Pygenomics: manipulating genomic intervals and data files in Python.

SUMMARY: We present pygenomics, a Python package for working with genomic intervals and bioinformatic data files. The package implements interval operations, provides both API and CLI, and supports reading and writing data in widely used bioinformatic formats, including BAM, BED, GFF3, and VCF. The source code of pygenomics is provided with in-source documentation and type annotations and adheres to the functional programming paradigm. These features facilitate seamless integration of pygenomics routines into scripts and pipelines. The package is implemented in pure Python using its standard library only and contains the property-based testing framework. Comparison of pygenomics with other Python bioinformatic packages with relation to features and performance is presented. The performance comparison covers operations with genomic intervals, read alignments, and genomic variants and demonstrates that pygenomics is suitable for computationally effective analysis. AVAILABILITY AND IMPLEMENTATION: The source code is available at https://gitlab.com/gtamazian/pygenomics.

Chromosome-length genome assemblies and cytogenomic analyses of pangolins reveal remarkable chromosome counts and plasticity.

We report the first chromosome-length genome assemblies for three species in the mammalian order Pholidota: the white-bellied, Chinese, and Sunda pangolins. Surprisingly, we observe extraordinary karyotypic plasticity within this order and, in female white-bellied pangolins, the largest number of chromosomes reported in a Laurasiatherian mammal: 2n = 114. We perform the first karyotype analysis of an African pangolin and report a Y-autosome fusion in white-bellied pangolins, resulting in 2n = 113 for males. We employ a novel strategy to confirm the fusion and identify the autosome involved by finding the pseudoautosomal region (PAR) in the female genome assembly and analyzing the 3D contact frequency between PAR sequences and the rest of the genome in male and female white-bellied pangolins. Analyses of genetic variability show that white-bellied pangolins have intermediate levels of genome-wide heterozygosity relative to Chinese and Sunda pangolins, consistent with two moderate declines of historical effective population size. Our results reveal a remarkable feature of pangolin genome biology and highlight the need for further studies of these unique and endangered mammals.

Chromosome-Length Assembly of the Baikal Seal (Pusa sibirica) Genome Reveals a Historically Large Population Prior to Isolation in Lake Baikal.

Pusa sibirica, the Baikal seal, is the only extant, exclusively freshwater, pinniped species. The pending issue is, how and when they reached their current habitat-the rift lake Baikal, more than three thousand kilometers away from the Arctic Ocean. To explore the demographic history and genetic diversity of this species, we generated a de novo chromosome-length assembly, and compared it with three closely related marine pinniped species. Multiple whole genome alignment of the four species compared with their karyotypes showed high conservation of chromosomal features, except for three large inversions on chromosome VI. We found the mean heterozygosity of the studied Baikal seal individuals was relatively low (0.61 SNPs/kbp), but comparable to other analyzed pinniped samples. Demographic reconstruction of seals revealed differing trajectories, yet remarkable variations in Ne occurred during approximately the same time periods. The Baikal seal showed a significantly more severe decline relative to other species. This could be due to the difference in environmental conditions encountered by the earlier populations of Baikal seals, as ice sheets changed during glacial-interglacial cycles. We connect this period to the time of migration to Lake Baikal, which occurred ~3-0.3 Mya, after which the population stabilized, indicating balanced habitat conditions.

Genome-wide association study reveals genetic variants associated with HIV-1C infection in a Botswana study population.

Although there have been many studies of gene variant association with different stages of HIV/AIDS progression in United States and European cohorts, few gene-association studies have assessed genic determinants in sub-Saharan African populations, which have the highest density of HIV infections worldwide. We carried out genome-wide association studies on 766 study participants at risk for HIV-1 subtype C (HIV-1C) infection in Botswana. Three gene associations (AP3B1, PTPRA, and NEO1) were shown to have significant association with HIV-1C acquisition. Each gene association was replicated within Botswana or in the United States-African American or United States-European American AIDS cohorts or in both. Each associated gene has a prior reported influence on HIV/AIDS pathogenesis. Thirteen previously discovered AIDS restriction genes were further replicated in the Botswana cohorts, extending our confidence in these prior AIDS restriction gene reports. This work presents an early step toward the identification of genetic variants associated with and affecting HIV acquisition or AIDS progression in the understudied HIV-1C afflicted Botswana population.

