôme; Bowie, Rauri C.K.; Dumbacher, John P.

2017-01-01

We report here the successful assembly of the complete mitochondrial genomes of the northern spotted owl (Strix occidentalis caurina) and the barred owl (S. varia). We utilized sequence data from two sequencing methodologies, Illumina paired-end sequence data with insert lengths ranging from approximately 250 nucleotides (nt) to 9,600 nt and read lengths from 100–375 nt and Sanger-derived sequences. We employed multiple assemblers and alignment methods to generate the final assemblies. The circular genomes of S. o. caurina and S. varia are comprised of 19,948 nt and 18,975 nt, respectively. Both code for two rRNAs, twenty-two tRNAs, and thirteen polypeptides. They both have duplicated control region sequences with complex repeat structures. We were not able to assemble the control regions solely using Illumina paired-end sequence data. By fully spanning the control regions, Sanger-derived sequences enabled accurate and complete assembly of these mitochondrial genomes. These are the first complete mitochondrial genome sequences of owls (Aves: Strigiformes) possessing duplicated control regions. We searched the nuclear genome of S. o. caurina for copies of mitochondrial genes and found at least nine separate stretches of nuclear copies of gene sequences originating in the mitochondrial genome (Numts). The Numts ranged from 226–19,522 nt in length and included copies of all mitochondrial genes except tRNAPro, ND6, and tRNAGlu. Strix occidentalis caurina and S. varia exhibited an average of 10.74% (8.68% uncorrected p-distance) divergence across the non-tRNA mitochondrial genes. PMID:29038757

The Peach v2.0 release: high-resolution linkage mapping and deep resequencing improve chromosome-scale assembly and contiguity.

PubMed

Verde, Ignazio; Jenkins, Jerry; Dondini, Luca; Micali, Sabrina; Pagliarani, Giulia; Vendramin, Elisa; Paris, Roberta; Aramini, Valeria; Gazza, Laura; Rossini, Laura; Bassi, Daniele; Troggio, Michela; Shu, Shengqiang; Grimwood, Jane; Tartarini, Stefano; Dettori, Maria Teresa; Schmutz, Jeremy

2017-03-11

The availability of the peach genome sequence has fostered relevant research in peach and related Prunus species enabling the identification of genes underlying important horticultural traits as well as the development of advanced tools for genetic and genomic analyses. The first release of the peach genome (Peach v1.0) represented a high-quality WGS (Whole Genome Shotgun) chromosome-scale assembly with high contiguity (contig L50 214.2 kb), large portions of mapped sequences (96%) and high base accuracy (99.96%). The aim of this work was to improve the quality of the first assembly by increasing the portion of mapped and oriented sequences, correcting misassemblies and improving the contiguity and base accuracy using high-throughput linkage mapping and deep resequencing approaches. Four linkage maps with 3,576 molecular markers were used to improve the portion of mapped and oriented sequences (from 96.0% and 85.6% of Peach v1.0 to 99.2% and 98.2% of v2.0, respectively) and enabled a more detailed identification of discernible misassemblies (10.4 Mb in total). The deep resequencing approach fixed 859 homozygous SNPs (Single Nucleotide Polymorphisms) and 1347 homozygous indels. Moreover, the assembled NGS contigs enabled the closing of 212 gaps with an improvement in the contig L50 of 19.2%. The improved high quality peach genome assembly (Peach v2.0) represents a valuable tool for the analysis of the genetic diversity, domestication, and as a vehicle for genetic improvement of peach and related Prunus species. Moreover, the important phylogenetic position of peach and the absence of recent whole genome duplication (WGD) events make peach a pivotal species for comparative genomics studies aiming at elucidating plant speciation and diversification processes.

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.

Finished genome assembly of warm spring isolate Francisella novicida DPG 3A-IS

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

Johnson, Shannon L.; Minogue, Timothy D.; Daligault, Hajnalka E.

2015-09-17

We sequenced the complete genome of Francisella novicida DPG 3A-IS to closed and finished status. This is a warm spring isolate recovered from Hobo Warm Spring (Utah, USA). The last assembly is available in NCBI under accession number CP012037.

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.

Opera: reconstructing optimal genomic scaffolds with high-throughput paired-end sequences.

PubMed

Gao, Song; Sung, Wing-Kin; Nagarajan, Niranjan

2011-11-01

Scaffolding, the problem of ordering and orienting contigs, typically using paired-end reads, is a crucial step in the assembly of high-quality draft genomes. Even as sequencing technologies and mate-pair protocols have improved significantly, scaffolding programs still rely on heuristics, with no guarantees on the quality of the solution. In this work, we explored the feasibility of an exact solution for scaffolding and present a first tractable solution for this problem (Opera). We also describe a graph contraction procedure that allows the solution to scale to large scaffolding problems and demonstrate this by scaffolding several large real and synthetic datasets. In comparisons with existing scaffolders, Opera simultaneously produced longer and more accurate scaffolds demonstrating the utility of an exact approach. Opera also incorporates an exact quadratic programming formulation to precisely compute gap sizes (Availability: http://sourceforge.net/projects/operasf/ ).

Opera: Reconstructing Optimal Genomic Scaffolds with High-Throughput Paired-End Sequences

PubMed Central

Gao, Song; Sung, Wing-Kin

2011-01-01

Abstract Scaffolding, the problem of ordering and orienting contigs, typically using paired-end reads, is a crucial step in the assembly of high-quality draft genomes. Even as sequencing technologies and mate-pair protocols have improved significantly, scaffolding programs still rely on heuristics, with no guarantees on the quality of the solution. In this work, we explored the feasibility of an exact solution for scaffolding and present a first tractable solution for this problem (Opera). We also describe a graph contraction procedure that allows the solution to scale to large scaffolding problems and demonstrate this by scaffolding several large real and synthetic datasets. In comparisons with existing scaffolders, Opera simultaneously produced longer and more accurate scaffolds demonstrating the utility of an exact approach. Opera also incorporates an exact quadratic programming formulation to precisely compute gap sizes (Availability: http://sourceforge.net/projects/operasf/). PMID:21929371

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.

Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites.

