Despite their importance in disease and evolution, highly identical segmental duplications (SDs) are among the last regions of the human reference genome (GRCh38) to be fully sequenced. Using a ...complete telomere-to-telomere human genome (T2T-CHM13), we present a comprehensive view of human SD organization. SDs account for nearly one-third of the additional sequence, increasing the genome-wide estimate from 5.4 to 7.0% 218 million base pairs (Mbp). An analysis of 268 human genomes shows that 91% of the previously unresolved T2T-CHM13 SD sequence (68.3 Mbp) better represents human copy number variation. Comparing long-read assemblies from human (
= 12) and nonhuman primate (
= 5) genomes, we systematically reconstruct the evolution and structural haplotype diversity of biomedically relevant and duplicated genes. This analysis reveals patterns of structural heterozygosity and evolutionary differences in SD organization between humans and other primates.
We have developed a computational method based on polyploid phasing of long sequence reads to resolve collapsed regions of segmental duplications within genome assemblies. Segmental Duplication ...Assembler (SDA; https://github.com/mvollger/SDA ) constructs graphs in which paralogous sequence variants define the nodes and long-read sequences provide attraction and repulsion edges, enabling the partition and assembly of long reads corresponding to distinct paralogs. We apply it to single-molecule, real-time sequence data from three human genomes and recover 33-79 megabase pairs (Mb) of duplications in which approximately half of the loci are diverged (<99.8%) compared to the reference genome. We show that the corresponding sequence is highly accurate (>99.9%) and that the diverged sequence corresponds to copy-number-variable paralogs that are absent from the human reference genome. Our method can be applied to other complex genomes to resolve the last gene-rich gaps, improve duplicate gene annotation, and better understand copy-number-variant genetic diversity at the base-pair level.
Abstract
Motivation
Highly contiguous de novo phased diploid genome assemblies are now feasible for large numbers of species and individuals. Methods are needed to validate assembly accuracy and ...detect misassemblies with orthologous sequencing data to allow for confident downstream analyses.
Results
We developed GAVISUNK, an open-source pipeline that detects misassemblies and produces a set of reliable regions genome-wide by assessing concordance of distances between unique k-mers in Pacific Biosciences high-fidelity assemblies and raw Oxford Nanopore Technologies reads.
Availability and implementation
GAVISUNK is available at https://github.com/pdishuck/GAVISUNK.
Supplementary information
Supplementary data are available at Bioinformatics online.
Abstract
TRP channel-associated factor 1/2 (TCAF1/TCAF2) proteins antagonistically regulate the cold-sensor protein TRPM8 in multiple human tissues. Understanding their significance has been ...complicated given the locus spans a gap-ridden region with complex segmental duplications in GRCh38. Using long-read sequencing, we sequence-resolve the locus, annotate full-length
TCAF
models in primate genomes, and show substantial human-specific
TCAF
copy number variation. We identify two human super haplogroups, H4 and H5, and establish that
TCAF
duplications originated ~1.7 million years ago but diversified only in
Homo sapiens
by recurrent structural mutations. Conversely, in all archaic-hominin samples the fixation for a specific H4 haplotype without duplication is likely due to positive selection. Here, our results of
TCAF
copy number expansion, selection signals in hominins, and differential
TCAF2
expression between haplogroups and high
TCAF2
and
TRPM8
expression in liver and prostate in modern-day humans imply
TCAF
diversification among hominins potentially in response to cold or dietary adaptations.
The prevalence of highly repetitive sequences within the human Y chromosome has prevented its complete assembly to date
and led to its systematic omission from genomic analyses. Here we present de ...novo assemblies of 43 Y chromosomes spanning 182,900 years of human evolution and report considerable diversity in size and structure. Half of the male-specific euchromatic region is subject to large inversions with a greater than twofold higher recurrence rate compared with all other chromosomes
. Ampliconic sequences associated with these inversions show differing mutation rates that are sequence context dependent, and some ampliconic genes exhibit evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, Yq12, is composed of alternating repeat arrays that show extensive variation in the number, size and distribution, but retain a 1:1 copy-number ratio. Finally, our data suggest that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kb away from the currently established
boundary. The availability of fully sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of traits with specific Y-chromosomal variants and garnering insights into the evolution and function of complex regions of the human genome.
