Peter H. Sudmant

A High-Coverage Genome Sequence from an Archaic Denisovan Individual

Ancient Genomics The Denisovans were archaic humans closely related to Neandertals, whose populations overlapped with the ancestors of modern-day humans. Using a single-stranded library preparation method, Meyer et al. (p. 222, published online 30 August) provide a detailed analysis of a high-quality Denisovan genome. The genomic sequence provides evidence for very low rates of heterozygosity in the Denisova, probably not because of recent inbreeding, but instead because of a small population size. The genome sequence also illuminates the relationships between humans and archaics, including Neandertals, and establishes a catalog of genetic changes within the human lineage. A close-up look provides clues to the relationships between modern humans, Denisovans, and Neandertals. We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.

The complete genome sequence of a Neanderthal from the Altai Mountains

We present a high-quality genome sequence of a Neanderthal woman from Siberia. We show that her parents were related at the level of half-siblings and that mating among close relatives was common among her recent ancestors. We also sequenced the genome of a Neanderthal from the Caucasus to low coverage. An analysis of the relationships and population history of available archaic genomes and 25 present-day human genomes shows that several gene flow events occurred among Neanderthals, Denisovans and early modern humans, possibly including gene flow into Denisovans from an unknown archaic group. Thus, interbreeding, albeit of low magnitude, occurred among many hominin groups in the Late Pleistocene. In addition, the high-quality Neanderthal genome allows us to establish a definitive list of substitutions that became fixed in modern humans after their separation from the ancestors of Neanderthals and Denisovans.

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

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.

Diversity of human copy number variation and multicopy genes

Evolution, Gene Number, and Disease Slight variations in the numbers of copies of genes influence human disease and other characters. Variants can be hard to detect when they lie in heavily duplicated and widely similar regions of sequence known as “dark matter.” Sudmant et al. (p. 641) have methods to tease apart the duplicated regions to reveal singly unique nucleotide identifiers. These have turned out to be among the most variable seen in different human population groups—most notably among genes for neurodevelopment and neurological diseases. Such polymorphisms can be genotyped with specificity and may help us understand how variation in copy number may affect human evolution and disease. Specific gene copies can be identified in regions of high copy number variability in the human genome. Copy number variants affect both disease and normal phenotypic variation, but those lying within heavily duplicated, highly identical sequence have been difficult to assay. By analyzing short-read mapping depth for 159 human genomes, we demonstrated accurate estimation of absolute copy number for duplications as small as 1.9 kilobase pairs, ranging from 0 to 48 copies. We identified 4.1 million “singly unique nucleotide” positions informative in distinguishing specific copies and used them to genotype the copy and content of specific paralogs within highly duplicated gene families. These data identify human-specific expansions in genes associated with brain development, reveal extensive population genetic diversity, and detect signatures consistent with gene conversion in the human species. Our approach makes ~1000 genes accessible to genetic studies of disease association.

Great ape genetic diversity and population history

Most great ape genetic variation remains uncharacterized; however, its study is critical for understanding population history, recombination, selection and susceptibility to disease. Here we sequence to high coverage a total of 79 wild- and captive-born individuals representing all six great ape species and seven subspecies and report 88.8 million single nucleotide polymorphisms. Our analysis provides support for genetically distinct populations within each species, signals of gene flow, and the split of common chimpanzees into two distinct groups: Nigeria–Cameroon/western and central/eastern populations. We find extensive inbreeding in almost all wild populations, with eastern gorillas being the most extreme. Inferred effective population sizes have varied radically over time in different lineages and this appears to have a profound effect on the genetic diversity at, or close to, genes in almost all species. We discover and assign 1,982 loss-of-function variants throughout the human and great ape lineages, determining that the rate of gene loss has not been different in the human branch compared to other internal branches in the great ape phylogeny. This comprehensive catalogue of great ape genome diversity provides a framework for understanding evolution and a resource for more effective management of wild and captive great ape populations.