PubMed

Boson, Bertrand; Denolly, Solène; Turlure, Fanny; Chamot, Christophe; Dreux, Marlène; Cosset, François-Loïc

2017-03-01

Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Next-generation sequencing of mixed genomic DNA allows efficient assembly of rearranged mitochondrial genomes in Amolops chunganensis and Quasipaa boulengeri

PubMed Central

Yuan, Siqi; Zheng, Yuchi; Zeng, Xiaomao

2016-01-01

Recent improvements in next-generation sequencing (NGS) technologies can facilitate the obtainment of mitochondrial genomes. However, it is not clear whether NGS could be effectively used to reconstruct the mitogenome with high gene rearrangement. These high rearrangements would cause amplification failure, and/or assembly and alignment errors. Here, we choose two frogs with rearranged gene order, Amolops chunganensis and Quasipaa boulengeri, to test whether gene rearrangements affect the mitogenome assembly and alignment by using NGS. The mitogenomes with gene rearrangements are sequenced through Illumina MiSeq genomic sequencing and assembled effectively by Trinity v2.1.0 and SOAPdenovo2. Gene order and contents in the mitogenome of A. chunganensis and Q. boulengeri are typical neobatrachian pattern except for rearrangements at the position of “WANCY” tRNA genes cluster. Further, the mitogenome of Q. boulengeri is characterized with a tandem duplication of trnM. Moreover, we utilize 13 protein-coding genes of A. chunganensis, Q. boulengeri and other neobatrachians to reconstruct the phylogenetic tree for evaluating mitochondrial sequence authenticity of A. chunganensis and Q. boulengeri. In this work, we provide nearly complete mitochondrial genomes of A. chunganensis and Q. boulengeri. PMID:27994980

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.

Integrated analyses using RNA-Seq data reveal viral genomes, single nucleotide variations, the phylogenetic relationship, and recombination for Apple stem grooving virus.

PubMed

Jo, Yeonhwa; Choi, Hoseong; Kim, Sang-Min; Kim, Sun-Lim; Lee, Bong Choon; Cho, Won Kyong

2016-08-09

Next-generation sequencing (NGS) provides many possibilities for plant virology research. In this study, we performed integrated analyses using plant transcriptome data for plant virus identification using Apple stem grooving virus (ASGV) as an exemplar virus. We used 15 publicly available transcriptome libraries from three different studies, two mRNA-Seq studies and a small RNA-Seq study. We de novo assembled nearly complete genomes of ASGV isolates Fuji and Cuiguan from apple and pear transcriptomes, respectively, and identified single nucleotide variations (SNVs) of ASGV within the transcriptomes. We demonstrated the application of NGS raw data to confirm viral infections in the plant transcriptomes. In addition, we compared the usability of two de novo assemblers, Trinity and Velvet, for virus identification and genome assembly. A phylogenetic tree revealed that ASGV and Citrus tatter leaf virus (CTLV) are the same virus, which was divided into two clades. Recombination analyses identified six recombination events from 21 viral genomes. Taken together, our in silico analyses using NGS data provide a successful application of plant transcriptomes to reveal extensive information associated with viral genome assembly, SNVs, phylogenetic relationships, and genetic recombination.

BAC-pool sequencing and analysis confirms growth-associated QTLs in the Asian seabass genome.

PubMed

Shen, Xueyan; Ngoh, Si Yan; Thevasagayam, Natascha May; Prakki, Sai Rama Sridatta; Bhandare, Pranjali; Tan, Andy Wee Kiat; Tan, Gui Quan; Singh, Siddharth; Phua, Norman Chun Han; Vij, Shubha; Orbán, László

2016-11-08

The Asian seabass is an important marine food fish that has been cultured for several decades in Asia Pacific. However, the lack of a high quality reference genome has hampered efforts to improve its selective breeding. A 3D BAC pool set generated in this study was screened using 22 SSR markers located on linkage group 2 which contains a growth-related QTL region. Seventy-two clones corresponding to 22 FPC contigs were sequenced by Illumina MiSeq technology. We co-assembled the MiSeq-derived scaffolds from each FPC contig with error-corrected PacBio reads, resulting in 187 sequences covering 9.7 Mb. Eleven genes annotated within this region were found to be potentially associated with growth and their tissue-specific expression was investigated. Correlation analysis demonstrated that SNPs in ctsb, skp1 and ppp2ca can be potentially used as markers for selecting fast-growing fingerlings. Conserved syntenies between seabass LG2 and five other teleosts were identified. This study i) provided a 10 Mb targeted genome assembly; ii) demonstrated NGS of BAC pools as a potential approach for mining candidates underlying QTLs of this species; iii) detected eleven genes potentially responsible for growth in the QTL region; and iv) identified useful SNP markers for selective breeding programs of Asian seabass.

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.

Genome Sequence of the Necrotrophic Plant Pathogen Alternaria brassicicola Abra43

PubMed Central

Belmas, Elodie; Briand, Martial; Kwasiborski, Anthony; Colou, Justine; N’Guyen, Guillaume; Iacomi, Béatrice; Grappin, Philippe; Campion, Claire; Simoneau, Philippe; Barret, Matthieu

2018-01-01

ABSTRACT Alternaria brassicicola causes dark spot (or black spot) disease, which is one of the most common and destructive fungal diseases of Brassicaceae spp. worldwide. Here, we report the draft genome sequence of strain Abra43. The assembly comprises 29 scaffolds, with an N50 value of 2.1 Mb. The assembled genome was 31,036,461 bp in length, with a G+C content of 50.85%. PMID:29439047

Genome Sequence of an Endophytic Fungus, Fusarium solani JS-169, Which Has Antifungal Activity.

PubMed

Kim, Jung A; Jeon, Jongbum; Park, Sook-Young; Kim, Ki-Tae; Choi, Gobong; Lee, Hyun-Jung; Kim, Yangsun; Yang, Hee-Sun; Yeo, Joo-Hong; Lee, Yong-Hwan; Kim, Soonok

2017-10-19

An endophytic fungus, Fusarium solani strain JS-169, isolated from a mulberry twig, showed considerable antifungal activity. Here, we report the draft genome sequence of this strain. The assembly comprises 17 scaffolds, with an N 50 value of 4.93 Mb. The assembled genome was 45,813,297 bp in length, with a G+C content of 49.91%. Copyright © 2017 Kim et al.

Design and characterization of a 52K SNP chip for goats.

PubMed

Tosser-Klopp, Gwenola; Bardou, Philippe; Bouchez, Olivier; Cabau, Cédric; Crooijmans, Richard; Dong, Yang; Donnadieu-Tonon, Cécile; Eggen, André; Heuven, Henri C M; Jamli, Saadiah; Jiken, Abdullah Johari; Klopp, Christophe; Lawley, Cynthia T; McEwan, John; Martin, Patrice; Moreno, Carole R; Mulsant, Philippe; Nabihoudine, Ibouniyamine; Pailhoux, Eric; Palhière, Isabelle; Rupp, Rachel; Sarry, Julien; Sayre, Brian L; Tircazes, Aurélie; Jun Wang; Wang, Wen; Zhang, Wenguang

2014-01-01

The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50-60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds (meat, milk and mixed): Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the de novo goat genome assembly. The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing (Illumina, Inc) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years.

