Haplotype-resolved diverse human genomes and integrated analysis of structural variation
Peter Ebert et al
Science 25 Feb 2021: eabf7117
https://science.sciencemag.org/conte...e.abf7117.full
Abstract
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 contig N50: 26 Mbp) integrate all forms of genetic variation even across complex loci. We identify 107,590 structural variants (SVs), of which 68% are not discovered by short-read sequencing, and 278 SV hotspots (spanning megabases of gene-rich sequence). We characterize 130 of the most active mobile element source elements and find that 63% of all SVs arise by homology-mediated mechanisms. This resource enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1,526 expression quantitative trait loci as well as SV candidates for adaptive selection within the human population.
Quotes from the article:
"The Human Genome Structural Variation Consortium (HGSVC) recently developed a method for phased genome assembly that combines long-read PacBio whole-genome sequencing (WGS) and Strand-seq data to produce fully phased diploid genome assemblies without dependency on parent–child trio data (Fig. 1A) (3). These phased assemblies enable a more complete sequence-resolved representation of variation in human genomes."
"Here, we present a resource consisting of phased genome assemblies, corresponding to 70 haplotypes (64 unrelated and 6 children) from a diverse panel of human genomes. We focus specifically on the discovery of novel SVs performing extensive orthogonal validation using supporting technologies with the goal of comprehensively understanding SV complexity, including in regions that cannot yet be resolved by long-read sequencing (fig. S1). Further, we genotype these newly defined SVs using a pangenome graph framework (12–14) into a diversity panel of human genomes now deeply sequenced (>30-fold) with short-read data from the 1000 Genomes Project (1000GP) (15, 16). These findings allow us to establish their population frequency, identify ancestral haplotypes, and discover new associations with respect to gene expression, splicing, and candidate disease loci. The work provides fundamental new insights into the structure, variation, and mutation of the human genome providing a framework for more systematic analyses of thousands of human genomes going forward."
"We have generated a diversity panel of phased long-read human genome assemblies that has significantly improved SV discovery and will serve as the basis to construct new population-specific references. Previous large-scale efforts have largely been inferential and biased when it comes to the detection of SVs. Here, we develop a method to discover all forms of genetic variation (PAV) directly by comparison of assembled human genomes. In contrast, SV discovery from the 1000GP was indirect and limited given the frequent proximity of SVs to repeat sequences inaccessible to short reads (15, 23). The 1000GP, for example, reported 69,000 SVs based on the analysis of 2,504 short-read sequenced genomes. In contrast, our analysis of 32 genomes (64 unrelated haplotypes) recovers 107,136 SVs, more than tripling the rate of discovery when compared to short-read Illumina SV analyses on the same samples (Fig. 2D). Recent large-scale short-read sequencing studies (5, 6), interrogating tens of thousands of samples, show even lower SV sensitivity reporting 5,000 to 10,000 SVs per sample, when compared to our phased-assembly approach, which identifies 23,000 to 28,000 SVs per sample. This lack of sensitivity for SV discovery from short reads also affects common variation (AF>5%) and we increase the amount of common SVs by 2.6-fold. The predominant source of this increase in sensitivity was among small SVs (<250 bp) localized to SDs and simple repeat sequences, where we observed a dramatic 8.4-fold increase in variant discovery (12,109 SVs per genome from long-read assembly, 1,444 per genome from Illumina short-read alignment; Fig. 5C). Notably, all discovered genetic variation is physically phased and therefore SVs are fully integrated with their flanking SNVs."
"Compared to previous reports based on short-read sequencing (25–27), a surprising finding has been the larger fraction of SVs (63%) now assigned to homology-based (>50 bp) mutation mechanisms, including HDR, NAHR and VNTR. Breakpoint characterization with short-read data apparently biased early reports toward relatively unique regions concluding that <30% of SVs were driven by homology-based mutational mechanisms (25–27). Since a majority of unresolved structural variation still maps to large repeats, including centromeres and SDs subject to NAHR, we conclude that homology-based mutational mechanisms will contribute even further and are, therefore, the most predominant mode shaping the SV germline mutational landscape. Notwithstanding, access to fully assembled retrotransposons and their flanking sequence provides the largest collection of annotated source elements for both L1 and SVA mobile elements. We find that 14% of SVA insertions are associated with transductions compared to 8% of L1s—a difference driven in part by the proclivity of SVAs to transduce sequences at their 5ʹ and 3ʹ ends. We find a surprisingly large number of L1 source elements (19%) with defective ORFs suggesting either trans-complementation (56) or polymorphisms leading to the recent demise of these active source elements. Of note, some of the youngest L1 copies (e.g., 6p22.1-1 and 2q24.1) have been reported to be rare polymorphisms able to mediate massive bursts of somatic retrotransposition in cancer genomes (57). This suggests that recently acquired hot L1s, which have not yet reached an equilibrium with our species, contribute disproportionately to disease-causing variation (58)."
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, usually caused by Mycobacterium tuberculosis bacteria, is the first cause of death from an infectious disease at the worldwide scale, yet the mode and tempo of TB pressure on humans remain unknown. The recent discovery that homozygotes for the P1104A polymorphism of TYK2 are at higher risk to develop clinical forms of TB provided the first evidence of a common, monogenic predisposition to TB, offering a unique opportunity to inform on human co-evolution with a deadly pathogen. Here, we investigate the history of human exposure to TB by determining the evolutionary trajectory of the TYK2 P1104A variant in Europe, where TB is considered to be the deadliest documented infectious disease. Leveraging a large dataset of 1,013 ancient human genomes and using an approximate Bayesian computation approach, we find that the P1104A variant originated in the common ancestors of West Eurasians ∼30,000 years ago. Furthermore, we show that, following large-scale population movements of Anatolian Neolithic farmers and Eurasian steppe herders into Europe, P1104A has markedly fluctuated in frequency over the last 10,000 years of European history, with a dramatic decrease in frequency after the Bronze Age. Our analyses indicate that such a frequency drop is attributable to strong negative selection starting ∼2,000 years ago, with a relative fitness reduction on homozygotes of 20%, among the highest in the human genome. Together, our results provide genetic evidence that TB has imposed a heavy burden on European health over the last two millennia.
