Linked-read Technology Research Articles

Haplotype information of large neuromuscular disease genes provided by linked-read sequencing has a potential to increase diagnostic yield.

Rare or novel missense variants in large genes such as TTN and NEB are frequent in the general population, which hampers the interpretation of putative disease-causing biallelic variants in patients with sporadic neuromuscular disorders. Often, when the first initial genetic analysis is performed, the reconstructed haplotype, i.e. phasing information of the variants is missing. Segregation analysis increases the diagnostic turnaround time and is not always possible if samples from family members are lacking. To overcome this difficulty, we investigated how well the linked-read technology succeeded to phase variants in these large genes, and whether it improved the identification of structural variants. Linked-read sequencing data of nemaline myopathy, distal myopathy, and proximal myopathy patients were analyzed for phasing, single nucleotide variants, and structural variants. Variant phasing was successful in the large muscle genes studied. The longest continuous phase blocks were gained using high-quality DNA samples with long DNA fragments. Homozygosity increased the number of phase blocks, especially in exome sequencing samples lacking intronic variation. In our cohort, linked-read sequencing added more information about the structural variation but did not lead to a molecular genetic diagnosis. The linked-read technology can support the clinical diagnosis of neuromuscular and other genetic disorders.

LRTK: a platform agnostic toolkit for linked-read analysis of both human genome and metagenome.

Linked-read sequencing technologies generate high-base quality short reads that contain extrapolative information on long-range DNA connectedness. These advantages of linked-read technologies are well known and have been demonstrated in many human genomic and metagenomic studies. However, existing linked-read analysis pipelines (e.g., Long Ranger) were primarily developed to process sequencing data from the human genome and are not suited for analyzing metagenomic sequencing data. Moreover, linked-read analysis pipelines are typically limited to 1 specific sequencing platform. To address these limitations, we present the Linked-Read ToolKit (LRTK), a unified and versatile toolkit for platform agnostic processing of linked-read sequencing data from both human genome and metagenome. LRTK provides functions to perform linked-read simulation, barcode sequencing error correction, barcode-aware read alignment and metagenome assembly, reconstruction of long DNA fragments, taxonomic classification and quantification, and barcode-assisted genomic variant calling and phasing. LRTK has the ability to process multiple samples automatically and provides users with the option to generate reproducible reports during processing of raw sequencing data and at multiple checkpoints throughout downstream analysis. We applied LRTK on linked reads from simulation, mock community, and real datasets for both human genome and metagenome. We showcased LRTK's ability to generate comparative performance results from preceding benchmark studies and to report these results in publication-ready HTML document plots. LRTK provides comprehensive and flexible modules along with an easy-to-use Python-based workflow for processing linked-read sequencing datasets, thereby filling the current gap in the field caused by platform-centric genome-specific linked-read data analysis tools.

BLR: a flexible pipeline for haplotype analysis of multiple linked-read technologies.

Linked-read sequencing promises a one-method approach for genome-wide insights including single nucleotide variants (SNVs), structural variants, and haplotyping. We introduce Barcode Linked Reads (BLR), an open-source haplotyping pipeline capable of handling millions of barcodes and data from multiple linked-read technologies including DBS, 10× Genomics, TELL-seq and stLFR. Running BLR on DBS linked-reads yielded megabase-scale phasing with low (<0.2%) switch error rates. Of 13616 protein-coding genes phased in the GIAB benchmark set (v4.2.1), 98.6% matched the BLR phasing. In addition, large structural variants showed concordance with HPRC-HG002 reference assembly calls. Compared to diploid assembly with PacBio HiFi reads, BLR phasing was more continuous when considering switch errors. We further show that integrating long reads at low coverage (∼10×) can improve phasing contiguity and reduce switch errors in tandem repeats. When compared to Long Ranger on 10× Genomics data, BLR showed an increase in phase block N50 with low switch-error rates. For TELL-Seq and stLFR linked reads, BLR generated longer or similar phase block lengths and low switch error rates compared to results presented in the original publications. In conclusion, BLR provides a flexible workflow for comprehensive haplotype analysis of linked reads from multiple platforms.