Design and Characterization of a 52K SNP Chip for Goats

PubMed Central

Tosser-Klopp, Gwenola; Bardou, Philippe; Bouchez, Olivier; Cabau, Cédric; Crooijmans, Richard; Dong, Yang; Donnadieu-Tonon, Cécile; Eggen, André; Heuven, Henri C. M.; Jamli, Saadiah; Jiken, Abdullah Johari; Klopp, Christophe; Lawley, Cynthia T.; McEwan, John; Martin, Patrice; Moreno, Carole R.; Mulsant, Philippe; Nabihoudine, Ibouniyamine; Pailhoux, Eric; Palhière, Isabelle; Rupp, Rachel; Sarry, Julien; Sayre, Brian L.; Tircazes, Aurélie; Jun Wang; Wang, Wen; Zhang, Wenguang

2014-01-01

The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50–60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds (meat, milk and mixed): Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the de novo goat genome assembly. The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing (Illumina, Inc) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years. PMID:24465974

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.

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.

Viral genome structures, charge, and sequences are optimal for capsid assembly

NASA Astrophysics Data System (ADS)

Hagan, Michael

2014-03-01

For many viruses, the spontaneous assembly of a capsid shell around the nu-cleic acid (NA) genome is an essential step in the viral life cycle. Capsid formation is a multicomponent, out-of-equilibrium assembly process for which kinetic effects and thermodynamic constraints compete to determine the outcome. Understand-ing how viral components drive highly efficient assembly under these constraints could promote biomedical efforts to block viral propagation, and would elucidate the factors controlling assembly in a wide range of systems containing proteins and polyelectrolytes. This talk will describe coarse-grained models of capsid proteins and NAs with which we investigate the dynamics and thermodynamics of virus assembly. In con-trast to recent theoretical models, we find that capsids spontaneously `overcharge' that is, the NA length which is kinetically and thermodynamically optimal possess-es a negative charge greater than the positive charge of the capsid. When applied to specific virus capsids, the calculated optimal NA lengths closely correspond to the natural viral genome lengths. These results suggest that the features included in this model (i.e. electrostatics, excluded volume, and NA tertiary structure) play key roles in determining assembly thermodynamics and consequently exert selec-tive pressure on viral evolution. I will then discuss mechanisms by which se-quence-specific interactions between NAs and capsid proteins promote selective encapsidation of the viral genome. This work was supported by NIH R01GM108021 and the Brandeis MRSEC NSF-MRSEC-0820492.

Long-read sequencing and de novo assembly of a Chinese genome

USDA-ARS?s Scientific Manuscript database

Short-read sequencing has enabled the de novo assembly of several individual human genomes, but with inherent limitations in characterizing repeat elements. Here we sequence a Chinese individual HX1 by single-molecule real-time (SMRT) long-read sequencing, construct a physical map by NanoChannel arr...

Assembly-Driven Metagenomics of a Hypersaline Microbial Ecosystem (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

ScienceCinema

Allen, Eric

2018-02-05

Eric Allen of Scripps and UC San Diego on Assembly-driven metagenomics of a hypersaline microbial ecosystem at the 8th Annual Genomics of Energy & Environment Meeting on March 27, 2013 in Walnut Creek, CA.

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...

Assembly-Driven Metagenomics of a Hypersaline Microbial Ecosystem (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

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

Allen, Eric

2013-03-01

Eric Allen of Scripps and UC San Diego on Assembly-driven metagenomics of a hypersaline microbial ecosystem at the 8th Annual Genomics of Energy & Environment Meeting on March 27, 2013 in Walnut Creek, CA.

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...

De novo genome assembly of Cercospora beticola for microsatellite marker development and validation

USDA-ARS?s Scientific Manuscript database

Cercospora leaf spot caused by Cercospora beticola is a significant threat to the production of sugar and table beet worldwide. A de novo genome assembly of C. beticola was used to develop eight polymorphic and reproducible microsatellite markers for population genetic analyses. These markers were u...

High-throughput physical mapping of chromosomes using automated in situ hybridization.

PubMed

George, Phillip; Sharakhova, Maria V; Sharakhov, Igor V

2012-06-28

Projects to obtain whole-genome sequences for 10,000 vertebrate species and for 5,000 insect and related arthropod species are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented. Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations. Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila, allows the user to visualize more details on chromosomes than the regular squashing technique. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

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.

PubMed

Barrero, Roberto A; Guerrero, Felix D; Black, Michael; McCooke, John; Chapman, Brett; Schilkey, Faye; Pérez de León, Adalberto A; Miller, Robert J; Bruns, Sara; Dobry, Jason; Mikhaylenko, Galina; Stormo, Keith; Bell, Callum; Tao, Quanzhou; Bogden, Robert; Moolhuijzen, Paula M; Hunter, Adam; Bellgard, Matthew I

2017-08-01

The genome of the cattle tick Rhipicephalus microplus, an ectoparasite with global distribution, is estimated to be 7.1Gbp in length and consists of approximately 70% repetitive DNA. We report the draft assembly of a tick genome that utilized a hybrid sequencing and assembly approach to capture the repetitive fractions of the genome. Our hybrid approach produced an assembly consisting of 2.0Gbp represented in 195,170 scaffolds with a N50 of 60,284bp. The Rmi v2.0 assembly is 51.46% repetitive with a large fraction of unclassified repeats, short interspersed elements, long interspersed elements and long terminal repeats. We identified 38,827 putative R. microplus gene loci, of which 24,758 were protein coding genes (≥100 amino acids). OrthoMCL comparative analysis against 11 selected species including insects and vertebrates identified 10,835 and 3,423 protein coding gene loci that are unique to R. microplus or common to both R. microplus and Ixodes scapularis ticks, respectively. We identified 191 microRNA loci, of which 168 have similarity to known miRNAs and 23 represent novel miRNA families. We identified the genomic loci of several highly divergent R. microplus esterases with sequence similarity to acetylcholinesterase. Additionally we report the finding of a novel cytochrome P450 CYP41 homolog that shows similar protein folding structures to known CYP41 proteins known to be involved in acaricide resistance. Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Packaging signals in two single-stranded RNA viruses imply a conserved assembly mechanism and geometry of the packaged genome.

