Chromosome-length genome assembly and karyotype of the endangered black-footed ferret (Mustela nigripes).

The black-footed ferret (Mustela nigripes) narrowly avoided extinction to become an oft-cited example of the benefits of intensive management, research, and collaboration to save a species through ex situ conservation breeding and reintroduction into its former range. However, the species remains at risk due to possible inbreeding, disease susceptibility, and multiple fertility challenges. Here, we report the de novo genome assembly of a male black-footed ferret generated through a combination of linked-read sequencing, optical mapping, and Hi-C proximity ligation. In addition, we report the karyotype for this species, which was used to anchor and assign chromosome numbers to the chromosome-length scaffolds. The draft assembly was ~2.5 Gb in length, with 95.6% of it anchored to 19 chromosome-length scaffolds, corresponding to the 2n = 38 chromosomes revealed by the karyotype. The assembly has contig and scaffold N50 values of 148.8 kbp and 145.4 Mbp, respectively, and is up to 96% complete based on BUSCO analyses. Annotation of the assembly, including evidence from RNA-seq data, identified 21,406 protein-coding genes and a repeat content of 37.35%. Phylogenomic analyses indicated that the black-footed ferret diverged from the European polecat/domestic ferret lineage 1.6 million yr ago. This assembly will enable research on the conservation genomics of black-footed ferrets and thereby aid in the further restoration of this endangered species.

Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

A tandem duplication of BRCA1 exons 1-19 through DHX8 exon 2 in four families with hereditary breast and ovarian cancer syndrome.

PURPOSE: The purpose of this study is to characterize a novel structural variant, a large duplication involving exons 1-19 of the BRCA1 gene in four independent families, and to provide diagnostically valuable information including the position of the breakpoints as well as clues to its clinical significance. METHODS: The duplication of exons 1-19 of the BRCA1 gene was initially detected by routine laboratory testing including MLPA analysis and next generation sequencing. For detailed characterization we performed array-comparative genome hybridization analysis, fluorescent in situ hybridization, next generation mapping, and long-distance PCR for break-point sequencing. RESULTS: Our data revealed a tandem duplication on chromosome 17 that encompassed 357 kb and included exons 1-19 of the BRCA1 gene and the genes NBR2, NBR1, TMEM106A, LOC100130581, ARL4D, MIR2117 up to parts of the DHX8 gene. This structural variant appeared as a tandem duplication with breakpoints in intron 19 of the BRCA1 gene and in intron 3 of the DHX8 gene (HGVS:chr17(hg19):g.41210776_41568516dup). Segregation analysis indicated that this structural rearrangement is phased in trans with a known pathogenic exon deletion of the BRCA1 gene in one family. CONCLUSIONS: The copy number variation initially recognized as duplication of exon 1-19 of the BRCA1 gene by MLPA analysis is a structural variation with breakpoints in the BRCA1 and DHX8 genes. Although currently to be classified as a variant of unknown significance, our family data indicates that this duplication may be a benign variation or at least of markedly reduced penetrance since it occurs in trans with another known fully pathogenic variant in the BRCA1 gene.

Structural Variation Detection and Analysis Using Bionano Optical Mapping.

The need to accurately identify the complete structural variation profile of genomes is becoming increasingly evident. In contrast to reference-based methods like sequencing or comparative methods like aCGH, optical mapping is a de novo assembly-based method that enables better realization of true genomic structure. It allows for independently detecting balanced and unbalanced structural variants (SVs) from separate alleles and for discovering de novo events. Here we show how Bionano Genome Mapping creates de novo assemblies from native and intact, megabase-scale DNA molecules and uses those assemblies to detect a wide range of structural variants.

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

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

A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

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

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.

Genome assembly and geospatial phylogenomics of the bed bug Cimex lectularius.

The common bed bug (Cimex lectularius) has been a persistent pest of humans for thousands of years, yet the genetic basis of the bed bug's basic biology and adaptation to dense human environments is largely unknown. Here we report the assembly, annotation and phylogenetic mapping of the 697.9-Mb Cimex lectularius genome, with an N50 of 971 kb, using both long and short read technologies. A RNA-seq time course across all five developmental stages and male and female adults generated 36,985 coding and noncoding gene models. The most pronounced change in gene expression during the life cycle occurs after feeding on human blood and included genes from the Wolbachia endosymbiont, which shows a simultaneous and coordinated host/commensal response to haematophagous activity. These data provide a rich genetic resource for mapping activity and density of C. lectularius across human hosts and cities, which can help track, manage and control bed bug infestations.

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

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.

Genome-Wide Structural Variation Detection by Genome Mapping on Nanochannel Arrays.

Comprehensive whole-genome structural variation detection is challenging with current approaches. With diploid cells as DNA source and the presence of numerous repetitive elements, short-read DNA sequencing cannot be used to detect structural variation efficiently. In this report, we show that genome mapping with long, fluorescently labeled DNA molecules imaged on nanochannel arrays can be used for whole-genome structural variation detection without sequencing. While whole-genome haplotyping is not achieved, local phasing (across >150-kb regions) is routine, as molecules from the parental chromosomes are examined separately. In one experiment, we generated genome maps from a trio from the 1000 Genomes Project, compared the maps against that derived from the reference human genome, and identified structural variations that are >5 kb in size. We find that these individuals have many more structural variants than those published, including some with the potential of disrupting gene function or regulation.

Rapid detection of structural variation in a human genome using nanochannel-based genome mapping technology.

BACKGROUND: Structural variants (SVs) are less common than single nucleotide polymorphisms and indels in the population, but collectively account for a significant fraction of genetic polymorphism and diseases. Base pair differences arising from SVs are on a much higher order (>100 fold) than point mutations; however, none of the current detection methods are comprehensive, and currently available methodologies are incapable of providing sufficient resolution and unambiguous information across complex regions in the human genome. To address these challenges, we applied a high-throughput, cost-effective genome mapping technology to comprehensively discover genome-wide SVs and characterize complex regions of the YH genome using long single molecules (>150 kb) in a global fashion. RESULTS: Utilizing nanochannel-based genome mapping technology, we obtained 708 insertions/deletions and 17 inversions larger than 1 kb. Excluding the 59 SVs (54 insertions/deletions, 5 inversions) that overlap with N-base gaps in the reference assembly hg19, 666 non-gap SVs remained, and 396 of them (60%) were verified by paired-end data from whole-genome sequencing-based re-sequencing or de novo assembly sequence from fosmid data. Of the remaining 270 SVs, 260 are insertions and 213 overlap known SVs in the Database of Genomic Variants. Overall, 609 out of 666 (90%) variants were supported by experimental orthogonal methods or historical evidence in public databases. At the same time, genome mapping also provides valuable information for complex regions with haplotypes in a straightforward fashion. In addition, with long single-molecule labeling patterns, exogenous viral sequences were mapped on a whole-genome scale, and sample heterogeneity was analyzed at a new level. CONCLUSION: Our study highlights genome mapping technology as a comprehensive and cost-effective method for detecting structural variation and studying complex regions in the human genome, as well as deciphering viral integration into the host genome.