A second unveiling: Haplotig masking of the eastern oyster genome improves population-level inference.

Genome assembly can be challenging for species that are characterized by high amounts of polymorphism, heterozygosity, and large effective population sizes. High levels of heterozygosity can result in genome mis-assemblies and a larger than expected genome size due to the haplotig versions of a single locus being assembled as separate loci. Here, we describe the first chromosome-level genome for the eastern oyster, Crassostrea virginica. Publicly released and annotated in 2017, the assembly has a scaffold N50 of 54 mb and is over 97.3% complete based on BUSCO analysis. The genome assembly for the eastern oyster is a critical resource for foundational research into molluscan adaptation to a changing environment and for selective breeding for the aquaculture industry. Subsequent resequencing data suggested the presence of haplotigs in the original assembly, and we developed a post hoc method to break up chimeric contigs and mask haplotigs in published heterozygous genomes and evaluated improvements to the accuracy of downstream analysis. Masking haplotigs had a large impact on SNP discovery and estimates of nucleotide diversity and had more subtle and nuanced effects on estimates of heterozygosity, population structure analysis, and outlier detection. We show that haplotig masking can be a powerful tool for improving genomic inference, and we present an open, reproducible resource for the masking of haplotigs in any published genome.

Whole Genome Analysis of SNV and Indel Polymorphism in Common Marmosets (Callithrix jacchus).

The common marmoset (Callithrix jacchus) is one of the most widely used nonhuman primate models of human disease. Owing to limitations in sequencing technology, early genome assemblies of this species using short-read sequencing suffered from gaps. In addition, the genetic diversity of the species has not yet been adequately explored. Using long-read genome sequencing and expert annotation, we generated a high-quality genome resource creating a 2.898 Gb marmoset genome in which most of the euchromatin portion is assembled contiguously (contig N50 = 25.23 Mbp, scaffold N50 = 98.2 Mbp). We then performed whole genome sequencing on 84 marmosets sampling the genetic diversity from several marmoset research centers. We identified a total of 19.1 million single nucleotide variants (SNVs), of which 11.9 million can be reliably mapped to orthologous locations in the human genome. We also observed 2.8 million small insertion/deletion variants. This dataset includes an average of 5.4 million SNVs per marmoset individual and a total of 74,088 missense variants in protein-coding genes. Of the 4956 variants orthologous to human ClinVar SNVs (present in the same annotated gene and with the same functional consequence in marmoset and human), 27 have a clinical significance of pathogenic and/or likely pathogenic. This important marmoset genomic resource will help guide genetic analyses of natural variation, the discovery of spontaneous functional variation relevant to human disease models, and the development of genetically engineered marmoset disease models.

A draft human pangenome reference.

Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals1. These assemblies cover more than 99% of the expected sequence in each genome and are more than 99% accurate at the structural and base pair levels. Based on alignments of the assemblies, we generate a draft pangenome that captures known variants and haplotypes and reveals new alleles at structurally complex loci. We also add 119 million base pairs of euchromatic polymorphic sequences and 1,115 gene duplications relative to the existing reference GRCh38. Roughly 90 million of the additional base pairs are derived from structural variation. Using our draft pangenome to analyse short-read data reduced small variant discovery errors by 34% and increased the number of structural variants detected per haplotype by 104% compared with GRCh38-based workflows, which enabled the typing of the vast majority of structural variant alleles per sample.

High resolution genomes of multiple Xiphophorus species provide new insights into microevolution, hybrid incompatibility, and epistasis.