PubMed

Dykeman, Eric C; Stockley, Peter G; Twarock, Reidun

2013-09-09

The current paradigm for assembly of single-stranded RNA viruses is based on a mechanism involving non-sequence-specific packaging of genomic RNA driven by electrostatic interactions. Recent experiments, however, provide compelling evidence for sequence specificity in this process both in vitro and in vivo. The existence of multiple RNA packaging signals (PSs) within viral genomes has been proposed, which facilitates assembly by binding coat proteins in such a way that they promote the protein-protein contacts needed to build the capsid. The binding energy from these interactions enables the confinement or compaction of the genomic RNAs. Identifying the nature of such PSs is crucial for a full understanding of assembly, which is an as yet untapped potential drug target for this important class of pathogens. Here, for two related bacterial viruses, we determine the sequences and locations of their PSs using Hamiltonian paths, a concept from graph theory, in combination with bioinformatics and structural studies. Their PSs have a common secondary structure motif but distinct consensus sequences and positions within the respective genomes. Despite these differences, the distributions of PSs in both viruses imply defined conformations for the packaged RNA genomes in contact with the protein shell in the capsid, consistent with a recent asymmetric structure determination of the MS2 virion. The PS distributions identified moreover imply a preferred, evolutionarily conserved assembly pathway with respect to the RNA sequence with potentially profound implications for other single-stranded RNA viruses known to have RNA PSs, including many animal and human pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Analysis and Functional Annotation of an Expressed Sequence Tag Collection for Tropical Crop Sugarcane

PubMed Central

Vettore, André L.; da Silva, Felipe R.; Kemper, Edson L.; Souza, Glaucia M.; da Silva, Aline M.; Ferro, Maria Inês T.; Henrique-Silva, Flavio; Giglioti, Éder A.; Lemos, Manoel V.F.; Coutinho, Luiz L.; Nobrega, Marina P.; Carrer, Helaine; França, Suzelei C.; Bacci, Maurício; Goldman, Maria Helena S.; Gomes, Suely L.; Nunes, Luiz R.; Camargo, Luis E.A.; Siqueira, Walter J.; Van Sluys, Marie-Anne; Thiemann, Otavio H.; Kuramae, Eiko E.; Santelli, Roberto V.; Marino, Celso L.; Targon, Maria L.P.N.; Ferro, Jesus A.; Silveira, Henrique C.S.; Marini, Danyelle C.; Lemos, Eliana G.M.; Monteiro-Vitorello, Claudia B.; Tambor, José H.M.; Carraro, Dirce M.; Roberto, Patrícia G.; Martins, Vanderlei G.; Goldman, Gustavo H.; de Oliveira, Regina C.; Truffi, Daniela; Colombo, Carlos A.; Rossi, Magdalena; de Araujo, Paula G.; Sculaccio, Susana A.; Angella, Aline; Lima, Marleide M.A.; de Rosa, Vicente E.; Siviero, Fábio; Coscrato, Virginia E.; Machado, Marcos A.; Grivet, Laurent; Di Mauro, Sonia M.Z.; Nobrega, Francisco G.; Menck, Carlos F.M.; Braga, Marilia D.V.; Telles, Guilherme P.; Cara, Frank A.A.; Pedrosa, Guilherme; Meidanis, João; Arruda, Paulo

2003-01-01

To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged. PMID:14613979

SWAP-Assembler 2: Optimization of De Novo Genome Assembler at Large Scale

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

Meng, Jintao; Seo, Sangmin; Balaji, Pavan

2016-08-16

In this paper, we analyze and optimize the most time-consuming steps of the SWAP-Assembler, a parallel genome assembler, so that it can scale to a large number of cores for huge genomes with the size of sequencing data ranging from terabyes to petabytes. According to the performance analysis results, the most time-consuming steps are input parallelization, k-mer graph construction, and graph simplification (edge merging). For the input parallelization, the input data is divided into virtual fragments with nearly equal size, and the start position and end position of each fragment are automatically separated at the beginning of the reads. Inmore » k-mer graph construction, in order to improve the communication efficiency, the message size is kept constant between any two processes by proportionally increasing the number of nucleotides to the number of processes in the input parallelization step for each round. The memory usage is also decreased because only a small part of the input data is processed in each round. With graph simplification, the communication protocol reduces the number of communication loops from four to two loops and decreases the idle communication time. The optimized assembler is denoted as SWAP-Assembler 2 (SWAP2). In our experiments using a 1000 Genomes project dataset of 4 terabytes (the largest dataset ever used for assembling) on the supercomputer Mira, the results show that SWAP2 scales to 131,072 cores with an efficiency of 40%. We also compared our work with both the HipMER assembler and the SWAP-Assembler. On the Yanhuang dataset of 300 gigabytes, SWAP2 shows a 3X speedup and 4X better scalability compared with the HipMer assembler and is 45 times faster than the SWAP-Assembler. The SWAP2 software is available at https://sourceforge.net/projects/swapassembler.« less

Insights into Conifer Giga-Genomes1

PubMed Central

De La Torre, Amanda R.; Birol, Inanc; Bousquet, Jean; Ingvarsson, Pär K.; Jansson, Stefan; Jones, Steven J.M.; Keeling, Christopher I.; MacKay, John; Nilsson, Ove; Ritland, Kermit; Street, Nathaniel; Yanchuk, Alvin; Zerbe, Philipp; Bohlmann, Jörg

2014-01-01

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world’s forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20–30 Gb) and the highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: white spruce (Picea glauca), Norway spruce (Picea abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared with other plant genomes and may represent a window into the past of seed plant genomes. This Update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes. PMID:25349325

Insights into conifer giga-genomes.

PubMed

De La Torre, Amanda R; Birol, Inanc; Bousquet, Jean; Ingvarsson, Pär K; Jansson, Stefan; Jones, Steven J M; Keeling, Christopher I; MacKay, John; Nilsson, Ove; Ritland, Kermit; Street, Nathaniel; Yanchuk, Alvin; Zerbe, Philipp; Bohlmann, Jörg

2014-12-01

Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world's forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20-30 Gb) and the highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: white spruce (Picea glauca), Norway spruce (Picea abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared with other plant genomes and may represent a window into the past of seed plant genomes. This Update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes. © 2014 American Society of Plant Biologists. All Rights Reserved.

Draft genome of the leopard gecko, Eublepharis macularius.

PubMed

Xiong, Zijun; Li, Fang; Li, Qiye; Zhou, Long; Gamble, Tony; Zheng, Jiao; Kui, Ling; Li, Cai; Li, Shengbin; Yang, Huanming; Zhang, Guojie

2016-10-26

Geckos are among the most species-rich reptile groups and the sister clade to all other lizards and snakes. Geckos possess a suite of distinctive characteristics, including adhesive digits, nocturnal activity, hard, calcareous eggshells, and a lack of eyelids. However, one gecko clade, the Eublepharidae, appears to be the exception to most of these 'rules' and lacks adhesive toe pads, has eyelids, and lays eggs with soft, leathery eggshells. These differences make eublepharids an important component of any investigation into the underlying genomic innovations contributing to the distinctive phenotypes in 'typical' geckos. We report high-depth genome sequencing, assembly, and annotation for a male leopard gecko, Eublepharis macularius (Eublepharidae). Illumina sequence data were generated from seven insert libraries (ranging from 170 to 20 kb), representing a raw sequencing depth of 136X from 303 Gb of data, reduced to 84X and 187 Gb after filtering. The assembled genome of 2.02 Gb was close to the 2.23 Gb estimated by k-mer analysis. Scaffold and contig N50 sizes of 664 and 20 kb, respectively, were comparable to the previously published Gekko japonicus genome. Repetitive elements accounted for 42 % of the genome. Gene annotation yielded 24,755 protein-coding genes, of which 93 % were functionally annotated. CEGMA and BUSCO assessment showed that our assembly captured 91 % (225 of 248) of the core eukaryotic genes, and 76 % of vertebrate universal single-copy orthologs. Assembly of the leopard gecko genome provides a valuable resource for future comparative genomic studies of geckos and other squamate reptiles.