We sequenced and assembled using multiple long-read sequencing technologies the genomes of chimpanzee, bonobo, gorilla, orangutan, gibbon, macaque, owl monkey, and marmoset. We identified 1,338,997 ...lineage-specific fixed structural variants (SVs) disrupting 1,561 protein-coding genes and 136,932 regulatory elements, including the most complete set of human-specific fixed differences. We estimate that 819.47 Mbp or ∼27% of the genome has been affected by SVs across primate evolution. We identify 1,607 structurally divergent regions wherein recurrent structural variation contributes to creating SV hotspots where genes are recurrently lost (e.g., CARD, C4, and OLAH gene families) and additional lineage-specific genes are generated (e.g., CKAP2, VPS36, ACBD7, and NEK5 paralogs), becoming targets of rapid chromosomal diversification and positive selection (e.g., RGPD gene family). High-fidelity long-read sequencing has made these dynamic regions of the genome accessible for sequence-level analyses within and between primate species.
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•Long-read sequence assembly of eight primate genomes•Atlas of lineage-specific and recurrent structural variation•Structurally divergent regions (SDRs) associate with lineage-specific genes•Recurrent duplications diversify primate genes and predispose to human disease
Analysis of high-quality, haplotype-resolved primate genomes provides a more complete understanding of lineage-specific, recurrent mutations and structurally divergent regions associated with primate adaptive evolution and human diseases.
The complete assembly of each human chromosome is essential for understanding human biology and evolution
. Here we use complementary long-read sequencing technologies to complete the linear assembly ...of human chromosome 8. Our assembly resolves the sequence of five previously long-standing gaps, including a 2.08-Mb centromeric α-satellite array, a 644-kb copy number polymorphism in the β-defensin gene cluster that is important for disease risk, and an 863-kb variable number tandem repeat at chromosome 8q21.2 that can function as a neocentromere. We show that the centromeric α-satellite array is generally methylated except for a 73-kb hypomethylated region of diverse higher-order α-satellites enriched with CENP-A nucleosomes, consistent with the location of the kinetochore. In addition, we confirm the overall organization and methylation pattern of the centromere in a diploid human genome. Using a dual long-read sequencing approach, we complete high-quality draft assemblies of the orthologous centromere from chromosome 8 in chimpanzee, orangutan and macaque to reconstruct its evolutionary history. Comparative and phylogenetic analyses show that the higher-order α-satellite structure evolved in the great ape ancestor with a layered symmetry, in which more ancient higher-order repeats locate peripherally to monomeric α-satellites. We estimate that the mutation rate of centromeric satellite DNA is accelerated by more than 2.2-fold compared to the unique portions of the genome, and this acceleration extends into the flanking sequence.
The divergence of chimpanzee and bonobo provides one of the few examples of recent hominid speciation
. Here we describe a fully annotated, high-quality bonobo genome assembly, which was constructed ...without guidance from reference genomes by applying a multiplatform genomics approach. We generate a bonobo genome assembly in which more than 98% of genes are completely annotated and 99% of the gaps are closed, including the resolution of about half of the segmental duplications and almost all of the full-length mobile elements. We compare the bonobo genome to those of other great apes
and identify more than 5,569 fixed structural variants that specifically distinguish the bonobo and chimpanzee lineages. We focus on genes that have been lost, changed in structure or expanded in the last few million years of bonobo evolution. We produce a high-resolution map of incomplete lineage sorting and estimate that around 5.1% of the human genome is genetically closer to chimpanzee or bonobo and that more than 36.5% of the genome shows incomplete lineage sorting if we consider a deeper phylogeny including gorilla and orangutan. We also show that 26% of the segments of incomplete lineage sorting between human and chimpanzee or human and bonobo are non-randomly distributed and that genes within these clustered segments show significant excess of amino acid replacement compared to the rest of the genome.
The rhesus macaque (
) is the most widely studied nonhuman primate (NHP) in biomedical research. We present an updated reference genome assembly (Mmul_10, contig N50 = 46 Mbp) that increases the ...sequence contiguity 120-fold and annotate it using 6.5 million full-length transcripts, thus improving our understanding of gene content, isoform diversity, and repeat organization. With the improved assembly of segmental duplications, we discovered new lineage-specific genes and expanded gene families that are potentially informative in studies of evolution and disease susceptibility. Whole-genome sequencing (WGS) data from 853 rhesus macaques identified 85.7 million single-nucleotide variants (SNVs) and 10.5 million indel variants, including potentially damaging variants in genes associated with human autism and developmental delay, providing a framework for developing noninvasive NHP models of human disease.
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