Because of diverged adaptative phenotypes, fish species of the genus Xiphophorus have contributed to a wide range of research for a century. Existing Xiphophorus genome assemblies are not at the chromosomal level and are prone to sequence gaps, thus hindering advancement of the intra- and inter-species differences for evolutionary, comparative, and translational biomedical studies. Herein, we assembled high-quality chromosome-level genome assemblies for three distantly related Xiphophorus species, namely, X. maculatus, X. couchianus, and X. hellerii Our overall goal is to precisely assess microevolutionary processes in the clade to ascertain molecular events that led to the divergence of the Xiphophorus species and to progress understanding of genetic incompatibility to disease. In particular, we measured intra- and inter-species divergence and assessed gene expression dysregulation in reciprocal interspecies hybrids among the three species. We found expanded gene families and positively selected genes associated with live bearing, a special mode of reproduction. We also found positively selected gene families are significantly enriched in nonpolymorphic transposable elements, suggesting the dispersal of these nonpolymorphic transposable elements has accompanied the evolution of the genes, possibly by incorporating new regulatory elements in support of the Britten-Davidson hypothesis. We characterized inter-specific polymorphisms, structural variants, and polymorphic transposable element insertions and assessed their association to interspecies hybridization-induced gene expression dysregulation related to specific disease states in humans.

Genome Assemblies across the Diverse Evolutionary Spectrum of Leishmania Protozoan Parasites.

We report the high-quality draft assemblies and gene annotations for 13 species and/or strains of the protozoan parasite genera Leishmania, Endotrypanum, and Crithidia, which span the phylogenetic diversity of the subfamily Leishmaniinae within the kinetoplastid order of the phylum Euglenazoa. These resources will support studies on the origins of parasitism.

A chromosome-level genome of Astyanax mexicanus surface fish for comparing population-specific genetic differences contributing to trait evolution.

Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss such as dusp26. We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3, in eye formation. We also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species.

Draft Genome Sequences of Two Polycyclic Tetramate Macrolactam Producers, Streptomyces sp. Strains JV180 and SP18CM02.

Here, we report the draft genome sequences of two related Streptomyces sp. strains, JV180 and SP18CM02. Despite their isolation from soils in Connecticut and Missouri (USA), respectively, they are strikingly similar in gene content. Both belong to the Streptomyces griseus clade and harbor several secondary metabolite biosynthetic gene clusters.

Sequence diversity analyses of an improved rhesus macaque genome enhance its biomedical utility.

The rhesus macaque (Macaca mulatta) is the most widely studied nonhuman primate (NHP) in biomedical research. We present an updated reference genome assembly (Mmul_10, contig N50 = 46 Mbp) that increases the sequence contiguity 120-fold and annotate it using 6.5 million full-length transcripts, thus improving our understanding of gene content, isoform diversity, and repeat organization. With the improved assembly of segmental duplications, we discovered new lineage-specific genes and expanded gene families that are potentially informative in studies of evolution and disease susceptibility. Whole-genome sequencing (WGS) data from 853 rhesus macaques identified 85.7 million single-nucleotide variants (SNVs) and 10.5 million indel variants, including potentially damaging variants in genes associated with human autism and developmental delay, providing a framework for developing noninvasive NHP models of human disease.

A new domestic cat genome assembly based on long sequence reads empowers feline genomic medicine and identifies a novel gene for dwarfism.

The domestic cat (Felis catus) numbers over 94 million in the USA alone, occupies households as a companion animal, and, like humans, suffers from cancer and common and rare diseases. However, genome-wide sequence variant information is limited for this species. To empower trait analyses, a new cat genome reference assembly was developed from PacBio long sequence reads that significantly improve sequence representation and assembly contiguity. The whole genome sequences of 54 domestic cats were aligned to the reference to identify single nucleotide variants (SNVs) and structural variants (SVs). Across all cats, 16 SNVs predicted to have deleterious impacts and in a singleton state were identified as high priority candidates for causative mutations. One candidate was a stop gain in the tumor suppressor FBXW7. The SNV is found in cats segregating for feline mediastinal lymphoma and is a candidate for inherited cancer susceptibility. SV analysis revealed a complex deletion coupled with a nearby potential duplication event that was shared privately across three unrelated cats with dwarfism and is found within a known dwarfism associated region on cat chromosome B1. This SV interrupted UDP-glucose 6-dehydrogenase (UGDH), a gene involved in the biosynthesis of glycosaminoglycans. Importantly, UGDH has not yet been associated with human dwarfism and should be screened in undiagnosed patients. The new high-quality cat genome reference and the compilation of sequence variation demonstrate the importance of these resources when searching for disease causative alleles in the domestic cat and for identification of feline biomedical models.

The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization.