MTG-Link: leveraging barcode information from linked-reads to assemble specific loci

BackgroundLocal assembly with short and long reads has proven to be very useful in many applications: reconstruction of the sequence of a locus of interest, gap-filling in draft assemblies, as well as alternative allele reconstruction of large Structural Variants. Whereas linked-read technologies have a great potential to assemble specific loci as they provide long-range information while maintaining the power and accuracy of short-read sequencing, there is a lack of local assembly tools for linked-read data.ResultsWe present MTG-Link, a novel local assembly tool dedicated to linked-reads. The originality of the method lies in its read subsampling step which takes advantage of the barcode information contained in linked-reads mapped in flanking regions. We validated our approach on several datasets from different linked-read technologies. We show that MTG-Link is able to assemble successfully large sequences, up to dozens of Kb. We also demonstrate that the read subsampling step of MTG-Link considerably improves the local assembly of specific loci compared to other existing short-read local assembly tools. Furthermore, MTG-Link was able to fully characterize large insertion variants and deletion breakpoints in a human genome and to reconstruct dark regions in clinically-relevant human genes. It also improved the contiguity of a 1.3 Mb locus of biological interest in several individual genomes of the mimetic butterfly Heliconius numata.ConclusionsMTG-Link is an efficient local assembly tool designed for different linked-read sequencing technologies. MTG-Link source code is available at https://github.com/anne-gcd/MTG-Link and as a Bioconda package.

Draft genome assemblies of the avian louse Brueelia nebulosa and its associates using long-read sequencing from an individual specimen.

Sequencing high molecular weight (HMW) DNA with long-read and linked-read technologies has promoted a major increase in more complete genome sequences for nonmodel organisms. Sequencing approaches that rely on HMW DNA have been limited to larger organisms or pools of multiple individuals, but recent advances have allowed for sequencing from individuals of small-bodied organisms. Here, we use HMW DNA sequencing with PacBio long reads and TELL-Seq linked reads to assemble and annotate the genome from a single individual feather louse (Brueelia nebulosa) from a European Starling (Sturnus vulgaris). We assembled a genome with a relatively high scaffold N50 (637 kb) and with BUSCO scores (96.1%) comparable to louse genomes assembled from pooled individuals. We annotated a number of genes (10,938) similar to the human louse (Pediculus humanus) genome. Additionally, calling phased variants revealed that the Brueelia genome is more heterozygous (∼1%) then expected for a highly obligate and dispersal-limited parasite. We also assembled and annotated the mitochondrial genome and primary endosymbiont (Sodalis) genome from the individual louse, which showed evidence for heteroplasmy in the mitogenome and a reduced genome size in the endosymbiont compared to its free-living relative. Our study is a valuable demonstration of the capability to obtain high-quality genomes from individual small, nonmodel organisms. Applying this approach to other organisms could greatly increase our understanding of the diversity and evolution of individual genomes.

Local assembly of long reads enables phylogenomics of transposable elements in a polyploid cell line.

Animal cell lines often undergo extreme genome restructuring events, including polyploidy and segmental aneuploidy that can impede de novo whole-genome assembly (WGA). In some species like Drosophila, cell lines also exhibit massive proliferation of transposable elements (TEs). To better understand the role of transposition during animal cell culture, we sequenced the genome of the tetraploid Drosophila S2R+ cell line using long-read and linked-read technologies. WGAs for S2R+ were highly fragmented and generated variable estimates of TE content across sequencing and assembly technologies. We therefore developed a novel WGA-independent bioinformatics method called TELR that identifies, locally assembles, and estimates allele frequency of TEs from long-read sequence data (https://github.com/bergmanlab/telr). Application of TELR to a ∼130x PacBio dataset for S2R+ revealed many haplotype-specific TE insertions that arose by transposition after initial cell line establishment and subsequent tetraploidization. Local assemblies from TELR also allowed phylogenetic analysis of paralogous TEs, which revealed that proliferation of TE families in vitro can be driven by single or multiple source lineages. Our work provides a model for the analysis of TEs in complex heterozygous or polyploid genomes that are recalcitrant to WGA and yields new insights into the mechanisms of genome evolution in animal cell culture.

