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Auto Tag: Structural variant

September 22, 2019  |  

Variation in human chromosome 21 ribosomal RNA genes characterized by TAR cloning and long-read sequencing.

Despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. We recovered ribosomal DNA segments from a single human chromosome 21 using transformation-associated recombination (TAR) cloning in yeast. Accurate long-read sequencing of 13 isolates covering ~0.82 Mb of the chromosome 21 rDNA complement revealed substantial variation among tandem repeat rDNA copies, several palindromic structures and potential errors in the previous reference sequence. These clones revealed 101 variant positions in the 45S transcription unit and 235 in the intergenic spacer sequence. Approximately 60% of the 45S variants were confirmed in independent whole-genome or RNA-seq data, with 47 of these further observed in mature 18S/28S rRNA sequences. TAR cloning and long-read sequencing enabled the accurate reconstruction of multiple rDNA units and a new, high-quality 44 838 bp rDNA reference sequence, which we have annotated with variants detected from chromosome 21 of a single individual. The large number of variants observed reveal heterogeneity in human rDNA, opening up the possibility of corresponding variations in ribosome dynamics.

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September 22, 2019  |  

A graph-based approach to diploid genome assembly.

Constructing high-quality haplotype-resolved de novo assemblies of diploid genomes is important for revealing the full extent of structural variation and its role in health and disease. Current assembly approaches often collapse the two sequences into one haploid consensus sequence and, therefore, fail to capture the diploid nature of the organism under study. Thus, building an assembler capable of producing accurate and complete diploid assemblies, while being resource-efficient with respect to sequencing costs, is a key challenge to be addressed by the bioinformatics community.We present a novel graph-based approach to diploid assembly, which combines accurate Illumina data and long-read Pacific Biosciences (PacBio) data. We demonstrate the effectiveness of our method on a pseudo-diploid yeast genome and show that we require as little as 50× coverage Illumina data and 10× PacBio data to generate accurate and complete assemblies. Additionally, we show that our approach has the ability to detect and phase structural variants.https://github.com/whatshap/whatshap.Supplementary data are available at Bioinformatics online.

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September 22, 2019  |  

Whole genome and transcriptome maps of the entirely black native Korean chicken breed Yeonsan Ogye.

Yeonsan Ogye (YO), an indigenous Korean chicken breed (Gallus gallus domesticus), has entirely black external features and internal organs. In this study, the draft genome of YO was assembled using a hybrid de novo assembly method that takes advantage of high-depth Illumina short reads (376.6X) and low-depth Pacific Biosciences (PacBio) long reads (9.7X).The contig and scaffold NG50s of the hybrid de novo assembly were 362.3 Kbp and 16.8 Mbp, respectively. The completeness (97.6%) of the draft genome (Ogye_1.1) was evaluated with single-copy orthologous genes using Benchmarking Universal Single-Copy Orthologs and found to be comparable to the current chicken reference genome (galGal5; 97.4%; contigs were assembled with high-depth PacBio long reads (50X) and scaffolded with short reads) and superior to other avian genomes (92%-93%; assembled with short read-only or hybrid methods). Compared to galGal4 and galGal5, the draft genome included 551 structural variations including the fibromelanosis (FM) locus duplication, related to hyperpigmentation. To comprehensively reconstruct transcriptome maps, RNA sequencing and reduced representation bisulfite sequencing data were analyzed from 20 tissues, including 4 black tissues (skin, shank, comb, and fascia). The maps included 15,766 protein-coding and 6,900 long noncoding RNA genes, many of which were tissue-specifically expressed and displayed tissue-specific DNA methylation patterns in the promoter regions.We expect that the resulting genome sequence and transcriptome maps will be valuable resources for studying domestic chicken breeds, including black-skinned chickens, as well as for understanding genomic differences between breeds and the evolution of hyperpigmented chickens and functional elements related to hyperpigmentation.

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September 22, 2019  |  

npInv: accurate detection and genotyping of inversions using long read sub-alignment.