Copy number variation detection and genotyping from exome sequence data

While exome sequencing is readily amenable to single-nucleotide variant discovery, the sparse and nonuniform nature of the exome capture reaction has hindered exome-based detection and characterization of genic copy number variation. We developed a novel method using singular value decomposition (SVD) normalization to discover rare genic copy number variants (CNVs) as well as genotype copy number polymorphic (CNP) loci with high sensitivity and specificity from exome sequencing data. We estimate the precision of our algorithm using 122 trios (366 exomes) and show that this method can be used to reliably predict (94% overall precision) both de novo and inherited rare CNVs involving three or more consecutive exons. We demonstrate that exome-based genotyping of CNPs strongly correlates with whole-genome data (median r(2) = 0.91), especially for loci with fewer than eight copies, and can estimate the absolute copy number of multi-allelic genes with high accuracy (78% call level). The resulting user-friendly computational pipeline, CoNIFER (copy number inference from exome reads), can reliably be used to discover disruptive genic CNVs missed by standard approaches and should have broad application in human genetic studies of disease.

Haplotype-resolved genome sequencing of a Gujarati Indian individual

Haplotype information is essential to the complete description and interpretation of genomes, genetic diversity and genetic ancestry. Although individual human genome sequencing is increasingly routine, nearly all such genomes are unresolved with respect to haplotype. Here we combine the throughput of massively parallel sequencing with the contiguity information provided by large-insert cloning to experimentally determine the haplotype-resolved genome of a South Asian individual. A single fosmid library was split into a modest number of pools, each providing ∼3% physical coverage of the diploid genome. Sequencing of each pool yielded reads overwhelmingly derived from only one homologous chromosome at any given location. These data were combined with whole-genome shotgun sequence to directly phase 94% of ascertained heterozygous single nucleotide polymorphisms (SNPs) into long haplotype blocks (N50 of 386 kilobases (kbp)). This method also facilitates the analysis of structural variation, for example, to anchor novel insertions to specific locations and haplotypes.

Reconstructing complex regions of genomes using long-read sequencing technology

Obtaining high-quality sequence continuity of complex regions of recent segmental duplication remains one of the major challenges of finishing genome assemblies. In the human and mouse genomes, this was achieved by targeting large-insert clones using costly and laborious capillary-based sequencing approaches. Sanger shotgun sequencing of clone inserts, however, has now been largely abandoned, leaving most of these regions unresolved in newer genome assemblies generated primarily by next-generation sequencing hybrid approaches. Here we show that it is possible to resolve regions that are complex in a genome-wide context but simple in isolation for a fraction of the time and cost of traditional methods using long-read single molecule, real-time (SMRT) sequencing and assembly technology from Pacific Biosciences (PacBio). We sequenced and assembled BAC clones corresponding to a 1.3-Mbp complex region of chromosome 17q21.31, demonstrating 99.994% identity to Sanger assemblies of the same clones. We targeted 44 differences using Illumina sequencing and find that PacBio and Sanger assemblies share a comparable number of validated variants, albeit with different sequence context biases. Finally, we targeted a poorly assembled 766-kbp duplicated region of the chimpanzee genome and resolved the structure and organization for a fraction of the cost and time of traditional finishing approaches. Our data suggest a straightforward path for upgrading genomes to a higher quality finished state.

Estimating the human mutation rate using autozygosity in a founder population

Knowledge of the rate and pattern of new mutation is critical to the understanding of human disease and evolution. We used extensive autozygosity in a genealogically well-defined population of Hutterites to estimate the human sequence mutation rate over multiple generations. We sequenced whole genomes from 5 parent-offspring trios and identified 44 segments of autozygosity. Using the number of meioses separating each pair of autozygous alleles and the 72 validated heterozygous single-nucleotide variants (SNVs) from 512 Mb of autozygous DNA, we obtained an SNV mutation rate of 1.20 × 10−8 (95% confidence interval 0.89–1.43 × 10−8) mutations per base pair per generation. The mutation rate for bases within CpG dinucleotides (9.72 × 10−8) was 9.5-fold that of non-CpG bases, and there was strong evidence (P = 2.67 × 10−4) for a paternal bias in the origin of new mutations (85% paternal). We observed a non-uniform distribution of heterozygous SNVs (both newly identified and known) in the autozygous segments (P = 0.001), which is suggestive of mutational hotspots or sites of long-range gene conversion.