Genome assembly of the Pendlebury's roundleaf bat, Hipposideros pendleburyi, revealed the expansion of Tc1/Mariner DNA transposons in Rhinolophoidea.

Bats (Chiroptera) constitute the second largest order of mammals and have several distinctive features, such as true self-powered flight and strong immunity. The Pendlebury’s roundleaf bat, Hipposideros pendleburyi, is endemic to Thailand and listed as a vulnerable species. We employed the 10× Genomics linked-read technology to obtain a genome assembly of H. pendleburyi. The assembly size was 2.17 Gb with a scaffold N50 length of 15,398,518 bases. Our phylogenetic analysis placed H. pendleburyi within the rhinolophoid clade of the suborder Yinpterochiroptera. A synteny analysis showed that H. pendleburyi shared conserved chromosome segments (up to 105 Mb) with Rhinolophus ferrumequinum and Phyllostomus discolor albeit having different chromosome numbers and belonging different families. We found positive selection signals in genes involved in inflammation, spermatogenesis and Wnt signalling. The analyses of transposable elements suggested the contraction of short interspersed nuclear elements (SINEs) and the accumulation of young mariner DNA transposons in the analysed hipposiderids. Distinct mariners were likely horizontally transferred to hipposiderid genomes over the evolution of this family. The lineage-specific profiles of SINEs and mariners might involve in the evolution of hipposiderids and be associated with the phylogenetic separations of these bats from other bat families.

Genome assembly of the numbat (Myrmecobius fasciatus), the only termitivorous marsupial.

The numbat (Myrmecobius fasciatus) is an endangered Australian marsupial, and the last surviving member of the Myrmecobiidae family. The numbat regularly undergoes torpor and is unique amongst marsupials as it is the only diurnal and termitivorous species. Here we sequenced the first draft genome of the numbat using 10× Genomics Chromium linked-read technology, resulting in a 3.42 Gbp genome with a scaffold N50 of 223 kbp. A global transcriptome from liver, lung and tongue was also generated to aid genome annotation, identifying 21,465 protein-coding genes. To investigate adaptation to the numbat's termitivorous diet and arid/semi-arid range, we interrogated the most highly expressed transcripts within the tongue and manually annotated taste, vomeronasal and aquaporin gene families. Antimicrobial proteins and proteins involved in digestion were highly expressed in the tongue, alongside umami taste receptors. However, sweet taste receptors were not expressed in this tissue, which combined with the putative contraction of the bitter taste receptor gene repertoire in the numbat genome, may indicate a potential evolutionary adaptation to their specialised termitivorous diet. Vomeronasal and aquaporin gene repertoires were similar to other marsupials. The draft numbat genome is a valuable tool for conservation and can be applied to population genetics/genomics studies and to investigate the unique biology of this interesting species.

De Novo Reference Assembly of the Upriver Orange Mangrove (Bruguiera sexangula) Genome.