Detection of genomic inversions remains challenging. Many existing methods primarily target inzversions with a non repetitive breakpoint, leaving inverted repeat (IR) mediated non-allelic homologous recombination (NAHR) inversions largely unexplored.We present npInv, a novel tool specifically for detecting and genotyping NAHR inversion using long read sub-alignment of long read sequencing data. We benchmark npInv with other tools in both simulation and real data. We use npInv to generate a whole-genome inversion map for NA12878 consisting of 30 NAHR inversions (of which 15 are novel), including all previously known NAHR mediated inversions in NA12878 with flanking IR less than 7kb. Our genotyping accuracy on this dataset was 94%. We used PCR to confirm the presence of two of these novel inversions. We show that there is a near linear relationship between the length of flanking IR and the minimum inversion size, without inverted repeats.The application of npInv shows high accuracy in both simulation and real data. The results give deeper insight into understanding inversion.

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September 22, 2019  |  

Using XCAVATOR and EXCAVATOR2 to Identify CNVs from WGS, WES, and TS Data.

Copy Number Variants (CNVs) are structural rearrangements contributing to phenotypic variation but also associated with many disease states. In recent years, the identification of CNVs from high-throughput sequencing experiments has become a common practice for both research and clinical purposes. Several computational methods have been developed so far. In this unit, we describe and give instructions on how to run two read count-based tools, XCAVATOR and EXCAVATOR2, which are tailored for the detection of both germline and somatic CNVs from different sequencing experiments (whole-genome, whole-exome, and targeted) in various disease contexts and population genetic studies. © 2018 by John Wiley & Sons, Inc.© 2018 John Wiley & Sons, Inc.

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September 22, 2019  |  

Validation of Genomic Structural Variants Through Long Sequencing Technologies.

Although numerous algorithms have been developed to identify large chromosomal rearrangements (i.e., genomic structural variants, SVs), there remains a dearth of approaches to evaluate their results. This is significant, as the accurate identification of SVs is still an outstanding problem whereby no single algorithm has been shown to be able to achieve high sensitivity and specificity across different classes of SVs. The method introduced in this chapter, VaPoR, is specifically designed to evaluate the accuracy of SV predictions using third-generation long sequences. This method uses a recurrence approach and collects direct evidence from raw reads thus avoiding computationally costly whole genome assembly. This chapter would describe in detail as how to apply this tool onto different data types.

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September 22, 2019  |  

Human copy number variants are enriched in regions of low mappability.

Copy number variants (CNVs) are known to affect a large portion of the human genome and have been implicated in many diseases. Although whole-genome sequencing (WGS) can help identify CNVs, most analytical methods suffer from limited sensitivity and specificity, especially in regions of low mappability. To address this, we use PopSV, a CNV caller that relies on multiple samples to control for technical variation. We demonstrate that our calls are stable across different types of repeat-rich regions and validate the accuracy of our predictions using orthogonal approaches. Applying PopSV to 640 human genomes, we find that low-mappability regions are approximately 5 times more likely to harbor germline CNVs, in stark contrast to the nearly uniform distribution observed for somatic CNVs in 95 cancer genomes. In addition to known enrichments in segmental duplication and near centromeres and telomeres, we also report that CNVs are enriched in specific types of satellite and in some of the most recent families of transposable elements. Finally, using this comprehensive approach, we identify 3455 regions with recurrent CNVs that were missing from existing catalogs. In particular, we identify 347 genes with a novel exonic CNV in low-mappability regions, including 29 genes previously associated with disease.

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September 22, 2019  |  

Whole-genome resequencing and pan-transcriptome reconstruction highlight the impact of genomic structural Variation on secondary metabolite gene clusters in the grapevine Esca pathogen Phaeoacremonium minimum.