Sturgeons seem to be frozen in time. The archaic characteristics of this ancient fish lineage place it in a key phylogenetic position at the base of the ~30,000 modern teleost fish species. Moreover, sturgeons are notoriously polyploid, providing unique opportunities to investigate the evolution of polyploid genomes. We assembled a high-quality chromosome-level reference genome for the sterlet, Acipenser ruthenus. Our analysis revealed a very low protein evolution rate that is at least as slow as in other deep branches of the vertebrate tree, such as that of the coelacanth. We uncovered a whole-genome duplication that occurred in the Jurassic, early in the evolution of the entire sturgeon lineage. Following this polyploidization, the rediploidization of the genome included the loss of whole chromosomes in a segmental deduplication process. While known adaptive processes helped conserve a high degree of structural and functional tetraploidy over more than 180 million years, the reduction of redundancy of the polyploid genome seems to have been remarkably random.

The quail genome: insights into social behaviour, seasonal biology and infectious disease response.

BACKGROUND: The Japanese quail (Coturnix japonica) is a popular domestic poultry species and an increasingly significant model species in avian developmental, behavioural and disease research. RESULTS: We have produced a high-quality quail genome sequence, spanning 0.93 Gb assigned to 33 chromosomes. In terms of contiguity, assembly statistics, gene content and chromosomal organisation, the quail genome shows high similarity to the chicken genome. We demonstrate the utility of this genome through three diverse applications. First, we identify selection signatures and candidate genes associated with social behaviour in the quail genome, an important agricultural and domestication trait. Second, we investigate the effects and interaction of photoperiod and temperature on the transcriptome of the quail medial basal hypothalamus, revealing key mechanisms of photoperiodism. Finally, we investigate the response of quail to H5N1 influenza infection. In quail lung, many critical immune genes and pathways were downregulated after H5N1 infection, and this may be key to the susceptibility of quail to H5N1. CONCLUSIONS: We have produced a high-quality genome of the quail which will facilitate further studies into diverse research questions using the quail as a model avian species.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

The Piranha Genome Provides Molecular Insight Associated to Its Unique Feeding Behavior.

The piranha enjoys notoriety due to its infamous predatory behavior but much is still not understood about its evolutionary origins and the underlying molecular mechanisms for its unusual feeding biology. We sequenced and assembled the red-bellied piranha (Pygocentrus nattereri) genome to aid future phenotypic and genetic investigations. The assembled draft genome is similar to other related fishes in repeat composition and gene count. Our evaluation of genes under positive selection suggests candidates for adaptations of piranhas' feeding behavior in neural functions, behavior, and regulation of energy metabolism. In the fasted brain, we find genes differentially expressed that are involved in lipid metabolism and appetite regulation as well as genes that may control the aggression/boldness behavior of hungry piranhas. Our first analysis of the piranha genome offers new insight and resources for the study of piranha biology and for feeding motivation and starvation in other organisms.

Dramatic changes in gene expression in different forms of Crithidia fasciculata reveal potential mechanisms for insect-specific adhesion in kinetoplastid parasites.

Kinetoplastids are a group of parasites that includes several medically-important species. These human-infective species are transmitted by insect vectors in which the parasites undergo specific developmental transformations. For each species, this includes a stage in which parasites adhere to insect tissue via a hemidesmosome-like structure. Although this structure has been described morphologically, it has never been molecularly characterized. We are using Crithidia fasciculata, an insect parasite that produces large numbers of adherent parasites inside its mosquito host, as a model kinetoplastid to investigate both the mechanism of adherence and the signals required for differentiation to an adherent form. An advantage of C. fasciculata is that adherent parasites can be generated both in vitro, allowing a direct comparison to cultured swimming forms, as well as in vivo within the mosquito. Using RNAseq, we identify genes associated with adherence in C. fasciculata. As almost all of these genes have orthologs in other kinetoplastid species, our findings may reveal shared mechanisms of adherence, allowing investigation of a crucial step in parasite development and disease transmission. In addition, dual-RNAseq allowed us to explore the interaction between the parasites and the mosquito. Although the infection is well-tolerated, anti-microbial peptides and other components of the mosquito innate immune system are upregulated. Our findings indicate that C. fasciculata is a powerful model system for probing kinetoplastid-insect interactions.