Upriver orange mangrove (Bruguiera sexangula) is a member of the most mangrove-rich taxon (Rhizophoraceae family) and is commonly distributed in the intertidal zones in tropical and subtropical latitudes. In this study, we employed the 10× Genomics linked-read technology to obtain a preliminary de novo assembly of the B. sexangula genome, which was further scaffolded to a pseudomolecule level using the Bruguiera parviflora genome as a reference. The final assembly of the B. sexangula genome contained 260 Mb with an N50 scaffold length of 11,020,310 bases. The assembly comprised 18 pseudomolecules (corresponding to the haploid chromosome number in B. sexangula), covering 204,645,832 bases or 78.6% of the 260-Mb assembly. We predicted a total of 23,978 protein-coding sequences, 17,598 of which were associated with gene ontology terms. Our gene prediction recovered 96.6% of the highly conserved orthologs based on the Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. The chromosome-level assembly presented in this work provides a valuable genetic resource to help strengthen our understanding of mangroves’ physiological and morphological adaptations to the intertidal zones.

A de novo reference assembly of the yellow mangrove Ceriops zippeliana genome.

Mangroves are of great ecological and economical importance, providing shelters for a wide range of species and nursery habitats for commercially important marine species. Ceriops zippeliana (yellow mangrove) belongs to Rhizophoraceae family and is commonly distributed in the tropical and subtropical coastal communities. In this study, we present a high-quality assembly of the C. zippeliana genome. We constructed an initial draft assembly of 240,139,412 bases with an N50 contig length of 564,761 bases using the 10x Genomics linked-read technology. This assembly was further scaffolded with RagTag using a chromosome-scale assembly of a closely related Ceriops species as a reference. The final assembly contained 243,228,612 bases with an N50 scaffold length of 10,559,178 Mb. The size of the final assembly was close to those estimated using DNA flow cytometry (248 Mb) and the k-mer distribution analysis (246 Mb). We predicted a total of 23,474 gene models and 21,724 protein-coding genes in the C. zippeliana genome, of which 16,002 were assigned gene ontology terms. We recovered 97.1% of the highly conserved orthologs based on the Benchmarking Universal Single-Copy Orthologs analysis. The phylogenetic analysis based on single-copy orthologous genes illustrated that C. zippeliana and Ceriops tagal diverged approximately 10.2 million years ago (MYA), and their last common ancestor and Kandelia obovata diverged approximately 29.9 MYA. The high-quality assembly of C. zippeliana presented in this work provides a useful genomic resource for studying mangroves’ unique adaptations to stressful intertidal habitats and for developing sustainable mangrove forest restoration and conservation programs.

A chromosome-scale reference genome assembly of yellow mangrove (Bruguiera parviflora) reveals a whole genome duplication event associated with the Rhizophoraceae lineage.

Mangrove forest ecosystems support a diverse flora and fauna of marine and terrestrial species and have important direct and indirect economic, ecological and social values to mankind. Yellow mangrove (Bruguiera parviflora) belongs to the Rhizophoraceae family and is widely distributed in the intertidal zones along sheltered coastal areas in tropical latitudes. Here, we present a high-quality, chromosome-level assembly of the B.parviflora genome. We employed the 10x Genomics linked-read technology to obtain a preliminary assembly, which was subsequently scaffolded using the long-range chromatin contact mapping technique (HiC) to obtain a final assembly containing 213,026,782 bases in 10,045 scaffolds with an N50 length of 10,906,948 bases. Our gene prediction recovered 96.5% of the highly conserved orthologues in the Embryophyta lineage based on the Benchmarking Universal Single-Copy Orthologues (BUSCO) analysis. We analysed the transversion rate at fourfold-degenerate sites from orthologous gene pairs and discovered evidence supporting a recent whole-genome duplication event in B.parviflora and other Rhizophoreae members. Comparative studies based on single-copy orthologous genes indicated that B.parviflora and Bruguiera gymnorrhiza diverged approximately 24.1 million years ago. The population structure analysis revealed that 63 B.parviflora accessions from different geographical regions in Thailand were an admixture of two subpopulations. The examination of alternative splicing events in B.parviflora showed that the most prevalent splicing mechanism was intron retention. This high-quality genome assembly together with the genetic diversity information obtained from the germplasm provide useful genomic resources for future studies on comparative phylogenetics and evolution of adaptive traits in mangrove species.

