Whole-genome Shotgun Assembly Research Articles

Sex-linked gene traffic underlies the acquisition of sexually dimorphic UV color vision in Heliconius butterflies

The acquisition of novel sexually dimorphic traits poses an evolutionary puzzle: How do new traits arise and become sex-limited? Recently acquired color vision, sexually dimorphic in animals like primates and butterflies, presents a compelling model for understanding how traits become sex-biased. For example, some Heliconius butterflies uniquely possess UV (ultraviolet) color vision, which correlates with the expression of two differentially tuned UV-sensitive rhodopsins, UVRh1 and UVRh2. To discover how such traits become sexually dimorphic, we studied Heliconius charithonia, which exhibits female-specific UVRh1 expression. We demonstrate that females, but not males, discriminate different UV wavelengths. Through whole-genome shotgun sequencing and assembly of the H. charithonia genome, we discovered that UVRh1 is present on the W chromosome, making it obligately female-specific. By knocking out UVRh1, we show that UVRh1 protein expression is absent in mutant female eye tissue, as in wild-type male eyes. A PCR survey of UVRh1 sex-linkage across the genus shows that species with female-specific UVRh1 expression lack UVRh1 gDNA in males. Thus, acquisition of sex linkage is sufficient to achieve female-specific expression of UVRh1, though this does not preclude other mechanisms, like cis-regulatory evolution from also contributing. Moreover, both this event, and mutations leading to differential UV opsin sensitivity, occurred early in the history of Heliconius. These results suggest a path for acquiring sexual dimorphism distinct from existing mechanistic models. We propose a model where gene traffic to heterosomes (the W or the Y) genetically partitions a trait by sex before a phenotype shifts (spectral tuning of UV sensitivity).

CARD 2023: expanded curation, support for machine learning,and resistome prediction at the Comprehensive Antibiotic Resistance Database.

The Comprehensive Antibiotic Resistance Database (CARD; card.mcmaster.ca) combines the Antibiotic Resistance Ontology (ARO) with curated AMR gene (ARG) sequences and resistance-conferring mutations to provide an informatics framework for annotation and interpretation of resistomes. As of version 3.2.4, CARD encompasses 6627 ontology terms, 5010 reference sequences, 1933 mutations, 3004 publications, and 5057 AMR detection models that can be used by the accompanying Resistance Gene Identifier (RGI) software to annotate genomic or metagenomic sequences. Focused curation enhancements since 2020 include expanded β-lactamase curation, incorporation of likelihood-based AMR mutations for Mycobacterium tuberculosis, addition of disinfectants and antiseptics plus their associated ARGs, and systematic curation of resistance-modifying agents. This expanded curation includes 180 new AMR gene families, 15 new drug classes, 1 new resistance mechanism, and two new ontological relationships: evolutionary_variant_of and is_small_molecule_inhibitor. In silico prediction of resistomes and prevalence statistics of ARGs has been expanded to 377 pathogens, 21,079 chromosomes, 2,662 genomic islands, 41,828 plasmidsand 155,606 whole-genome shotgun assemblies, resulting in collation of 322,710 unique ARG allele sequences. New features include the CARD:Live collection of community submitted isolate resistome data and the introduction of standardized 15 character CARD Short Names for ARGs to support machine learning efforts.

Genomic, morphological, and biochemical analyses of a multi-metal resistant but multi-drug susceptible strain of Bordetella petrii from hospital soil

Contamination of soil by antibiotics and heavy metals originating from hospital facilities has emerged as a major cause for the development of resistant microbes. We collected soil samples surrounding a hospital effluent and measured the resistance of bacterial isolates against multiple antibiotics and heavy metals. One strain BMCSI 3 was found to be sensitive to all tested antibiotics. However, it was resistant to many heavy metals and metalloids like cadmium, chromium, copper, mercury, arsenic, and others. This strain was motile and potentially spore-forming. Whole-genome shotgun assembly of BMCSI 3 produced 4.95 Mb genome with 4,638 protein-coding genes. The taxonomic and phylogenetic analysis revealed it, to be a Bordetella petrii strain. Multiple genomic islands carrying mobile genetic elements; coding for heavy metal resistant genes, response regulators or transcription factors, transporters, and multi-drug efflux pumps were identified from the genome. A comparative genomic analysis of BMCSI 3 with annotated genomes of other free-living B. petrii revealed the presence of multiple transposable elements and several genes involved in stress response and metabolism. This study provides insights into how genomic reorganization and plasticity results in evolution of heavy metals resistance by acquiring genes from its natural environment.