Draft de novo Genome Assembly of the Elusive Jaguarundi, Puma yagouaroundi.

The Puma lineage within the family Felidae consists of 3 species that last shared a common ancestor around 4.9 million years ago. Whole-genome sequences of 2 species from the lineage were previously reported: the cheetah (Acinonyx jubatus) and the mountain lion (Puma concolor). The present report describes a whole-genome assembly of the remaining species, the jaguarundi (Puma yagouaroundi). We sequenced the genome of a male jaguarundi with 10X Genomics linked reads and assembled the whole-genome sequence. The assembled genome contains a series of scaffolds that reach the length of chromosome arms and is similar in scaffold contiguity to the genome assemblies of cheetah and puma, with a contig N50 = 100.2 kbp and a scaffold N50 = 49.27 Mbp. We assessed the assembled sequence of the jaguarundi genome using BUSCO, aligned reads of the sequenced individual and another published female jaguarundi to the assembled genome, annotated protein-coding genes, repeats, genomic variants and their effects with respect to the protein-coding genes, and analyzed differences of the 2 jaguarundis from the reference mitochondrial genome. The jaguarundi genome assembly and its annotation were compared in quality, variants, and features to the previously reported genome assemblies of puma and cheetah. Computational analyzes used in the study were implemented in transparent and reproducible way to allow their further reuse and modification.

Comparison of genomic diversity and structure of sable antelope (Hippotragus niger) in zoos, conservation centers, and private ranches in North America.

As we enter the sixth mass extinction, many species that are no longer self-sustaining in their natural habitat will require ex situ management. Zoos have finite resources for ex situ management, and there is a need for holistic conservation programs between the public and private sector. Ex situ populations of sable antelope, Hippotragus niger, have existed in zoos and privately owned ranches in North America since the 1910s. Unknown founder representation and relatedness has made the genetic management of this species challenging within zoos, while populations on privately owned ranches are managed independently and retain minimal-to-no pedigree history. Consequences of such challenges include an increased risk of inbreeding and a loss of genetic diversity. Here, we developed and applied a customized targeted sequence capture panel based on 5,000 genomewide single-nucleotide polymorphisms to investigate the genomic diversity present in these uniquely managed populations. We genotyped 111 sable antelope: 23 from zoos, 43 from a single conservation center, and 45 from ranches. We found significantly higher genetic diversity and significantly lower inbreeding in herds housed in zoos and conservation centers, when compared to those in privately owned ranches, likely due to genetic-based breeding recommendations implemented in the former populations. Genetic clustering was strong among all three populations, possibly as a result of genetic drift. We propose that the North American ex situ population of sable antelope would benefit from a metapopulation management system, to halt genetic drift, reduce the occurrence of inbreeding, and enable sustainable population sizes to be managed ex situ.

Chromosomal-level genome assembly of the scimitar-horned oryx: Insights into diversity and demography of a species extinct in the wild.

Captive populations provide a valuable insurance against extinctions in the wild. However, they are also vulnerable to the negative impacts of inbreeding, selection and drift. Genetic information is therefore considered a critical aspect of conservation management. Recent developments in sequencing technologies have the potential to improve the outcomes of management programmes; however, the transfer of these approaches to applied conservation has been slow. The scimitar-horned oryx (Oryx dammah) is a North African antelope that has been extinct in the wild since the early 1980s and is the focus of a large-scale and long-term reintroduction project. To enable the selection of suitable founder individuals, facilitate post-release monitoring and improve captive breeding management, comprehensive genomic resources are required. Here, we used 10X Chromium sequencing together with Hi-C contact mapping to develop a chromosomal-level genome assembly for the species. The resulting assembly contained 29 chromosomes with a scaffold N50 of 100.4 Mb, and displayed strong chromosomal synteny with the cattle genome. Using resequencing data from six additional individuals, we demonstrated relatively high genetic diversity in the scimitar-horned oryx compared to other mammals, despite it having experienced a strong founding event in captivity. Additionally, the level of diversity across populations varied according to management strategy. Finally, we uncovered a dynamic demographic history that coincided with periods of climate variation during the Pleistocene. Overall, our study provides a clear example of how genomic data can uncover valuable insights into captive populations and contributes important resources to guide future management decisions of an endangered species.