Local selection signals in the genome of blue tits emphasize regulatory and neuronal evolution.

Understanding the genomic landscape of adaptation is central to understanding microevolution in wild populations. Genomic targets of selection and the underlying genomic mechanisms of adaptation can be elucidated by genome-wide scans for past selective sweeps or by scans for direct fitness associations. We sequenced and assembled 150haplotypes of 75 blue tits (Cyanistes caeruleus) of a single Central European population by a linked-read technology. We used these genome data in combination with coalescent simulations (i) to estimate an historical effective population size of ~250,000, which recently declined to ~10,000, and (ii) to identify genome-wide distributed selective sweeps of beneficial variants probably originating from standing genetic variation (soft sweeps). The genes linked to these soft sweeps, but also those linked to hard sweeps based on new beneficial mutants, showed a significant enrichment for functions associated with gene expression and transcription regulation. This emphasizes the importance of regulatory evolution in the population's adaptive history. Soft sweeps were further enriched for genes related to axon and synapse development, indicating the significance of neuronal connectivity changes in the brain potentially linked to behavioural adaptations. A previous scan of heterozygosity-fitness correlations revealed a consistent negative effect on arrival date at the breeding site for a single microsatellite in the MDGA2gene. Here, we used the haplotype structure around this microsatellite to explain the effect as a local and direct outbreeding effect of a gene involved in synapse development.

Chromosome-scale haplotype-phased genome assemblies of the male and female lines of wild asparagus (Asparagus kiusianus), a dioecious plant species.

Asparagus kiusianus is a disease-resistant dioecious plant species and a wild relative of garden asparagus (Asparagus officinalis). To enhance A. kiusianus genomic resources, advance plant science, and facilitate asparagus breeding, we determined the genome sequences of the male and female lines of A. kiusianus. Genome sequence reads obtained with a linked-read technology were assembled into four haplotype-phased contig sequences (∼1.6 Gb each) for the male and female lines. The contig sequences were aligned onto the chromosome sequences of garden asparagus to construct pseudomolecule sequences. Approximately 55,000 potential protein-encoding genes were predicted in each genome assembly, and ∼70% of the genome sequence was annotated as repetitive. Comparative analysis of the genomes of the two species revealed structural and sequence variants between the two species as well as between the male and female lines of each species. Genes with high sequence similarity with the male-specific sex determinant gene in A. officinalis, MSE1/AoMYB35/AspTDF1, were presented in the genomes of the male line but absent from the female genome assemblies. Overall, the genome sequence assemblies, gene sequences, and structural and sequence variants determined in this study will reveal the genetic mechanisms underlying sexual differentiation in plants, and will accelerate disease-resistance breeding in garden asparagus.

QuickDeconvolution: fast and scalable deconvolution of linked-read sequencing data.

Recently introduced, linked-read technologies, such as the 10× chromium system, use microfluidics to tag multiple short reads from the same long fragment (50-200 kb) with a small sequence, called a barcode. They are inexpensive and easy to prepare, combining the accuracy of short-read sequencing with the long-range information of barcodes. The same barcode can be used for several different fragments, which complicates the analyses. We present QuickDeconvolution (QD), a new software for deconvolving a set of reads sharing a barcode, i.e. separating the reads from the different fragments. QD only takes sequencing data as input, without the need for a reference genome. We show that QD outperforms existing software in terms of accuracy, speed and scalability, making it capable of deconvolving previously inaccessible data sets. In particular, we demonstrate here the first example in the literature of a successfully deconvoluted animal sequencing dataset, a 33-Gb Drosophila melanogaster dataset. We show that the taxonomic assignment of linked reads can be improved by deconvoluting reads with QD before taxonomic classification. Code and instructions are available on https://github.com/RolandFaure/QuickDeconvolution. Supplementary data are available at Bioinformatics Advances online.