From NGS assembly challenges to instability of fungal mitochondrial genomes: A case study in genome complexity.

PubMed

Misas, Elizabeth; Muñoz, José Fernando; Gallo, Juan Esteban; McEwen, Juan Guillermo; Clay, Oliver Keatinge

2016-04-01

The presence of repetitive or non-unique DNA persisting over sizable regions of a eukaryotic genome can hinder the genome's successful de novo assembly from short reads: ambiguities in assigning genome locations to the non-unique subsequences can result in premature termination of contigs and thus overfragmented assemblies. Fungal mitochondrial (mtDNA) genomes are compact (typically less than 100 kb), yet often contain short non-unique sequences that can be shown to impede their successful de novo assembly in silico. Such repeats can also confuse processes in the cell in vivo. A well-studied example is ectopic (out-of-register, illegitimate) recombination associated with repeat pairs, which can lead to deletion of functionally important genes that are located between the repeats. Repeats that remain conserved over micro- or macroevolutionary timescales despite such risks may indicate functionally or structurally (e.g., for replication) important regions. This principle could form the basis of a mining strategy for accelerating discovery of function in genome sequences. We present here our screening of a sample of 11 fully sequenced fungal mitochondrial genomes by observing where exact k-mer repeats occurred several times; initial analyses motivated us to focus on 17-mers occurring more than three times. Based on the diverse repeats we observe, we propose that such screening may serve as an efficient expedient for gaining a rapid but representative first insight into the repeat landscapes of sparsely characterized mitochondrial chromosomes. Our matching of the flagged repeats to previously reported regions of interest supports the idea that systems of persisting, non-trivial repeats in genomes can often highlight features meriting further attention. Copyright © 2016 Elsevier Ltd. All rights reserved.

Whole-Genome Comparison Reveals Novel Genetic Elements That Characterize the Genome of Industrial Strains of Saccharomyces cerevisiae

PubMed Central

Borneman, Anthony R.; Desany, Brian A.; Riches, David; Affourtit, Jason P.; Forgan, Angus H.; Pretorius, Isak S.; Egholm, Michael; Chambers, Paul J.

2011-01-01

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species. PMID:21304888

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.

Genome-wide comparative analysis of four Indian Drosophila species.

PubMed

Mohanty, Sujata; Khanna, Radhika

2017-12-01

Comparative analysis of multiple genomes of closely or distantly related Drosophila species undoubtedly creates excitement among evolutionary biologists in exploring the genomic changes with an ecology and evolutionary perspective. We present herewith the de novo assembled whole genome sequences of four Drosophila species, D. bipectinata, D. takahashii, D. biarmipes and D. nasuta of Indian origin using Next Generation Sequencing technology on an Illumina platform along with their detailed assembly statistics. The comparative genomics analysis, e.g. gene predictions and annotations, functional and orthogroup analysis of coding sequences and genome wide SNP distribution were performed. The whole genome of Zaprionus indianus of Indian origin published earlier by us and the genome sequences of previously sequenced 12 Drosophila species available in the NCBI database were included in the analysis. The present work is a part of our ongoing genomics project of Indian Drosophila species.

Single molecule sequencing-guided scaffolding and correction of draft assemblies.

PubMed

Zhu, Shenglong; Chen, Danny Z; Emrich, Scott J

2017-12-06

Although single molecule sequencing is still improving, the lengths of the generated sequences are inevitably an advantage in genome assembly. Prior work that utilizes long reads to conduct genome assembly has mostly focused on correcting sequencing errors and improving contiguity of de novo assemblies. We propose a disassembling-reassembling approach for both correcting structural errors in the draft assembly and scaffolding a target assembly based on error-corrected single molecule sequences. To achieve this goal, we formulate a maximum alternating path cover problem. We prove that this problem is NP-hard, and solve it by a 2-approximation algorithm. Our experimental results show that our approach can improve the structural correctness of target assemblies in the cost of some contiguity, even with smaller amounts of long reads. In addition, our reassembling process can also serve as a competitive scaffolder relative to well-established assembly benchmarks.

Consensus pan-genome assembly of the specialised wine bacterium Oenococcus oeni.

PubMed

Sternes, Peter R; Borneman, Anthony R

2016-04-27

Oenococcus oeni is a lactic acid bacterium that is specialised for growth in the ecological niche of wine, where it is noted for its ability to perform the secondary, malolactic fermentation that is often required for many types of wine. Expanding the understanding of strain-dependent genetic variations in its small and streamlined genome is important for realising its full potential in industrial fermentation processes. Whole genome comparison was performed on 191 strains of O. oeni; from this rich source of genomic information consensus pan-genome assemblies of the invariant (core) and variable (flexible) regions of this organism were established. Genetic variation in amino acid biosynthesis and sugar transport and utilisation was found to be common between strains. Furthermore, we characterised previously-unreported intra-specific genetic variations in the natural competence of this microbe. By assembling a consensus pan-genome from a large number of strains, this study provides a tool for researchers to readily compare protein-coding genes across strains and infer functional relationships between genes in conserved syntenic regions. This establishes a foundation for further genetic, and thus phenotypic, research of this industrially-important species.

Northern Spotted Owl (Strix occidentalis caurina) Genome: Divergence with the Barred Owl (Strix varia) and Characterization of Light-Associated Genes

PubMed Central

Henderson, James B.; Wall, Jeffrey D.; Emerling, Christopher A.; Fuchs, Jérôme; Runckel, Charles; Mindell, David P.; Bowie, Rauri C. K.; DeRisi, Joseph L.; Dumbacher, John P.

