In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 ...insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity.
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•We sequence resolve and annotate 99,604 common human structural variants•55% of VNTRs map to the end of chromosomes and correlate with double-strand breaks•Alternate alleles facilitate accurate genotyping with short reads and new associations•We patch the reference and add diversity needed for developing a pan human genome
Long-read sequencing allows generation of a large catalog of human structural variants and the development of an algorithm for genotyping SVs from short-read data, clarifying the spectrum and importance of structural variation in the human genome.
High-quality genome assemblies and sophisticated algorithms have increased sensitivity for a wide range of variant types, and breakpoint accuracy for structural variants (SVs, ≥50 bp) has improved to ...near base pair precision. Despite these advances, many SV breakpoint locations are subject to systematic bias affecting variant representation. To understand why SV breakpoints are inconsistent across samples, we reanalyzed 64 phased haplotypes constructed from long-read assemblies released by the Human Genome Structural Variation Consortium (HGSVC). We identify 882 SV insertions and 180 SV deletions with variable breakpoints not anchored in tandem repeats (TRs) or segmental duplications (SDs). SVs called from aligned sequencing reads increase breakpoint disagreements by 2×-16×. Sequence accuracy had a minimal impact on breakpoints, but we observe a strong effect of ancestry. We confirm that SNP and indel polymorphisms are enriched at shifted breakpoints and are also absent from variant callsets. Breakpoint homology increases the likelihood of imprecise SV calls and the distance they are shifted, and tandem duplications are the most heavily affected SVs. Because graph genome methods normalize SV calls across samples, we investigated graphs generated by two different methods and find the resulting breakpoints are subject to other technical biases affecting breakpoint accuracy. The breakpoint inconsistencies we characterize affect ∼5% of the SVs called in a human genome and can impact variant interpretation and annotation. These limitations underscore a need for algorithm development to improve SV databases, mitigate the impact of ancestry on breakpoints, and increase the value of callsets for investigating breakpoint features.
We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current ...Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.
Typical genotyping workflows map reads to a reference genome before identifying genetic variants. Generating such alignments introduces reference biases and comes with substantial computational ...burden. Furthermore, short-read lengths limit the ability to characterize repetitive genomic regions, which are particularly challenging for fast k-mer-based genotypers. In the present study, we propose a new algorithm, PanGenie, that leverages a haplotype-resolved pangenome reference together with k-mer counts from short-read sequencing data to genotype a wide spectrum of genetic variation-a process we refer to as genome inference. Compared with mapping-based approaches, PanGenie is more than 4 times faster at 30-fold coverage and achieves better genotype concordances for almost all variant types and coverages tested. Improvements are especially pronounced for large insertions (≥50 bp) and variants in repetitive regions, enabling the inclusion of these classes of variants in genome-wide association studies. PanGenie efficiently leverages the increasing amount of haplotype-resolved assemblies to unravel the functional impact of previously inaccessible variants while being faster compared with alignment-based workflows.
Abstract
Motivation
The standard protocol for detecting variation in DNA is to map millions of short sequence reads to a known reference and find loci that differ. While this approach works well, it ...cannot be applied where the sample contains dense variants or is too distant from known references. De novo assembly or hybrid methods can recover genomic variation, but the cost of computation is often much higher. We developed a novel k-mer algorithm and software implementation, Kestrel, capable of characterizing densely packed SNPs and large indels without mapping, assembly or de Bruijn graphs.
Results
When applied to mosaic penicillin binding protein (PBP) genes in Streptococcus pneumoniae, we found near perfect concordance with assembled contigs at a fraction of the CPU time. Multilocus sequence typing (MLST) with this approach was able to bypass de novo assemblies. Kestrel has a very low false-positive rate when applied to the whole genome, and while Kestrel identified many variants missed by other methods, limitations of a purely k-mer based approach affect overall sensitivity.
Availability and implementation
Source code and documentation for a Java implementation of Kestrel can be found at https://github.com/paudano/kestrel. All test code for this publication is located at https://github.com/paudano/kescases.
Supplementary information
Supplementary data are available at Bioinformatics online.
The sequence and assembly of human genomes using long‐read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, ...continuity, and gene annotation of genome assemblies generated from either high‐fidelity (HiFi) or continuous long‐read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5‐fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective standalone technology for de novo assembly of human genomes.
Transposable elements constitute about half of human genomes, and their role in generating human variation through retrotransposition is broadly studied and appreciated. Structural variants mediated ...by transposons, which we call transposable element-mediated rearrangements (TEMRs), are less well studied, and the mechanisms leading to their formation as well as their broader impact on human diversity are poorly understood. Here, we identify 493 unique TEMRs across the genomes of three individuals. While homology directed repair is the dominant driver of TEMRs, our sequence-resolved TEMR resource allows us to identify complex inversion breakpoints, triplications or other high copy number polymorphisms, and additional complexities. TEMRs are enriched in genic loci and can create potentially important risk alleles such as a deletion in TRIM65, a known cancer biomarker and therapeutic target. These findings expand our understanding of this important class of structural variation, the mechanisms responsible for their formation, and establish them as an important driver of human diversity.
Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent-child trio data. We present 64 assembled ...haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average minimum contig length needed to cover 50% of the genome: 26 million base pairs) integrate all forms of genetic variation, even across complex loci. We identified 107,590 structural variants (SVs), of which 68% were not discovered with short-read sequencing, and 278 SV hotspots (spanning megabases of gene-rich sequence). We characterized 130 of the most active mobile element source elements and found that 63% of all SVs arise through homology-mediated mechanisms. This resource enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1526 expression quantitative trait loci as well as SV candidates for adaptive selection within the human population.
Human genomes are typically assembled as consensus sequences that lack information on parental haplotypes. Here we describe a reference-free workflow for diploid de novo genome assembly that combines ...the chromosome-wide phasing and scaffolding capabilities of single-cell strand sequencing
with continuous long-read or high-fidelity
sequencing data. Employing this strategy, we produced a completely phased de novo genome assembly for each haplotype of an individual of Puerto Rican descent (HG00733) in the absence of parental data. The assemblies are accurate (quality value > 40) and highly contiguous (contig N50 > 23 Mbp) with low switch error rates (0.17%), providing fully phased single-nucleotide variants, indels and structural variants. A comparison of Oxford Nanopore Technologies and Pacific Biosciences phased assemblies identified 154 regions that are preferential sites of contig breaks, irrespective of sequencing technology or phasing algorithms.
Complex structural variants (CSVs) encompass multiple breakpoints and are often missed or misinterpreted. We developed SVision, a deep-learning-based multi-object-recognition framework, to ...automatically detect and haracterize CSVs from long-read sequencing data. SVision outperforms current callers at identifying the internal structure of complex events and has revealed 80 high-quality CSVs with 25 distinct structures from an individual genome. SVision directly detects CSVs without matching known structures, allowing sensitive detection of both common and previously uncharacterized complex rearrangements.
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