The Ascomycete fungus Phaeoacremonium minimum is one of the primary causal agents of Esca, a widespread and damaging grapevine trunk disease. Variation in virulence among Pm. minimum isolates has been reported, but the underlying genetic basis of the phenotypic variability remains unknown. The goal of this study was to characterize intraspecific genetic diversity and explore its potential impact on virulence functions associated with secondary metabolism, cellular transport, and cell wall decomposition. We generated a chromosome-scale genome assembly, using single molecule real-time sequencing, and resequenced the genomes and transcriptomes of multiple isolates to identify sequence and structural polymorphisms. Numerous insertion and deletion events were found for a total of about 1 Mbp in each isolate. Structural variation in this extremely gene dense genome frequently caused presence/absence polymorphisms of multiple adjacent genes, mostly belonging to biosynthetic clusters associated with secondary metabolism. Because of the observed intraspecific diversity in gene content due to structural variation we concluded that a transcriptome reference developed from a single isolate is insufficient to represent the virulence factor repertoire of the species. We therefore compiled a pan-transcriptome reference of Pm. minimum comprising a non-redundant set of 15,245 protein-coding sequences. Using naturally infected field samples expressing Esca symptoms, we demonstrated that mapping of meta-transcriptomics data on a multi-species reference that included the Pm. minimum pan-transcriptome allows the profiling of an expanded set of virulence factors, including variable genes associated with secondary metabolism and cellular transport.

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September 22, 2019  |  

Ring synthetic chromosome V SCRaMbLE.

Structural variations (SVs) exert important functional impacts on biological phenotypic diversity. Here we show a ring synthetic yeast chromosome V (ring_synV) can be used to continuously generate complex genomic variations and improve the production of prodeoxyviolacein (PDV) by applying Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) in haploid yeast cells. The SCRaMbLE of ring_synV generates aneuploid yeast strains with increased PDV productivity, and we identify aneuploid chromosome I, III, VI, XII, XIII, and ring_synV. The neochromosome of SCRaMbLEd ring_synV generated more unbalanced forms of variations, including duplication, insertions, and balanced forms of translocations and inversions than its linear form. Furthermore, of the 29 novel SVs detected, 11 prompted the PDV biosynthesis; and the deletion of uncharacterized gene YER182W is related to the improvement of the PDV. Overall, the SCRaMbLEing ring_synV embraces the evolution of the genome by modifying the chromosome number, structure, and organization, identifying targets for phenotypic comprehension.

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September 22, 2019  |  

Structural variants exhibit allelic heterogeneity and shape variation in complex traits

Despite extensive effort to reveal the genetic basis of complex phenotypic variation, studies typically explain only a fraction of trait heritability. It has been hypothesized that individually rare hidden structural variants (SVs) could account for a significant fraction of variation in complex traits. To investigate this hypothesis, we assembled 14 Drosophila melanogaster genomes and systematically identified more than 20,000 euchromatic SVs, of which ~40% are invisible to high specificity short read genotyping approaches. SVs are common in Drosophila genes, with almost one third of diploid individuals harboring an SV in genes larger than 5kb, and nearly a quarter harboring multiple SVs in genes larger than 10kb. We show that SV alleles are rarer than amino acid polymorphisms, implying that they are more strongly deleterious. A number of functionally important genes harbor previously hidden structural variants that likely affect complex phenotypes (e.g., Cyp6g1, Drsl5, Cyp28d1&2, InR, and Gss1&2). Furthermore, SVs are overrepresented in quantitative trait locus candidate genes from eight Drosophila Synthetic Population Resource (DSPR) mapping experiments. We conclude that SVs are pervasive in genomes, are frequently present as heterogeneous allelic series, and can act as rare alleles of large effect.

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September 22, 2019  |  

Parliament2: Fast structural variant calling using optimized combinations of callers

Here we present Parliament2: a structural variant caller which combines multiple best-in-class structural variant callers to create a highly accurate callset. This captures more events than the individual callers achieve independently. Parliament2 uses a call-overlap-genotype approach that is highly extensible to new methods and presents users the choice to run some or all of Breakdancer, Breakseq, CNVnator, Delly, Lumpy, and Manta to run. Parliament2 applies an additional parallelization framework to speed certain callers and executes these in parallel, taking advantage of the different resource requirements to complete structural variant calling much faster than running the programs individually. Parliament2 is available as a Docker container, which pre-installs all required dependencies. This allows users to run any caller with easy installation and execution. This Docker container can easily be deployed in cloud or local environments and is available as an app on DNAnexus.

