SUMMARY 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.

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.

We combined de novo mutation (DNM) data from 10,927 cases of developmental delay and autism to identify 253 candidate neurodevelopmental disease genes with an excess of missense and/or likely gene-disruptive mutations. Of these genes, 124 reach exome-wide significance (p < 5 × 10 −7 ) for DNM. Intersecting these results with copy number variation morbidity data shows an enrichment for genomic disorder regions (30/253, LR+ 1.85, p = 0.0017). We identify genes with an excess of missense DNMs overlapping deletion syndromes (e.g., KIF1A and the 2q37 deletion) as well as duplication syndromes, such as recurrent MAPK3 missense mutations within the chromosome 16p11.2 duplication, recurrent CHD4 missense DNMs in the 12p13 duplication region, and recurrent WDFY4 missense DNMs in the 10q11.23 duplication region. Network analyses of genes showing an excess of DNMs highlights functional networks, including cell-specific enrichments in the D1+ and D2+ spiny neurons of the striatum.

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 sequencing1,2 with continuous long-read or high-fidelity3 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.

Next-generation sequencing (NGS) is now more accessible to clinicians and researchers. As a result, our understanding of the genetics of neurodevelopmental disorders (NDDs) has rapidly advanced over the past few years. NGS has led to the discovery of new NDD genes with an excess of recurrent de novo mutations (DNMs) when compared to controls. Development of large-scale databases of normal and disease variation has given rise to metrics exploring the relative tolerance of individual genes to human mutation. Genetic etiology and diagnosis rates have improved, which have led to the discovery of new pathways and tissue types relevant to NDDs. In this review, we highlight several key findings based on the discovery of recurrent DNMs ranging from copy number variants to point mutations. We explore biases and patterns of DNM enrichment and the role of mosaicism and secondary mutations in variable expressivity. We discuss the benefit of whole-genome sequencing (WGS) over whole-exome sequencing (WES) to understand more complex, multifactorial cases of NDD and explain how this improved understanding aids diagnosis and management of these disorders. Comprehensive assessment of the DNM landscape across the genome using WGS and other technologies will lead to the development of novel functional and bioinformatics approaches to interpret DNMs and drive new insights into NDD biology.

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Short tandem repeats (STRs) and variable number tandem repeats (VNTRs) are important sources of natural and disease-causing variation, yet they have been problematic to resolve in reference genomes and genotype with short-read technology. We created a framework to model the evolution and instability of STRs and VNTRs in apes. We phased and assembled 3 ape genomes (chimpanzee, gorilla, and orangutan) using long-read and 10x Genomics linked-read sequence data for 21,442 human tandem repeats discovered in 6 haplotype-resolved assemblies of Yoruban, Chinese, and Puerto Rican origin. We define a set of 1,584 STRs/VNTRs expanded specifically in humans, including large tandem repeats affecting coding and noncoding portions of genes (e.g., MUC3A, CACNA1C). We show that short interspersed nuclear element–VNTR–Alu (SVA) retrotransposition is the main mechanism for distributing GC-rich human-specific tandem repeat expansions throughout the genome but with a bias against genes. In contrast, we observe that VNTRs not originating from retrotransposons have a propensity to cluster near genes, especially in the subtelomere. Using tissue-specific expression from human and chimpanzee brains, we identify genes where transcript isoform usage differs significantly, likely caused by cryptic splicing variation within VNTRs. Using single-cell expression from cerebral organoids, we observe a strong effect for genes associated with transcription profiles analogous to intermediate progenitor cells. Finally, we compare the sequence composition of some of the largest human-specific repeat expansions and identify 52 STRs/VNTRs with at least 40 uninterrupted pure tracts as candidates for genetically unstable regions associated with disease.

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.