Genome-wide sequence analyses of ethnic populations across Russia.

The Russian Federation is the largest and one of the most ethnically diverse countries in the world, however no centralized reference database of genetic variation exists to date. Such data are crucial for medical genetics and essential for studying population history. The Genome Russia Project aims at filling this gap by performing whole genome sequencing and analysis of peoples of the Russian Federation. Here we report the characterization of genome-wide variation of 264 healthy adults, including 60 newly sequenced samples. People of Russia carry known and novel genetic variants of adaptive, clinical and functional consequence that in many cases show allele frequency divergence from neighboring populations. Population genetics analyses revealed six phylogeographic partitions among indigenous ethnicities corresponding to their geographic locales. This study presents a characterization of population-specific genomic variation in Russia with results important for medical genetics and for understanding the dynamic population history of the world's largest country.

Evolutionary Switches Structural Transitions via Coarse-Grained Models.

Transitions between different conformational states are ubiquitous in proteins. A vast class of conformation-changing proteins includes evolutionary switches, which vary their conformation as an effect of few mutations or weak environmental variations. However, modeling those processes is extremely difficult due to the need of efficiently exploring a vast conformational space to look for the actual transition path. In this study, we report a strategy that simplifies this task attacking the complexity on several sides. We first apply a minimalist coarse-grained model to the protein, based on an empirical force field with a partial structural bias toward one or both the reference structures. We then explore the transition paths by means of stochastic molecular dynamics and select representative structures by means of a principal path-based clustering algorithm. We finally compare this trajectory with that produced by independent methods adopting a morphing-oriented approach. Our analysis indicates that the minimalist model returns trajectories capable of exploring intermediate states with physical meaning, retaining a very low computational cost, which can allow systematic and extensive exploration of the multistable proteins transition pathways.

Structural Transition States Explored With Minimalist Coarse Grained Models: Applications to Calmodulin.

Transitions between different conformational states are ubiquitous in proteins, being involved in signaling, catalysis, and other fundamental activities in cells. However, modeling those processes is extremely difficult, due to the need of efficiently exploring a vast conformational space in order to seek for the actual transition path for systems whose complexity is already high in the stable states. Here we report a strategy that simplifies this task attacking the complexity on several sides. We first apply a minimalist coarse-grained model to Calmodulin, based on an empirical force field with a partial structural bias, to explore the transition paths between the apo-closed state and the Ca-bound open state of the protein. We then select representative structures along the trajectory based on a structural clustering algorithm and build a cleaned-up trajectory with them. We finally compare this trajectory with that produced by the online tool MinActionPath, by minimizing the action integral using a harmonic network model, and with that obtained by the PROMPT morphing method, based on an optimal mass transportation-type approach including physical constraints. The comparison is performed both on the structural and energetic level, using the coarse-grained and the atomistic force fields upon reconstruction. Our analysis indicates that this method returns trajectories capable of exploring intermediate states with physical meaning, retaining a very low computational cost, which can allow systematic and extensive exploration of the multi-stable proteins transition pathways.

Whole Genome Sequencing and Re-sequencing of the Sable Antelope (Hippotragus niger): A Resource for Monitoring Diversity in ex Situ and in Situ Populations.