Toward a methodology for evaluating DNA variants in nuclear families.

The genetic underpinnings of most pediatric-cancer cases are unknown. Population-based studies use large sample sizes but have accounted for only a small proportion of the estimated heritability of pediatric cancers. Pedigree-based studies are infeasible for most human populations. One alternative is to collect genetic data from a single nuclear family and use inheritance patterns within the family to filter candidate variants. This approach can be applied to common and rare variants, including those that are private to a given family or to an affected individual. We evaluated this approach using genetic data from three nuclear families with 5, 4, and 7 children, respectively. Only one child in each nuclear family had been diagnosed with cancer, and neither parent had been affected. Diagnoses for the affected children were benign low-grade astrocytoma, Wilms tumor (stage 2), and Burkitt's lymphoma, respectively. We used whole-genome sequencing to profile normal cells from each family member and a linked-read technology for genomic phasing. For initial variant filtering, we used global minor allele frequencies, deleteriousness scores, and functional-impact annotations. Next, we used genetic variation in the unaffected siblings as a guide to filter the remaining variants. As a way to evaluate our ability to detect variant(s) that may be relevant to disease status, the corresponding author blinded the primary author to affected status; the primary author then assigned a risk score to each child. Based on this evidence, the primary author predicted which child had been affected in each family. The primary author's prediction was correct for the child who had been diagnosed with a Wilms tumor; the child with Burkitt's lymphoma had the second-highest risk score among the seven children in that family. This study demonstrates a methodology for filtering and evaluating candidate genomic variants and genes within nuclear families that may merit further exploration.

A spinach genome assembly with remarkable completeness, and its use for rapid identification of candidate genes for agronomic traits.

Spinach (Spinacia oleracea) is grown as a nutritious leafy vegetable worldwide. To accelerate spinach breeding efficiency, a high-quality reference genome sequence with great completeness and continuity is needed as a basic infrastructure. Here, we used long-read and linked-read technologies to construct a de novo spinach genome assembly, designated SOL_r1.1, which was comprised of 287 scaffolds (total size: 935.7 Mb; N50 = 11.3 Mb) with a low proportion of undetermined nucleotides (Ns = 0.34%) and with high gene completeness (BUSCO complete 96.9%). A genome-wide survey of resistance gene analogues identified 695 genes encoding nucleotide-binding site domains, receptor-like protein kinases, receptor-like proteins and transmembrane-coiled coil domains. Based on a high-density double-digest restriction-site associated DNA sequencing-based linkage map, the genome assembly was anchored to six pseudomolecules representing ∼73.5% of the whole genome assembly. In addition, we used SOL_r1.1 to identify quantitative trait loci for bolting timing and fruit/seed shape, which harbour biologically plausible candidate genes, such as homologues of the FLOWERING LOCUS T and EPIDERMAL PATTERNING FACTOR-LIKE genes. The new genome assembly, SOL_r1.1, will serve as a useful resource for identifying loci associated with important agronomic traits and for developing molecular markers for spinach breeding/selection programs.

De novo chromosome-level assembly of the Centella asiatica genome

Centella asiatica is a herbaceous, perennial species indigenous to India and Southeast Asia. C. asiatica possesses several medicinal properties: anti-aging, anti-inflammatory, wound healing and memory enhancing. The lack of available genomics resources significantly impedes the improvement of C. asiatica varieties through molecular breeding. Here, we combined the 10× Genomics linked-read technology and the long-range HiC technique to obtain the genome assembly. The final assembly contained nine pseudomolecules, corresponding to the haploid chromosome number in C. asiatica. These nine chromosomes covered 402,536,584 bases or 93.6% of the 430-Mb assembly. Comparative genomics analyses based on single-copy orthologous genes showed that C. asiatica and the common ancestor of Coriandrum sativum (coriander) and Daucus carota (carrot) diverged about 48 million years ago. This assembly provides a valuable reference genome for future molecular studies, varietal development through marker-assisted breeding and comparative genomics studies in C. asiatica.