Clonal polymorphism and high heterozygosity in the celibate genome of the Amazon molly.

The extreme rarity of asexual vertebrates in nature is generally explained by genomic decay due to absence of meiotic recombination, thus leading to extinction of such lineages. We explore features of a vertebrate asexual genome, the Amazon molly, Poecilia formosa, and find few signs of genetic degeneration but unique genetic variability and ongoing evolution. We uncovered a substantial clonal polymorphism and, as a conserved feature from its interspecific hybrid origin, a 10-fold higher heterozygosity than in the sexual parental species. These characteristics seem to be a principal reason for the unpredicted fitness of this asexual vertebrate. Our data suggest that asexual vertebrate lineages are scarce not because they are at a disadvantage, but because the genomic combinations required to bypass meiosis and to make up a functioning hybrid genome are rarely met in nature.

The Genome and Adult Somatic Transcriptome of the Mormyrid Electric Fish Paramormyrops kingsleyae.

Several studies have begun to elucidate the genetic and developmental processes underlying major vertebrate traits. Few of these traits have evolved repeatedly in vertebrates, preventing the analysis of molecular mechanisms underlying these traits comparatively. Electric organs have evolved multiple times among vertebrates, presenting a unique opportunity to understand the degree of constraint and repeatability of the evolutionary processes underlying novel vertebrate traits. As there is now a completed genome sequence representing South American electric eels, we were motivated to obtain genomic sequence from a linage that independently evolved electric organs to facilitate future comparative analyses of the evolution and development of electric organs. We report here the sequencing and de novo assembly of the genome of the mormyrid Paramormyrops kingsleyae using short-read sequencing. In addition, we have completed a somatic transcriptome from 11 tissues to construct a gene expression atlas of predicted genes from this assembly, enabling us to identify candidate housekeeping genes as well as genes differentially expressed in the major somatic tissues of the mormyrid electric fish. We anticipate that this resource will greatly facilitate comparative studies on the evolution and development of electric organs and electroreceptors.

A 3-way hybrid approach to generate a new high-quality chimpanzee reference genome (Pan_tro_3.0).

The chimpanzee is arguably the most important species for the study of human origins. A key resource for these studies is a high-quality reference genome assembly; however, as with most mammalian genomes, the current iteration of the chimpanzee reference genome assembly is highly fragmented. In the current iteration of the chimpanzee reference genome assembly (Pan_tro_2.1.4), the sequence is scattered across more then 183 000 contigs, incorporating more than 159 000 gaps, with a genome-wide contig N50 of 51 Kbp. In this work, we produce an extensive and diverse array of sequencing datasets to rapidly assemble a new chimpanzee reference that surpasses previous iterations in bases represented and organized in large scaffolds. To this end, we show substantial improvements over the current release of the chimpanzee genome (Pan_tro_2.1.4) by several metrics, such as increased contiguity by >750% and 300% on contigs and scaffolds, respectively, and closure of 77% of gaps in the Pan_tro_2.1.4 assembly gaps spanning >850 Kbp of the novel coding sequence based on RNASeq data. We further report more than 2700 genes that had putatively erroneous frame-shift predictions to human in Pan_tro_2.1.4 and show a substantial increase in the annotation of repetitive elements. We apply a simple 3-way hybrid approach to considerably improve the reference genome assembly for the chimpanzee, providing a valuable resource for the study of human origins. Furthermore, we produce extensive sequencing datasets that are all derived from the same cell line, generating a broad non-human benchmark dataset.

The Novel Evolution of the Sperm Whale Genome.

The sperm whale, made famous by Moby Dick, is one of the most fascinating of all ocean-dwelling species given their unique life history, novel physiological adaptations to hunting squid at extreme ocean depths, and their position as one of the earliest branching toothed whales (Odontoceti). We assembled the sperm whale (Physeter macrocephalus) genome and resequenced individuals from multiple ocean basins to identify new candidate genes for adaptation to an aquatic environment and infer demographic history. Genes crucial for skin integrity appeared to be particularly important in both the sperm whale and other cetaceans. We also find sperm whales experienced a steep population decline during the early Pleistocene epoch. These genomic data add new comparative insight into the evolution of whales.