Genome-wide assessment of genetic diversity has the potential to increase the ability to understand admixture, inbreeding, kinship and erosion of genetic diversity affecting both captive (ex situ) and wild (in situ) populations of threatened species. The sable antelope (Hippotragus niger), native to the savannah woodlands of sub-Saharan Africa, is a species that is being managed ex situ in both public (zoo) and private (ranch) collections in the United States. Our objective was to develop whole genome sequence resources that will serve as a foundation for characterizing the genetic status of ex situ populations of sable antelope relative to populations in the wild. Here we report the draft genome assembly of a male sable antelope, a member of the subfamily Hippotraginae (Bovidae, Cetartiodactyla, Mammalia). The 2.596 Gb draft genome consists of 136,528 contigs with an N50 of 45.5 Kbp and 16,927 scaffolds with an N50 of 4.59 Mbp. De novo annotation identified 18,828 protein-coding genes and repetitive sequences encompassing 46.97% of the genome. The discovery of single nucleotide variants (SNVs) was assisted by the re-sequencing of seven additional captive and wild individuals, representing two different subspecies, leading to the identification of 1,987,710 bi-allelic SNVs. Assembly of the mitochondrial genomes revealed that each individual was defined by a unique haplotype and these data were used to infer the mitochondrial gene tree relative to other hippotragine species. The sable antelope genome constitutes a valuable resource for assessing genome-wide diversity and evolutionary potential, thereby facilitating long-term conservation of this charismatic species.

Genomes of Three Closely Related Caribbean Amazons Provide Insight for Species History and Conservation.

Islands have been used as model systems for studies of speciation and extinction since Darwin published his observations about finches found on the Galapagos. Amazon parrots inhabiting the Greater Antillean Islands represent a fascinating model of species diversification. Unfortunately, many of these birds are threatened as a result of human activity and some, like the Puerto Rican parrot, are now critically endangered. In this study we used a combination of de novo and reference-assisted assembly methods, integrating it with information obtained from related genomes to perform genome reconstruction of three amazon species. First, we used whole genome sequencing data to generate a new de novo genome assembly for the Puerto Rican parrot (Amazona vittata). We then improved the obtained assembly using transcriptome data from Amazona ventralis and used the resulting sequences as a reference to assemble the genomes Hispaniolan (A. ventralis) and Cuban (Amazona leucocephala) parrots. Finally, we, annotated genes and repetitive elements, estimated genome sizes and current levels of heterozygosity, built models of demographic history and provided interpretation of our findings in the context of parrot evolution in the Caribbean.

Analytical "bake-off" of whole genome sequencing quality for the Genome Russia project using a small cohort for autoimmune hepatitis.

A comparative analysis of whole genome sequencing (WGS) and genotype calling was initiated for ten human genome samples sequenced by St. Petersburg State University Peterhof Sequencing Center and by three commercial sequencing centers outside of Russia. The sequence quality, efficiency of DNA variant and genotype calling were compared with each other and with DNA microarrays for each of ten study subjects. We assessed calling of SNPs, indels, copy number variation, and the speed of WGS throughput promised. Twenty separate QC analyses showed high similarities among the sequence quality and called genotypes. The ten genomes tested by the centers included eight American patients afflicted with autoimmune hepatitis (AIH), plus one case's unaffected parents, in a prelude to discovering genetic influences in this rare disease of unknown etiology. The detailed internal replication and parallel analyses allowed the observation of two of eight AIH cases carrying a rare allele genotype for a previously described AIH-associated gene (FTCD), plus multiple occurrences of known HLA-DRB1 alleles associated with AIH (HLA-DRB1-03:01:01, 13:01:01 and 7:01:01). We also list putative SNVs in other genes as suggestive in AIH influence.

Robust forensic matching of confiscated horns to individual poached African rhinoceros.