Global diversity, population stratification, and selection of human copy-number variation.

Duplications and deletions in the human genome Duplications and deletions can lead to variation in copy number for genes and genomic loci among humans. Such variants can reveal evolutionary patterns and have implications for human health. Sudmant et al. examined copy-number variation across 236 individual genomes from 125 human populations. Deletions were under more selection, whereas duplications showed more population-specific structure. Interestingly, Oceanic populations retain large duplications postulated to have originated in an ancient Denisovan lineage. Science, this issue 10.1126/science.aab3761 Copy-number variation reveals how selection affects the human genome across the globe. INTRODUCTION Most studies of human genetic variation have focused on single-nucleotide variants (SNVs). However, copy-number variants (CNVs) affect more base pairs of DNA among humans, and yet our understanding of CNV diversity among human populations is limited. RATIONALE We aimed to understand the pattern, selection, and diversity of copy-number variation by analyzing deeply sequenced genomes representing the diversity of all humans. We compared the selective constraints of deletions versus duplications to understand population stratification in the context of the ancestral human genome and to assess differences in CNV load between African and non-African populations. RESULTS We sequenced 236 individual genomes from 125 distinct human populations and identified 14,467 autosomal CNVs and 545 X-linked CNVs with a sequence read-depth approach. Deletions exhibit stronger selective pressure and are better phylogenetic markers of population relationships than duplication polymorphisms. We identified 1036 population-stratified copy-number–variable regions, 295 of which intersect coding regions and 199 of which exhibit extreme signatures of differentiation. Duplicated loci were 1.8-fold more likely to be stratified than deletions but were poorly correlated with flanking genetic diversity. Among these, we highlight a duplication polymorphism restricted to modern Oceanic populations yet also present in the genome of the archaic Denisova hominin. This 225–kilo–base pair (kbp) duplication includes two microRNA genes and is almost fixed among human Papuan-Bougainville genomes. The data allowed us to reconstruct the ancestral human genome and create a more accurate evolutionary framework for the gain and loss of sequences during human evolution. We identified 571 loci that segregate in the human population and another 2026 loci of fixed-copy 2 in all human genomes but absent from the reference genome. The total deletion and duplication load between African and non-African population groups showed no difference after we account for ancestral sequences missing from the human reference. However, we did observe that the relative number of base pairs affected by CNVs compared to single-nucleotide polymorphisms is higher among non-Africans than Africans. CONCLUSION Deletions, duplications, and CNVs have shaped, to different extents, the genetic diversity of human populations by the combined forces of mutation, selection, and demography. Figure Global human CNV diversity and archaic introgression of a chromosome 16 duplication. (Left) The geographic coordinates of populations sampled are indicated on a world map (colored dots). The pie charts show the continental population allele frequency of a single ~225-kbp duplication polymorphism found exclusively among Oceanic populations and an archaic Denisova. (Right) The ancestral structure of this duplication locus (1) and the Denisova duplication structure (2) are shown in relation to their position on chromosome 16. We estimate that the duplication emerged ~440 thousand years ago (ka) in the Denisova and then introgressed into ancestral Papuan populations ~40 ka. In order to explore the diversity and selective signatures of duplication and deletion human copy-number variants (CNVs), we sequenced 236 individuals from 125 distinct human populations. We observed that duplications exhibit fundamentally different population genetic and selective signatures than deletions and are more likely to be stratified between human populations. Through reconstruction of the ancestral human genome, we identify megabases of DNA lost in different human lineages and pinpoint large duplications that introgressed from the extinct Denisova lineage now found at high frequency exclusively in Oceanic populations. We find that the proportion of CNV base pairs to single-nucleotide–variant base pairs is greater among non-Africans than it is among African populations, but we conclude that this difference is likely due to unique aspects of non-African population history as opposed to differences in CNV load.