The practical use of genome sequencing data in the management of a feline colony pedigree.

BACKGROUND: A higher prevalence of inherited disorders among companion animals are often rooted in their historical restricted artificial selection for a variety of observed phenotypes that eventually decreased genetic diversity. Cats have been afflicted with many inherited diseases due to domestication and intense breed selection. Advances in sequencing technology have generated a more comprehensive way to access genetic information from an individual, allowing identification of putative disease-causing variants and in practice a means to avoid their spread and thus better pedigree management. We examine variants in three domestic shorthair cats and then calculated overall genetic diversity to extrapolate the benefits of this data for breeding programs within a feline colony. RESULTS: We generated whole genome sequence (WGS) data for three related cats that belong to a large feline pedigree colony. Genome-wide coverage ranged from 27-32X, from which we identified 18 million variants in total. Previously known disease-causing variants were screened in our cats, but none carry any of these known disease alleles. Loss of function (LoF) variants, that are in genes associated with a detrimental phenotype in human or mice were chosen for further evaluation on the comparative impact inferred. A set of LoF variants were observed in four genes, each with predicted detrimental phenotypes as a result. However, none of our cats displayed the expected disease phenotypes. Inbreeding coefficients and runs of homozygosity were also evaluated as a measure of genetic diversity. We find low inbreeding coefficients and total runs of homozygosity, thus suggesting pedigree management of genetic relatedness is acceptable. CONCLUSIONS: The use of WGS of a small sampling among a large feline colony has enabled us to identify possible disease-causing variants, their genotype state and measure pedigree management of genetic diversity. We contend a limited but strategic sampling of feline colony individuals using WGS can inform veterinarians of future health anomalies and guide breeding practices to ensure healthy genetic diversity.

The Biomphalaria glabrata DNA methylation machinery displays spatial tissue expression, is differentially active in distinct snail populations and is modulated by interactions with Schistosoma mansoni.

BACKGROUND: The debilitating human disease schistosomiasis is caused by infection with schistosome parasites that maintain a complex lifecycle alternating between definitive (human) and intermediate (snail) hosts. While much is known about how the definitive host responds to schistosome infection, there is comparably less information available describing the snail's response to infection. METHODOLOGY/PRINCIPLE FINDINGS: Here, using information recently revealed by sequencing of the Biomphalaria glabrata intermediate host genome, we provide evidence that the predicted core snail DNA methylation machinery components are associated with both intra-species reproduction processes and inter-species interactions. Firstly, methyl-CpG binding domain protein (Bgmbd2/3) and DNA methyltransferase 1 (Bgdnmt1) genes are transcriptionally enriched in gonadal compared to somatic tissues with 5-azacytidine (5-AzaC) treatment significantly inhibiting oviposition. Secondly, elevated levels of 5-methyl cytosine (5mC), DNA methyltransferase activity and 5mC binding in pigmented hybrid- compared to inbred (NMRI)- B. glabrata populations indicate a role for the snail's DNA methylation machinery in maintaining hybrid vigour or heterosis. Thirdly, locus-specific detection of 5mC by bisulfite (BS)-PCR revealed 5mC within an exonic region of a housekeeping protein-coding gene (Bg14-3-3), supporting previous in silico predictions and whole genome BS-Seq analysis of this species' genome. Finally, we provide preliminary evidence for parasite-mediated host epigenetic reprogramming in the schistosome/snail system, as demonstrated by the increase in Bgdnmt1 and Bgmbd2/3 transcript abundance following Bge (B. glabrata embryonic cell line) exposure to parasite larval transformation products (LTP). CONCLUSIONS/SIGNIFICANCE: The presence of a functional DNA methylation machinery in B. glabrata as well as the modulation of these gene products in response to schistosome products, suggests a vital role for DNA methylation during snail development/oviposition and parasite interactions. Further deciphering the role of this epigenetic process during Biomphalaria/Schistosoma co-evolutionary biology may reveal key factors associated with disease transmission and, moreover, enable the discovery of novel lifecycle intervention strategies.