Black and white rhinoceros (Diceros bicornis and Ceratotherium simum) are iconic African species that are classified by the International Union for the Conservation of Nature (IUCN) as Critically Endangered and Near Threatened (http://www.iucnredlist.org/), respectively [1]. At the end of the 19th century, Southern white rhinoceros (Ceratotherium simum simum) numbers had declined to fewer than 50 animals in the Hluhluwe-iMfolozi region of the KwaZulu-Natal (KZN) province of South Africa, mainly due to uncontrolled hunting [2,3]. Efforts by the Natal Parks Board facilitated an increase in population to over 20,000 in 2015 through aggressive conservation management [2]. Black rhinoceros (Diceros bicornis) populations declined from several hundred thousand in the early 19th century to ∼65,000 in 1970 and to ∼2,400 by 1995 [1] with subsequent genetic reduction, also due to hunting, land clearances and later poaching [4]. In South Africa, rhinoceros poaching incidents have increased from 13 in 2007 to 1,215 in 2014 [1]. This has occurred despite strict trade bans on rhinoceros products and strict enforcement in recent years.

PGD: a pangolin genome hub for the research community.

Pangolins (order Pholidota) are the only mammals covered by scales. We have recently sequenced and analyzed the genomes of two critically endangered Asian pangolin species, namely the Malayan pangolin (Manis javanica) and the Chinese pangolin (Manis pentadactyla). These complete genome sequences will serve as reference sequences for future research to address issues of species conservation and to advance knowledge in mammalian biology and evolution. To further facilitate the global research effort in pangolin biology, we developed the Pangolin Genome Database (PGD), as a future hub for hosting pangolin genomic and transcriptomic data and annotations, and with useful analysis tools for the research community. Currently, the PGD provides the reference pangolin genome and transcriptome data, gene sequences and functional information, expressed transcripts, pseudogenes, genomic variations, organ-specific expression data and other useful annotations. We anticipate that the PGD will be an invaluable platform for researchers who are interested in pangolin and mammalian research. We will continue updating this hub by including more data, annotation and analysis tools particularly from our research consortium.Database URL: http://pangolin-genome.um.edu.my.

Pangolin genomes and the evolution of mammalian scales and immunity.

Pangolins, unique mammals with scales over most of their body, no teeth, poor vision, and an acute olfactory system, comprise the only placental order (Pholidota) without a whole-genome map. To investigate pangolin biology and evolution, we developed genome assemblies of the Malayan (Manis javanica) and Chinese (M. pentadactyla) pangolins. Strikingly, we found that interferon epsilon (IFNE), exclusively expressed in epithelial cells and important in skin and mucosal immunity, is pseudogenized in all African and Asian pangolin species that we examined, perhaps impacting resistance to infection. We propose that scale development was an innovation that provided protection against injuries or stress and reduced pangolin vulnerability to infection. Further evidence of specialized adaptations was evident from positively selected genes involving immunity-related pathways, inflammation, energy storage and metabolism, muscular and nervous systems, and scale/hair development. Olfactory receptor gene families are significantly expanded in pangolins, reflecting their well-developed olfaction system. This study provides insights into mammalian adaptation and functional diversification, new research tools and questions, and perhaps a new natural IFNE-deficient animal model for studying mammalian immunity.

Chromosomer: a reference-based genome arrangement tool for producing draft chromosome sequences.

BACKGROUND: As the number of sequenced genomes rapidly increases, chromosome assembly is becoming an even more crucial step of any genome study. Since de novo chromosome assemblies are confounded by repeat-mediated artifacts, reference-assisted assemblies that use comparative inference have become widely used, prompting the development of several reference-assisted assembly programs for prokaryotic and eukaryotic genomes. FINDINGS: We developed Chromosomer - a reference-based genome arrangement tool, which rapidly builds chromosomes from genome contigs or scaffolds using their alignments to a reference genome of a closely related species. Chromosomer does not require mate-pair libraries and it offers a number of auxiliary tools that implement common operations accompanying the genome assembly process. CONCLUSIONS: Despite implementing a straightforward alignment-based approach, Chromosomer is a useful tool for genomic analysis of species without chromosome maps. Putative chromosome assemblies by Chromosomer can be used in comparative genomic analysis, genomic variation assessment, potential linkage group inference and other kinds of analysis involving contig or scaffold mapping to a high-quality assembly.

Genomic legacy of the African cheetah, Acinonyx jubatus.