A Crowdsourced Gameplay for Whole-Genome Assembly via Short Reads

Next-generation sequencing has revolutionized the field of genomics by producing accurate, rapid and cost-effective genome analysis with the use of high throughput sequencing technologies. This has intensified the need for accurate and performance efficient genome assemblers to assemble a large set of short reads produced by next-generation sequencing technology. Genome assembly is an NP-hard problem that is computationally challenging. Therefore, the current methods that rely on heuristic and approximation algorithms to assemble genomes prevent them from arriving at the most accurate solution. This paper presents a novel approach by gamifying whole-genome shotgun assembly from next-generation sequencing data; we present "Geno", a human-computing game designed with the aim of improving the accuracy of whole-genome shotgun assembly. We evaluate the feasibility of crowdsourcing the problem of whole-genome shotgun assembly by breaking the problem into small subtasks. The evaluation results, for single-cell Escherichia coli K-12 substr. MG1655 with a read length of 25 bp that produced 144,867 game instances of mean 25 sequences per instance at 40x coverage indicate the feasibility of sub-tasking the problem of genome assembly to be solved using crowdsourcing.<br /><br />

Whole-Genome Restriction Mapping by "Subhaploid"-Based RAD Sequencing: An Efficient and Flexible Approach for Physical Mapping and Genome Scaffolding.

Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based “in vitro” linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of “subhaploid” fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning tree algorithm is developed for grouping and ordering of genome-wide markers and is implemented in a user-friendly, integrated software package (AMMO). We perform extensive analyses to validate the power and accuracy of our approach in the model plant Arabidopsis thaliana and human. We also demonstrate the utility of RadMap for enhancing the contiguity of a variety of whole-genome shotgun assemblies generated using either short Illumina reads (300 bp) or long PacBio reads (6–14 kb), with up to 15-fold improvement of N50 (∼816 kb-3.7 Mb) and high scaffolding accuracy (98.1–98.5%). RadMap outperforms BioNano and Hi-C when input assembly is highly fragmented (contig N50 = 54 kb). RadMap can capture wide-range contiguity information and provide an efficient and flexible tool for high-resolution physical mapping and scaffolding of highly fragmented assemblies.

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

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 51128 SNPs, which was then applied to five bi-parental RIL populations to produce a consensus genetic map containing 37372 SNPs. This genetic map enabled the anchoring of 100Mb of WGS and 420Mb 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.

Editorial: Marching Toward 100% Whole Genome Sequencing.

Editorial: Marching Toward 100% Whole Genome Sequencing.

Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes.

SummaryHierarchical shotgun sequencing remains the method of choice for assembling high‐quality reference sequences of complex plant genomes. The efficient exploitation of current high‐throughput technologies and powerful computational facilities for large‐insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole‐genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high‐quality assemblies of a large number of clones to assemble map‐based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.

The Release 6 reference sequence of the Drosophila melanogaster genome

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

Genetic anchoring of whole-genome shotgun assemblies.

The recent advances in sequencing throughput and genome assembly algorithms have established whole-genome shotgun (WGS) assemblies as the cornerstone of the genomic infrastructure for many species. WGS assemblies can be constructed with comparative ease and give a comprehensive representation of the gene space even of large and complex genomes. One major obstacle in utilizing WGS assemblies for important research applications such as gene isolation or comparative genomics has been the lack of chromosomal positioning and contextualization of short sequence contigs. Assigning chromosomal locations to sequence contigs required the construction and integration of genome-wide physical maps and dense genetic linkage maps as well as synteny to model species. Recently, methods to rapidly construct ultra-dense linkage maps encompassing millions of genetic markers from WGS sequencing data of segregating populations have made possible the direct assignment of genetic positions to short sequence contigs. Here, we review recent developments in the integration of WGS assemblies and sequence-based linkage maps, discuss challenges for further improvement of the methodology and outline possible applications building on genetically anchored WGS assemblies.

Sequencing and Assembly of the 22-Gb Loblolly Pine Genome

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.

Unique features of the loblolly pine (Pinus taeda L.) megagenome revealed through sequence annotation.

The largest genus in the conifer family Pinaceae is Pinus, with over 100 species. The size and complexity of their genomes (∼20–40 Gb, 2n = 24) have delayed the arrival of a well-annotated reference sequence. In this study, we present the annotation of the first whole-genome shotgun assembly of loblolly pine (Pinus taeda L.), which comprises 20.1 Gb of sequence. The MAKER-P annotation pipeline combined evidence-based alignments and ab initio predictions to generate 50,172 gene models, of which 15,653 are classified as high confidence. Clustering these gene models with 13 other plant species resulted in 20,646 gene families, of which 1554 are predicted to be unique to conifers. Among the conifer gene families, 159 are composed exclusively of loblolly pine members. The gene models for loblolly pine have the highest median and mean intron lengths of 24 fully sequenced plant genomes. Conifer genomes are full of repetitive DNA, with the most significant contributions from long-terminal-repeat retrotransposons. In depth analysis of the tandem and interspersed repetitive content yielded a combined estimate of 82%.

