An integrated map of structural variation in 2,504 human genomes

© 2015 Macmillan Publishers Limited. All rights reserved. Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association

Systematic Inference of Copy-Number Genotypes from Personal Genome Sequencing Data Reveals Extensive Olfactory Receptor Gene Content Diversity

Copy-number variations (CNVs) are widespread in the human genome, but comprehensive assignments of integer locus copy-numbers (i.e., copy-number genotypes) that, for example, enable discrimination of homozygous from heterozygous CNVs, have remained challenging. Here we present CopySeq, a novel computational approach with an underlying statistical framework that analyzes the depth-of-coverage of high-throughput DNA sequencing reads, and can incorporate paired-end and breakpoint junction analysis based CNV-analysis approaches, to infer locus copy-number genotypes. We benchmarked CopySeq by genotyping 500 chromosome 1 CNV regions in 150 personal genomes sequenced at low-coverage. The assessed copy-number genotypes were highly concordant with our performed qPCR experiments (Pearson correlation coefficient 0.94), and with the published results of two microarray platforms (95–99% concordance). We further demonstrated the utility of CopySeq for analyzing gene regions enriched for segmental duplications by comprehensively inferring copy-number genotypes in the CNV-enriched >800 olfactory receptor (OR) human gene and pseudogene loci. CopySeq revealed that OR loci display an extensive range of locus copy-numbers across individuals, with zero to two copies in some OR loci, and two to nine copies in others. Among genetic variants affecting OR loci we identified deleterious variants including CNVs and SNPs affecting ∼15% and ∼20% of the human OR gene repertoire, respectively, implying that genetic variants with a possible impact on smell perception are widespread. Finally, we found that for several OR loci the reference genome appears to represent a minor-frequency variant, implying a necessary revision of the OR repertoire for future functional studies. CopySeq can ascertain genomic structural variation in specific gene families as well as at a genome-wide scale, where it may enable the quantitative evaluation of CNVs in genome-wide association studies involving high-throughput sequencing

A Comprehensive Map of Mobile Element Insertion Polymorphisms in Humans

As a consequence of the accumulation of insertion events over evolutionary time, mobile elements now comprise nearly half of the human genome. The Alu, L1, and SVA mobile element families are still duplicating, generating variation between individual genomes. Mobile element insertions (MEI) have been identified as causes for genetic diseases, including hemophilia, neurofibromatosis, and various cancers. Here we present a comprehensive map of 7,380 MEI polymorphisms from the 1000 Genomes Project whole-genome sequencing data of 185 samples in three major populations detected with two detection methods. This catalog enables us to systematically study mutation rates, population segregation, genomic distribution, and functional properties of MEI polymorphisms and to compare MEI to SNP variation from the same individuals. Population allele frequencies of MEI and SNPs are described, broadly, by the same neutral ancestral processes despite vastly different mutation mechanisms and rates, except in coding regions where MEI are virtually absent, presumably due to strong negative selection. A direct comparison of MEI and SNP diversity levels suggests a differential mobile element insertion rate among populations

An Improved Protocol for Sequencing of Repetitive Genomic Regions and Structural Variations Using Mutagenesis and Next Generation Sequencing

<div><p>The rise of Next Generation Sequencing (NGS) technologies has transformed <em>de novo</em> genome sequencing into an accessible research tool, but obtaining high quality eukaryotic genome assemblies remains a challenge, mostly due to the abundance of repetitive elements. These also make it difficult to study nucleotide polymorphism in repetitive regions, including certain types of structural variations. One solution proposed for resolving such regions is Sequence Assembly aided by Mutagenesis (SAM), which relies on the fact that introducing enough random mutations breaks the repetitive structure, making assembly possible. Sequencing many different mutated copies permits the sequence of the repetitive region to be inferred by consensus methods. However, this approach relies on molecular cloning in order to isolate and amplify individual mutant copies, making it hard to scale-up the approach for use in conjunction with high-throughput sequencing technologies. To address this problem, we propose NG-SAM, a modified version of the SAM protocol that relies on PCR and dilution steps only, coupled to a NGS workflow. NG-SAM therefore has the potential to be scaled-up, e.g. using emerging microfluidics technologies. We built a realistic simulation pipeline to study the feasibility of NG-SAM, and our results suggest that under appropriate experimental conditions the approach might be successfully put into practice. Moreover, our simulations suggest that NG-SAM is capable of reconstructing robustly a wide range of potential target sequences of varying lengths and repetitive structures.</p> </div

DELLY: structural variant discovery by integrated paired‐end and split‐read analysis. Bioinformatics 28:i333‐i339.

ABSTRACT Motivation: The discovery of genomic structural variants (SVs) at high sensitivity and specificity is an essential requirement for characterizing naturally occurring variation and for understanding pathological somatic rearrangements in personal genome sequencing data. Of particular interest are integrated methods that accurately identify simple and complex rearrangements in heterogeneous sequencing datasets at single-nucleotide resolution, as an optimal basis for investigating the formation mechanisms and functional consequences of SVs. Results: We have developed an SV discovery method, called DELLY, that integrates short insert paired-ends, long-range matepairs and split-read alignments to accurately delineate genomic rearrangements at single-nucleotide resolution. DELLY is suitable for detecting copy-number variable deletion and tandem duplication events as well as balanced rearrangements such as inversions or reciprocal translocations. DELLY, thus, enables to ascertain the full spectrum of genomic rearrangements, including complex events. On simulated data, DELLY compares favorably to other SV prediction methods across a wide range of sequencing parameters. On real data, DELLY reliably uncovers SVs from the 1000 Genomes Project and cancer genomes, and validation experiments of randomly selected deletion loci show a high specificity

Performance of NG-SAM in simulated experiments.

<p>The hexagons are colored according to the mean of the metrics from all covered simulated experiments. White areas represent unexplored parameter space. <b>A</b>. The percentage of successful simulated experiments in the first simulation setting, as a function of length and number of repetitive units. The black circle [at the point (3813, 3)] marks the repetitive structure of the target region used in the second simulation setting. The dashed line corresponds to target regions with a total size of 10 kb. <b>B</b>. Percentage of correctly reconstructed bases in the successful experiments from the first simulation setting, as a function of length and number of repetitive units in the target sequence (black circle and dashed line as in <b>A</b>). <b>C</b>. The percentage of successful simulated experiments in the second simulation setting, as a function of the dilution factors ( and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043359#pone-0043359-g002" target="_blank">Figure 2</a>). The black circle corresponds to the dilution factors used in the first simulation setting. <b>D</b>. Percentage of correctly reconstructed bases in the second simulation setting as a function of the dilution factors. Black circle as in <b>C</b>; see text for further details.</p

Overview of the simulated NG-SAM protocol.

<p>The numbering corresponds to the steps enumerated above in the main text. The trapezoids shaded in light blue represent PCR amplifications (with – being the number of cycles), while the rectangles shaded in yellow represent sampling of molecules by dilution. – are the number of molecules present in the various stages of the simulated experiment, with unique variants symbolised by different coloured dots. and are the dilution factors corresponding to the first and second dilution steps. The black lines represent the “lineages" of the molecules sampled by the second dilution, traced back to the initial molecule pool of size . The steps <b>A</b>–<b>C</b> correspond to the mutagenic PCR, dilution and cleanup PCR steps of the mutagenic protocol. simNGS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043359#pone.0043359-simNGS1" target="_blank">[35]</a> is a software for simulating Illumina sequencing and Velvet <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043359#pone.0043359-Zerbino1" target="_blank">[8]</a> is a short read assembler.</p