2017-01-01

Abstract We report here the assembly of a northern spotted owl (Strix occidentalis caurina) genome. We generated Illumina paired-end sequence data at 90× coverage using nine libraries with insert lengths ranging from ∼250 to 9,600 nt and read lengths from 100 to 375 nt. The genome assembly is comprised of 8,108 scaffolds totaling 1.26 × 109 nt in length with an N50 length of 3.98 × 106 nt. We calculated the genome-wide fixation index (FST) of S. o. caurina with the closely related barred owl (Strix varia) as 0.819. We examined 19 genes that encode proteins with light-dependent functions in our genome assembly as well as in that of the barn owl (Tyto alba). We present genomic evidence for loss of three of these in S. o. caurina and four in T. alba. We suggest that most light-associated gene functions have been maintained in owls and their loss has not proceeded to the same extent as in other dim-light-adapted vertebrates. PMID:28992302

Complete Sequence and Analysis of Coconut Palm (Cocos nucifera) Mitochondrial Genome.

PubMed

Aljohi, Hasan Awad; Liu, Wanfei; Lin, Qiang; Zhao, Yuhui; Zeng, Jingyao; Alamer, Ali; Alanazi, Ibrahim O; Alawad, Abdullah O; Al-Sadi, Abdullah M; Hu, Songnian; Yu, Jun

2016-01-01

Coconut (Cocos nucifera L.), a member of the palm family (Arecaceae), is one of the most economically important crops in tropics, serving as an important source of food, drink, fuel, medicine, and construction material. Here we report an assembly of the coconut (C. nucifera, Oman local Tall cultivar) mitochondrial (mt) genome based on next-generation sequencing data. This genome, 678,653bp in length and 45.5% in GC content, encodes 72 proteins, 9 pseudogenes, 23 tRNAs, and 3 ribosomal RNAs. Within the assembly, we find that the chloroplast (cp) derived regions account for 5.07% of the total assembly length, including 13 proteins, 2 pseudogenes, and 11 tRNAs. The mt genome has a relatively large fraction of repeat content (17.26%), including both forward (tandem) and inverted (palindromic) repeats. Sequence variation analysis shows that the Ti/Tv ratio of the mt genome is lower as compared to that of the nuclear genome and neutral expectation. By combining public RNA-Seq data for coconut, we identify 734 RNA editing sites supported by at least two datasets. In summary, our data provides the second complete mt genome sequence in the family Arecaceae, essential for further investigations on mitochondrial biology of seed plants.

Verdant: automated annotation, alignment and phylogenetic analysis of whole chloroplast genomes.

PubMed

McKain, Michael R; Hartsock, Ryan H; Wohl, Molly M; Kellogg, Elizabeth A

2017-01-01

Chloroplast genomes are now produced in the hundreds for angiosperm phylogenetics projects, but current methods for annotation, alignment and tree estimation still require some manual intervention reducing throughput and increasing analysis time for large chloroplast systematics projects. Verdant is a web-based software suite and database built to take advantage a novel annotation program, annoBTD. Using annoBTD, Verdant provides accurate annotation of chloroplast genomes without manual intervention. Subsequent alignment and tree estimation can incorporate newly annotated and publically available plastomes and can accommodate a large number of taxa. Verdant sharply reduces the time required for analysis of assembled chloroplast genomes and removes the need for pipelines and software on personal hardware. Verdant is available at: http://verdant.iplantcollaborative.org/plastidDB/ It is implemented in PHP, Perl, MySQL, Javascript, HTML and CSS with all major browsers supported. mrmckain@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

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.

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.

Draft genome sequence of Lactobacillus mali KCTC 3596.

PubMed

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

2011-09-01

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

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.

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

PubMed

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

2018-01-01

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

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

PubMed Central

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

2018-01-01

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

Complete nucleotide sequence and annotation of the temperate corynephage ϕ16 genome.

PubMed

Lobanova, Juliya S; Gak, Evgueni R; Andreeva, Irina G; Rybak, Konstantin V; Krylov, Alexander A; Mashko, Sergey V

2017-08-01

The complete genome of ϕ16, a temperate corynephage from Corynebacterium glutamicum ATCC 21792, was sequenced and annotated (GenBank: KY250482). The electron microscopy study of ϕ16 virion confirmed that it belongs to the family Siphoviridae. The ϕ16 genome consists of a linear double-stranded DNA molecule of 58,200 bp (G+C = 52.2%) with protruding cohesive 3'-ends of 14 nt. Four major structural proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting technique. Using bioinformatics analysis, 101 putative ORFs and 5 tRNA genes were predicted. Only 27 putative gene products could be assigned to known biological functions. The ϕ16 genome was divided into functional modules. Seven putative promoters and eight putative unidirectional intrinsic terminators were predicted. One site of putative «-1» programmed ribosomal frameshifting was proposed in the phage tail assembly genome region. C. glutamicum genetic tools could be broadened by exploiting the known integrase gene (gp33) and the newly identified excisionase gene (gp47), participating in site-specific recombination between ϕ16-attP/attB.

Draft genome resources for the phytopathogenic fungi Monilinia fructicola, M. fructigena, M. polystroma and M. laxa, the causal agents of brown rot.

PubMed

Rivera, Yazmin; Zeller, Kurt; Srivastava, Subodh K; Sutherland, Jeremy; Galvez, Marco E; Nakhla, Mark K; Poniatowska, Anna; Schnabel, Guido; Sundin, George W; Abad, Gloria

2018-05-03

Fungi in the genus Monilinia are known to cause devastating brown rot disease of stone and pome fruits. Here, we report the draft genome assemblies of four important phytopathogenic species: Monilinia fructicola, Monilinia fructigena, Monilinia polystroma, and Monilinia laxa. The draft genome assemblies were 39 Mb (M. fructigena), 42 Mb (M. laxa), 43 Mb (M. fructicola), and 45 Mb (M. polystroma) with as few as 550 contigs (M. laxa). These are the first draft genome resources publicly available for M. laxa, M. fructigena, and M. polystroma.

Modulation of Root Microbiome Community Assembly by the Plant Immune Response (2013 DOE JGI Genomics of Energy and Environment 8th Annual User Meeting)

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

Lebeis, Sarah

2013-03-01

Sarah Lebeis of University of North Carolina on Modulation of root microbiome community assembly by the plant immune response at the 8th Annual Genomics of Energy Environment Meeting on March 28, 2013 in Walnut Creek, CA.

Finished Prokaryotic Genome Assemblies from a Low-cost Combination of Short and Long Reads (Seventh Annual Sequencing, Finishing, Analysis in the Future (SFAF) Meeting 2012)

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

Yin, Shuangye

2012-06-01

Shuangye Yin on "Finished prokaryotic genome assemblies from a low-cost combination of short and long reads"; at the 2012 Sequencing, Finishing, Analysis in the Future Meeting held June 5-7, 2012 in Santa Fe, New Mexico.

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...

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.