The yellow mealworm (Tenebrio molitor) genome: a resource for the emerging insects as food and feed industry

The yellow mealworm, the larval stage of the darkling beetle Tenebrio molitor, shows great promise as an alternative source of animal protein. Herein we present the 312 Mb draft genome assembled using 10x Genomics linked-read technology to inform research efforts and to provide resources to optimise yellow mealworm for mass production and consumption. The genome with a contig N50 of 39,478 bp contains 89% of conserved arthropod genes among the &gt; 20,000 genes assembled (complete and partial genes). This draft assembly represents a valuable resource to understanding T. molitor biology as a means of producing alternative, sustainable protein for the growing population and in the face of changing climates.

OMICs AND AI APPROACHES FOR MUSCLE DISEASES: O.06 Haplotype and variant phasing of large muscle genes with linked-read sequencing

Molecular genetic analysis of large genes is demanding although high-throughput sequencing (HTS) has facilitated the task immensely. Ultra-rare or novel missense variants in large genes such as nebulin and titin are very common in the general population, which makes variant interpretation difficult in sporadic cases. The interpretation of rare heterozygous variants is especially difficult when samples from family members are lacking and thus, segregation analysis is not possible. Short-read HTS collapses a diploid genome to a single sequence, but the linked-read technology provides phase information of continuous DNA fragments and resolves the haplotypes of patients. Long-range information is achieved by linking the barcoded short-read sequences to each other in the data-analysis step, allowing multiple variants in the same gene to be phased. Link-read technology enables the identification of complex structural variation and repeat expansions using the HTS data. Linked-read whole exome (WES) and whole genome sequencing (WGS) have the potential to improve the sequencing quality of genes harboring segmental duplications, such as nebulin and titin. We have linked-read WES and WGS data of 30 individuals. For this study, we analyzed 10 genes, with the longest coding regions in the human genome associated with skeletal muscle disorders. Our aim was to investigate how well the linked-read technology succeeded to detect phase variants in these large genes, and whether this facilitated variant classification by adding crucial information. We also discuss the technical limitations of the method, such as quality standards of DNA samples, which need to be considered to obtain the best possible results. Our preliminary results indicate that the linked-read technology is likely to improve the diagnostics of neuromuscular disorders.

A chromosome-scale assembly of the black gram (Vigna mungo) genome.

Black gram (Vigna mungo) is an important short duration grain legume crop. Black gram seeds provide an inexpensive source of dietary protein. Here, we applied the 10X Genomics linked-read technology to obtain a de novo whole genome assembly of V.mungo cultivated variety Chai Nat 80 (CN80). The preliminary assembly contained 12,228 contigs and had an N50 length of 5.2Mb. Subsequent scaffolding using the long-range Chicago and HiC techniques yielded the first high-quality, chromosome-level assembly of 499Mb comprising 11 pseudomolecules. Comparative genomics analyses based on sequence information from single-copy orthologous genes revealed that black gram and mungbean (Vignaradiata) diverged about 2.7 million years ago . The transversion rate (4DTv) analysis in V.mungo revealed no evidence supporting a recent genome-wide duplication event observed in the tetraploid créole bean (Vigna reflexo-pilosa). The proportion of repetitive elements in the black gram genome is slightly lower than the numbers reported for related Vigna species. The majority of long terminal repeat retrotransposons appeared to integrate into the genome within the last five million years. We also examined alternative splicing events in V.mungo using full-length transcript sequences. While intron retention was the most prevalent mode of alternative splicing in several plant species, alternative 3' acceptor site selection represented the majority of events in black gram. Our high-quality genome assembly along with the genomic variation information from the germplasm provides valuable resources for accelerating the development of elite varieties through marker-assisted breeding and for future comparative genomics and phylogenetic studies in legume species.