BACKGROUND: Patterns of genetic and genomic variance are informative in inferring population history for human, model species and endangered populations. RESULTS: Here the genome sequence of wild-born African cheetahs reveals extreme genomic depletion in SNV incidence, SNV density, SNVs of coding genes, MHC class I and II genes, and mitochondrial DNA SNVs. Cheetah genomes are on average 95 % homozygous compared to the genomes of the outbred domestic cat (24.08 % homozygous), Virunga Mountain Gorilla (78.12 %), inbred Abyssinian cat (62.63 %), Tasmanian devil, domestic dog and other mammalian species. Demographic estimators impute two ancestral population bottlenecks: one >100,000 years ago coincident with cheetah migrations out of the Americas and into Eurasia and Africa, and a second 11,084-12,589 years ago in Africa coincident with late Pleistocene large mammal extinctions. MHC class I gene loss and dramatic reduction in functional diversity of MHC genes would explain why cheetahs ablate skin graft rejection among unrelated individuals. Significant excess of non-synonymous mutations in AKAP4 (p<0.02), a gene mediating spermatozoon development, indicates cheetah fixation of five function-damaging amino acid variants distinct from AKAP4 homologues of other Felidae or mammals; AKAP4 dysfunction may cause the cheetah's extremely high (>80 %) pleiomorphic sperm. CONCLUSIONS: The study provides an unprecedented genomic perspective for the rare cheetah, with potential relevance to the species' natural history, physiological adaptations and unique reproductive disposition.

Modeling conformational redox-switch modulation of human succinic semialdehyde dehydrogenase.

Succinic semialdehyde dehydrogenase (SSADH) converts succinic semialdehyde (SSA) to succinic acid in the mitochondrial matrix and is involved in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). The molecular structure of human SSADH revealed the intrinsic regulatory mechanism--redox-switch modulation--by which large conformational changes are brought about in the catalytic loop through disulfide bonding. The crystal structures revealed two SSADH conformations, and computational modeling of transformation between them can provide substantial insights into detailed dynamic redox modulation. On the basis of these two clear crystal structures, we modeled the conformational motion between these structures in silico. For that purpose, we proposed and used a geometry-based coarse-grained mathematical model of long-range protein motion and the related modeling algorithm. The algorithm is based on solving the special optimization problem, which is similar to the classical Monge-Kantorovich mass transportation problem. The modeled transformation was supported by another morphing method based on a completely different framework. The result of the modeling facilitates better interpretation and understanding of the SSADH biological role.

Annotated features of domestic cat - Felis catus genome.

BACKGROUND: Domestic cats enjoy an extensive veterinary medical surveillance which has described nearly 250 genetic diseases analogous to human disorders. Feline infectious agents offer powerful natural models of deadly human diseases, which include feline immunodeficiency virus, feline sarcoma virus and feline leukemia virus. A rich veterinary literature of feline disease pathogenesis and the demonstration of a highly conserved ancestral mammal genome organization make the cat genome annotation a highly informative resource that facilitates multifaceted research endeavors. FINDINGS: Here we report a preliminary annotation of the whole genome sequence of Cinnamon, a domestic cat living in Columbia (MO, USA), bisulfite sequencing of Boris, a male cat from St. Petersburg (Russia), and light 30× sequencing of Sylvester, a European wildcat progenitor of cat domestication. The annotation includes 21,865 protein-coding genes identified by a comparative approach, 217 loci of endogenous retrovirus-like elements, repetitive elements which comprise about 55.7% of the whole genome, 99,494 new SNVs, 8,355 new indels, 743,326 evolutionary constrained elements, and 3,182 microRNA homologues. The methylation sites study shows that 10.5% of cat genome cytosines are methylated. An assisted assembly of a European wildcat, Felis silvestris silvestris, was performed; variants between F. silvestris and F. catus genomes were derived and compared to F. catus. CONCLUSIONS: The presented genome annotation extends beyond earlier ones by closing gaps of sequence that were unavoidable with previous low-coverage shotgun genome sequencing. The assembly and its annotation offer an important resource for connecting the rich veterinary and natural history of cats to genome discovery.