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

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.

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes.

High-density linkage mapping in a pine tree reveals a genomic region associated with inbreeding depression and provides clues to the extent and distribution of meiotic recombination

BackgroundThe availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies.ResultsIn this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable.ConclusionThis study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.

Whole-Genome Shotgun Assembly and Analysis of the Genome of Streptomyces mobaraensis DSM 40847, a Strain for Industrial Production of Microbial Transglutaminase

Here, we report the draft annotated genome sequence of Streptomyces mobaraensis strain DSM 40847, which is used in industry to produce microbial transglutaminase. The genome sequence will allow for the characterization of the molecular mechanisms underlying the beneficial properties of this organism.

So, you want to sequence a genome...

Anyone who has attempted to identify the responsible gene or mutation underlying a disease or mutant phenotype will know the importance of an accurate reference genome assembly. For complex vertebrate genomes, however, generating such an assembly is not trivial, even with new sequencing technologies. In 2007, a mid-point in the zebrafish genome sequencing project, I was asked to lead the project to completion. At that point we were faced with a highly fragmented physical assembly and lacked genetic maps of sufficient density and resolution to produce an accurate assembly. A high-quality reference genome assembly is generally made up of a large set of minimally overlapping large-insert genomic clones, each of which has been sequenced to completion, with a minimal number of gaps and with no artificially duplicated regions. These high-quality reference genome assemblies, such as the current human reference genome (http://www.genomereference.org), are essential for modern molecular genetic studies. For many species, however, only lower quality whole-genome shotgun assemblies are available. When one considers, for example, only the protein-coding genes, this quality of genome sequence is often not sufficient to determine the complete gene count or comprehensive set of accurate gene models. It is important, for the best application of the genomic information, that the reference genome be complete and accurately assembled. While high-throughput short-read sequencing using the current generation of machines will yield high quality for bacterial, and other small, genomes, it is not possible to completely and accurately assemble the large, complex genomes of vertebrates without other long-range contiguity information. Experience with the zebrafish genome [1] may provide some useful guidance for anyone embarking on a genome-sequencing project for new species with a complex genome. The human, mouse and zebrafish reference genomes were assembled using old-school approaches, where the long-range contiguity was derived from genetic or genomic mapping and not derived directly from sequencing reads or read-pairs. The maps used were accurate physical maps of overlapping genomic DNA fragments or high-resolution genetic maps with a high density of short sequence markers, but such maps are expensive and time-consuming to generate. There are some good possibilities for cheaper, easier way to generate accurate maps, but there are several issues to consider.

A physical, genetic and functional sequence assembly of the barley genome

Barley (Hordeum vulgare L.) is among the world's earliest domesticated and most important crop plants. It is diploid with a large haploid genome of 5.1 gigabases (Gb). Here we present an integrated and ordered physical, genetic and functional sequence resource that describes the barley gene-space in a structured whole-genome context. We developed a physical map of 4.98 Gb, with more than 3.90 Gb anchored to a high-resolution genetic map. Projecting a deep whole-genome shotgun assembly, complementary DNA and deep RNA sequence data onto this framework supports 79,379 transcript clusters, including 26,159 'high-confidence' genes with homology support from other plant genomes. Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation. Our data provide a platform for both genome-assisted research and enabling contemporary crop improvement.

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

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.

HaploMerger: reconstructing allelic relationships for polymorphic diploid genome assemblies.

Whole-genome shotgun assembly has been a long-standing issue for highly polymorphic genomes, and the advent of next-generation sequencing technologies has made the issue more challenging than ever. Here we present an automated pipeline, HaploMerger, for reconstructing allelic relationships in a diploid assembly. HaploMerger combines a LASTZ-ChainNet alignment approach with a novel graph-based structure, which helps to untangle allelic relationships between two haplotypes and guides the subsequent creation of reference haploid assemblies. The pipeline provides flexible parameters and schemes to improve the contiguity, continuity, and completeness of the reference assemblies. We show that HaploMerger produces efficient and accurate results in simulations and has advantages over manual curation when applied to real polymorphic assemblies (e.g., 4%–5% heterozygosity). We also used HaploMerger to analyze the diploid assembly of a single Chinese amphioxus (Branchiostoma belcheri) and compared the resulting haploid assemblies with EST sequences, which revealed that the two haplotypes are not only divergent but also highly complementary to each other. Taken together, we have demonstrated that HaploMerger is an effective tool for analyzing and exploiting polymorphic genome assemblies.