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April 21, 2020  |  

Haplotype-aware diplotyping from noisy long reads.

Current genotyping approaches for single-nucleotide variations rely on short, accurate reads from second-generation sequencing devices. Presently, third-generation sequencing platforms are rapidly becoming more widespread, yet approaches for leveraging their long but error-prone reads for genotyping are lacking. Here, we introduce a novel statistical framework for the joint inference of haplotypes and genotypes from noisy long reads, which we term diplotyping. Our technique takes full advantage of linkage information provided by long reads. We validate hundreds of thousands of candidate variants that have not yet been included in the high-confidence reference set of the Genome-in-a-Bottle effort.


April 21, 2020  |  

Systematic analysis of dark and camouflaged genes reveals disease-relevant genes hiding in plain sight.

The human genome contains “dark” gene regions that cannot be adequately assembled or aligned using standard short-read sequencing technologies, preventing researchers from identifying mutations within these gene regions that may be relevant to human disease. Here, we identify regions with few mappable reads that we call dark by depth, and others that have ambiguous alignment, called camouflaged. We assess how well long-read or linked-read technologies resolve these regions.Based on standard whole-genome Illumina sequencing data, we identify 36,794 dark regions in 6054 gene bodies from pathways important to human health, development, and reproduction. Of these gene bodies, 8.7% are completely dark and 35.2% are =?5% dark. We identify dark regions that are present in protein-coding exons across 748 genes. Linked-read or long-read sequencing technologies from 10x Genomics, PacBio, and Oxford Nanopore Technologies reduce dark protein-coding regions to approximately 50.5%, 35.6%, and 9.6%, respectively. We present an algorithm to resolve most camouflaged regions and apply it to the Alzheimer’s Disease Sequencing Project. We rescue a rare ten-nucleotide frameshift deletion in CR1, a top Alzheimer’s disease gene, found in disease cases but not in controls.While we could not formally assess the association of the CR1 frameshift mutation with Alzheimer’s disease due to insufficient sample-size, we believe it merits investigating in a larger cohort. There remain thousands of potentially important genomic regions overlooked by short-read sequencing that are largely resolved by long-read technologies.


April 21, 2020  |  

Experimental validation of in silico predicted RAD locus frequencies using genomic resources and short read data from a model marine mammal.

Restriction site-associated DNA sequencing (RADseq) has revolutionized the study of wild organisms by allowing cost-effective genotyping of thousands of loci. However, for species lacking reference genomes, it can be challenging to select the restriction enzyme that offers the best balance between the number of obtained RAD loci and depth of coverage, which is crucial for a successful outcome. To address this issue, PredRAD was recently developed, which uses probabilistic models to predict restriction site frequencies from a transcriptome assembly or other sequence resource based on either GC content or mono-, di- or trinucleotide composition. This program generates predictions that are broadly consistent with estimates of the true number of restriction sites obtained through in silico digestion of available reference genome assemblies. However, in practice the actual number of loci obtained could potentially differ as incomplete enzymatic digestion or patchy sequence coverage across the genome might lead to some loci not being represented in a RAD dataset, while erroneous assembly could potentially inflate the number of loci. To investigate this, we used genome and transcriptome assemblies together with RADseq data from the Antarctic fur seal (Arctocephalus gazella) to compare PredRAD predictions with empirical estimates of the number of loci obtained via in silico digestion and from de novo assemblies.PredRAD yielded consistently higher predicted numbers of restriction sites for the transcriptome assembly relative to the genome assembly. The trinucleotide and dinucleotide models also predicted higher frequencies than the mononucleotide or GC content models. Overall, the dinucleotide and trinucleotide models applied to the transcriptome and the genome assemblies respectively generated predictions that were closest to the number of restriction sites estimated by in silico digestion. Furthermore, the number of de novo assembled RAD loci mapping to restriction sites was similar to the expectation based on in silico digestion.Our study reveals generally high concordance between PredRAD predictions and empirical estimates of the number of RAD loci. This further supports the utility of PredRAD, while also suggesting that it may be feasible to sequence and assemble the majority of RAD loci present in an organism’s genome.


April 21, 2020  |  

Retrotranspositional landscape of Asian rice revealed by 3000 genomes.

The recent release of genomic sequences for 3000 rice varieties provides access to the genetic diversity at species level for this crop. We take advantage of this resource to unravel some features of the retrotranspositional landscape of rice. We develop software TRACKPOSON specifically for the detection of transposable elements insertion polymorphisms (TIPs) from large datasets. We apply this tool to 32 families of retrotransposons and identify more than 50,000 TIPs in the 3000 rice genomes. Most polymorphisms are found at very low frequency, suggesting that they may have occurred recently in agro. A genome-wide association study shows that these activations in rice may be triggered by external stimuli, rather than by the alteration of genetic factors involved in transposable element silencing pathways. Finally, the TIPs dataset is used to trace the origin of rice domestication. Our results suggest that rice originated from three distinct domestication events.


April 21, 2020  |  

Origin and recent expansion of an endogenous gammaretroviral lineage in domestic and wild canids.

Vertebrate genomes contain a record of retroviruses that invaded the germlines of ancestral hosts and are passed to offspring as endogenous retroviruses (ERVs). ERVs can impact host function since they contain the necessary sequences for expression within the host. Dogs are an important system for the study of disease and evolution, yet no substantiated reports of infectious retroviruses in dogs exist. Here, we utilized Illumina whole genome sequence data to assess the origin and evolution of a recently active gammaretroviral lineage in domestic and wild canids.We identified numerous recently integrated loci of a canid-specific ERV-Fc sublineage within Canis, including 58 insertions that were absent from the reference assembly. Insertions were found throughout the dog genome including within and near gene models. By comparison of orthologous occupied sites, we characterized element prevalence across 332 genomes including all nine extant canid species, revealing evolutionary patterns of ERV-Fc segregation among species as well as subpopulations.Sequence analysis revealed common disruptive mutations, suggesting a predominant form of ERV-Fc spread by trans complementation of defective proviruses. ERV-Fc activity included multiple circulating variants that infected canid ancestors from the last 20 million to within 1.6 million years, with recent bursts of germline invasion in the sublineage leading to wolves and dogs.


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