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

Automatic Tool for Local Assembly Structures

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

Whole community shotgun sequencing of total DNA (i.e. metagenomics) and total RNA (i.e. metatranscriptomics) has provided a wealth of information in the microbial community structure, predicted functions, metabolic networks, and is even able to reconstruct complete genomes directly. Here we present ATLAS (Automatic Tool for Local Assembly Structures) a comprehensive pipeline for assembly, annotation, genomic binning of metagenomic and metatranscriptomic data with an integrated framework for Multi-Omics. This will provide an open source tool for the Multi-Omic community at large.

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

Complete mitochondrial genome sequence of Indian medium carp, Labeo gonius (Hamilton, 1822) and its comparison with other related carp species.

PubMed

Behera, Bijay Kumar; Kumari, Kavita; Baisvar, Vishwamitra Singh; Rout, Ajaya Kumar; Pakrashi, Sudip; Paria, Prasenjet; Jena, J K

2017-01-01

In the present study, the complete mitochondrial genome sequence of Labeo gonius is reported using PGM sequencer (Ion Torrent). The complete mitogenome of L. gonius is obtained by the de novo sequences assembly of genomic reads using the Torrent Mapping Alignment Program (TMAP) which is 16 614 bp in length. The mitogenome of L. gonius comprised of 13 protein-coding genes, 22 tRNAs, 2 rRNA genes, and D-loop as control region along with gene order and organization, being similar to most of other fish mitogenomes of NCBI databases. The mitogenome in the present study has 99% similarity to the complete mitogenome sequence of Labeo fimbriatus, as reported earlier. The phylogenetic analysis of Cypriniformes depicted that their mitogenomes are closely related to each other. The complete mitogenome sequence of L. gonius would be helpful in understanding the population genetics, phylogenetics, and evolution of Indian Carps.

Genome survey and high-density genetic map construction provide genomic and genetic resources for the Pacific White Shrimp Litopenaeus vannamei

PubMed Central

Yu, Yang; Zhang, Xiaojun; Yuan, Jianbo; Li, Fuhua; Chen, Xiaohan; Zhao, Yongzhen; Huang, Long; Zheng, Hongkun; Xiang, Jianhai

2015-01-01

The Pacific white shrimp Litopenaeus vannamei is the dominant crustacean species in global seafood mariculture. Understanding the genome and genetic architecture is useful for deciphering complex traits and accelerating the breeding program in shrimp. In this study, a genome survey was conducted and a high-density linkage map was constructed using a next-generation sequencing approach. The genome survey was used to identify preliminary genome characteristics and to generate a rough reference for linkage map construction. De novo SNP discovery resulted in 25,140 polymorphic markers. A total of 6,359 high-quality markers were selected for linkage map construction based on marker coverage among individuals and read depths. For the linkage map, a total of 6,146 markers spanning 4,271.43 cM were mapped to 44 sex-averaged linkage groups, with an average marker distance of 0.7 cM. An integration analysis linked 5,885 genome scaffolds and 1,504 BAC clones to the linkage map. Based on the high-density linkage map, several QTLs for body weight and body length were detected. This high-density genetic linkage map reveals basic genomic architecture and will be useful for comparative genomics research, genome assembly and genetic improvement of L. vannamei and other penaeid shrimp species. PMID:26503227

An improved assembly of the loblolly pine mega-genome using long-read single-molecule sequencing.

PubMed

Zimin, Aleksey V; Stevens, Kristian A; Crepeau, Marc W; Puiu, Daniela; Wegrzyn, Jill L; Yorke, James A; Langley, Charles H; Neale, David B; Salzberg, Steven L

2017-01-01

The 22-gigabase genome of loblolly pine (Pinus taeda) is one of the largest ever sequenced. The draft assembly published in 2014 was built entirely from short Illumina reads, with lengths ranging from 100 to 250 base pairs (bp). The assembly was quite fragmented, containing over 11 million contigs whose weighted average (N50) size was 8206 bp. To improve this result, we generated approximately 12-fold coverage in long reads using the Single Molecule Real Time sequencing technology developed at Pacific Biosciences. We assembled the long and short reads together using the MaSuRCA mega-reads assembly algorithm, which produced a substantially better assembly, P. taeda version 2.0. The new assembly has an N50 contig size of 25 361, more than three times as large as achieved in the original assembly, and an N50 scaffold size of 107 821, 61% larger than the previous assembly. © The Author 2017. Published by Oxford University Press.

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

Erratum to: An improved assembly of the loblolly pine mega-genome using long-read single-molecule sequencing.

PubMed

Zimin, Aleksey V; Stevens, Kristian A; Crepeau, Marc W; Puiu, Daniela; Wegrzyn, Jill L; Yorke, James A; Langley, Charles H; Neale, David B; Salzberg, Steven L

2017-10-01

The 22-gigabase genome of loblolly pine (Pinus taeda) is one of the largest ever sequenced. The draft assembly published in 2014 was built entirely from short Illumina reads, with lengths ranging from 100 to 250 base pairs (bp). The assembly was quite fragmented, containing over 11 million contigs whose weighted average (N50) size was 8206 bp. To improve this result, we generated approximately 12-fold coverage in long reads using the Single Molecule Real Time sequencing technology developed at Pacific Biosciences. We assembled the long and short reads together using the MaSuRCA mega-reads assembly algorithm, which produced a substantially better assembly, P. taeda version 2.0. The new assembly has an N50 contig size of 25 361, more than three times as large as achieved in the original assembly, and an N50 scaffold size of 107 821, 61% larger than the previous assembly. © The Authors 2017. Published by Oxford University Press.

High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development.

PubMed

Daccord, Nicolas; Celton, Jean-Marc; Linsmith, Gareth; Becker, Claude; Choisne, Nathalie; Schijlen, Elio; van de Geest, Henri; Bianco, Luca; Micheletti, Diego; Velasco, Riccardo; Di Pierro, Erica Adele; Gouzy, Jérôme; Rees, D Jasper G; Guérif, Philippe; Muranty, Hélène; Durel, Charles-Eric; Laurens, François; Lespinasse, Yves; Gaillard, Sylvain; Aubourg, Sébastien; Quesneville, Hadi; Weigel, Detlef; van de Weg, Eric; Troggio, Michela; Bucher, Etienne

2017-07-01

Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development.

A comprehensive and quantitative exploration of thousands of viral genomes

PubMed Central

Mahmoudabadi, Gita

2018-01-01

The complete assembly of viral genomes from metagenomic datasets (short genomic sequences gathered from environmental samples) has proven to be challenging, so there are significant blind spots when we view viral genomes through the lens of metagenomics. One approach to overcoming this problem is to leverage the thousands of complete viral genomes that are publicly available. Here we describe our efforts to assemble a comprehensive resource that provides a quantitative snapshot of viral genomic trends – such as gene density, noncoding percentage, and abundances of functional gene categories – across thousands of viral genomes. We have also developed a coarse-grained method for visualizing viral genome organization for hundreds of genomes at once, and have explored the extent of the overlap between bacterial and bacteriophage gene pools. Existing viral classification systems were developed prior to the sequencing era, so we present our analysis in a way that allows us to assess the utility of the different classification systems for capturing genomic trends. PMID:29624169

A comprehensive and quantitative exploration of thousands of viral genomes.