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September 22, 2019  |  

Generic accelerated sequence alignment in SeqAn using vectorization and multi-threading.

Pairwise sequence alignment is undoubtedly a central tool in many bioinformatics analyses. In this paper, we present a generically accelerated module for pairwise sequence alignments applicable for a broad range of applications. In our module, we unified the standard dynamic programming kernel used for pairwise sequence alignments and extended it with a generalized inter-sequence vectorization layout, such that many alignments can be computed simultaneously by exploiting SIMD (single instruction multiple data) instructions of modern processors. We then extended the module by adding two layers of thread-level parallelization, where we (a) distribute many independent alignments on multiple threads and (b) inherently parallelize a single alignment computation using a work stealing approach producing a dynamic wavefront progressing along the minor diagonal.We evaluated our alignment vectorization and parallelization on different processors, including the newest Intel® Xeon® (Skylake) and Intel® Xeon PhiTM (KNL) processors, and use cases. The instruction set AVX512-BW (Byte and Word), available on Skylake processors, can genuinely improve the performance of vectorized alignments. We could run single alignments 1600 times faster on the Xeon PhiTM and 1400 times faster on the Xeon® than executing them with our previous sequential alignment module.The module is programmed in C++?using the SeqAn (Reinert et al., 2017) library and distributed with version 2.4 under the BSD license. We support SSE4, AVX2, AVX512 instructions and included UME: SIMD, a SIMD-instruction wrapper library, to extend our module for further instruction sets. We thoroughly test all alignment components with all major C++?compilers on various platforms.Supplementary data are available at Bioinformatics online.

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September 22, 2019  |  

Multisite de novo mutations in human offspring after paternal exposure to ionizing radiation.

A genome-wide evaluation of the effects of ionizing radiation on mutation induction in the mouse germline has identified multisite de novo mutations (MSDNs) as marker for previous exposure. Here we present the results of a small pilot study of whole genome sequencing in offspring of soldiers who served in radar units on weapon systems that were emitting high-frequency radiation. We found cases of exceptionally high MSDN rates as well as an increased mean in our cohort: While a MSDN mutation is detected in average in 1 out of 5 offspring of unexposed controls, we observed 12 MSDNs in altogether 18 offspring, including a family with 6 MSDNs in 3 offspring. Moreover, we found two translocations, also resulting from neighboring mutations. Our findings indicate that MSDNs might be suited in principle for the assessment of DNA damage from ionizing radiation also in humans. However, as exact person-related dose values in risk groups are usually not available, the interpretation of MSDNs in single families would benefit from larger molecular epidemiologic studies on this new biomarker.

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September 22, 2019  |  

Deletions linked to PROG1 gene participate in plant architecture domestication in Asian and African rice.

Improving the yield by modifying plant architecture was a key step during crop domestication. Here, we show that a 110-kb deletion on the short arm of chromosome 7 in Asian cultivated rice (Oryza sativa), which is closely linked to the previously identified PROSTRATE GROWTH 1 (PROG1) gene, harbors a tandem repeat of seven zinc-finger genes. Three of these genes regulate the plant architecture, suggesting that the deletion also promoted the critical transition from the prostrate growth and low yield of wild rice (O. rufipogon) to the erect growth and high yield of Asian cultivated rice. We refer to this locus as RICE PLANT ARCHITECTURE DOMESTICATION (RPAD). Further, a similar but independent 113-kb deletion is detected at the RPAD locus in African cultivated rice. These results indicate that the deletions, eliminating a tandem repeat of zinc-finger genes, may have been involved in the parallel domestication of plant architecture in Asian and African rice.

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