PubMed

Mahmoudabadi, Gita; Phillips, Rob

2018-04-19

The complete assembly of viral genomes from metagenomic datasets (short genomic sequences gathered from environmental samples) has proven to be challenging, so there are significant blind spots when we view viral genomes through the lens of metagenomics. One approach to overcoming this problem is to leverage the thousands of complete viral genomes that are publicly available. Here we describe our efforts to assemble a comprehensive resource that provides a quantitative snapshot of viral genomic trends - such as gene density, noncoding percentage, and abundances of functional gene categories - across thousands of viral genomes. We have also developed a coarse-grained method for visualizing viral genome organization for hundreds of genomes at once, and have explored the extent of the overlap between bacterial and bacteriophage gene pools. Existing viral classification systems were developed prior to the sequencing era, so we present our analysis in a way that allows us to assess the utility of the different classification systems for capturing genomic trends. © 2018, Mahmoudabadi et al.

Comparison of bacterial genome assembly software for MinION data and their applicability to medical microbiology.

PubMed

Judge, Kim; Hunt, Martin; Reuter, Sandra; Tracey, Alan; Quail, Michael A; Parkhill, Julian; Peacock, Sharon J

2016-09-01

Translating the Oxford Nanopore MinION sequencing technology into medical microbiology requires on-going analysis that keeps pace with technological improvements to the instrument and release of associated analysis software. Here, we use a multidrug-resistant Enterobacter kobei isolate as a model organism to compare open source software for the assembly of genome data, and relate this to the time taken to generate actionable information. Three software tools (PBcR, Canu and miniasm) were used to assemble MinION data and a fourth (SPAdes) was used to combine MinION and Illumina data to produce a hybrid assembly. All four had a similar number of contigs and were more contiguous than the assembly using Illumina data alone, with SPAdes producing a single chromosomal contig. Evaluation of the four assemblies to represent the genome structure revealed a single large inversion in the SPAdes assembly, which also incorrectly integrated a plasmid into the chromosomal contig. Almost 50 %, 80 % and 90 % of MinION pass reads were generated in the first 6, 9 and 12 h, respectively. Using data from the first 6 h alone led to a less accurate, fragmented assembly, but data from the first 9 or 12 h generated similar assemblies to that from 48 h sequencing. Assemblies were generated in 2 h using Canu, indicating that going from isolate to assembled data is possible in less than 48 h. MinION data identified that genes responsible for resistance were carried by two plasmids encoding resistance to carbapenem and to sulphonamides, rifampicin and aminoglycosides, respectively.

Multi-Omics Driven Assembly and Annotation of the Sandalwood (Santalum album) Genome.

PubMed

Mahesh, Hirehally Basavarajegowda; Subba, Pratigya; Advani, Jayshree; Shirke, Meghana Deepak; Loganathan, Ramya Malarini; Chandana, Shankara Lingu; Shilpa, Siddappa; Chatterjee, Oishi; Pinto, Sneha Maria; Prasad, Thottethodi Subrahmanya Keshava; Gowda, Malali

2018-04-01

Indian sandalwood ( Santalum album ) is an important tropical evergreen tree known for its fragrant heartwood-derived essential oil and its valuable carving wood. Here, we applied an integrated genomic, transcriptomic, and proteomic approach to assemble and annotate the Indian sandalwood genome. Our genome sequencing resulted in the establishment of a draft map of the smallest genome for any woody tree species to date (221 Mb). The genome annotation predicted 38,119 protein-coding genes and 27.42% repetitive DNA elements. In-depth proteome analysis revealed the identities of 72,325 unique peptides, which confirmed 10,076 of the predicted genes. The addition of transcriptomic and proteogenomic approaches resulted in the identification of 53 novel proteins and 34 gene-correction events that were missed by genomic approaches. Proteogenomic analysis also helped in reassigning 1,348 potential noncoding RNAs as bona fide protein-coding messenger RNAs. Gene expression patterns at the RNA and protein levels indicated that peptide sequencing was useful in capturing proteins encoded by nuclear and organellar genomes alike. Mass spectrometry-based proteomic evidence provided an unbiased approach toward the identification of proteins encoded by organellar genomes. Such proteins are often missed in transcriptome data sets due to the enrichment of only messenger RNAs that contain poly(A) tails. Overall, the use of integrated omic approaches enhanced the quality of the assembly and annotation of this nonmodel plant genome. The availability of genomic, transcriptomic, and proteomic data will enhance genomics-assisted breeding, germplasm characterization, and conservation of sandalwood trees. © 2018 American Society of Plant Biologists. All Rights Reserved.

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

Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants.

PubMed

Jo, Jihoon; Oh, Jooseong; Lee, Hyun-Gwan; Hong, Hyun-Hee; Lee, Sung-Gwon; Cheon, Seongmin; Kern, Elizabeth M A; Jin, Soyeong; Cho, Sung-Jin; Park, Joong-Ki; Park, Chungoo

2017-01-01

The Japanese sea cucumber (Apostichopus japonicus Selenka 1867) is an economically important species as a source of seafood and ingredient in traditional medicine. It is mainly found off the coasts of northeast Asia. Recently, substantial exploitation and widespread biotic diseases in A. japonicus have generated increasing conservation concern. However, the genomic knowledge base and resources available for researchers to use in managing this natural resource and to establish genetically based breeding systems for sea cucumber aquaculture are still in a nascent stage. A total of 312 Gb of raw sequences were generated using the Illumina HiSeq 2000 platform and assembled to a final size of 0.66 Gb, which is about 80.5% of the estimated genome size (0.82 Gb). We observed nucleotide-level heterozygosity within the assembled genome to be 0.986%. The resulting draft genome assembly comprising 132 607 scaffolds with an N50 value of 10.5 kb contains a total of 21 771 predicted protein-coding genes. We identified 6.6-14.5 million heterozygous single nucleotide polymorphisms in the assembled genome of the three natural color variants (green, red, and black), resulting in an estimated nucleotide diversity of 0.00146. We report the first draft genome of A. japonicus and provide a general overview of the genetic variation in the three major color variants of A. japonicus. These data will help provide a comprehensive view of the genetic, physiological, and evolutionary relationships among color variants in A. japonicus, and will be invaluable resources for sea cucumber genomic research. © The Author 2017. Published by Oxford University Press.