Verkko: telomere-to-telomere assembly of diploid chromosomes

Rautiainen, Mikko; Nurk, Sergey; Walenz, Brian P.; Logsdon, Glennis A.; Porubský, David; Rhie, Arang; Eichler, Evan E.; Phillippy, Adam M.; Koren, Sergey

doi:10.1101/2022.06.24.497523

Cited by 45 publications

(58 citation statements)

References 82 publications

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“…This haplotype-specific analysis confirmed the copy number and integrity of >94% of all tested SDs. As a final control for potential haplotype-phasing errors, we generated deep ONT and HiFi data from a second hydatidiform mole (CHM1) where a single paternal haplotype was present (Chaisson et al 2015; Vollger et al 2020) and show that across our many analyses the results from the CHM1 Verkko assembly are consistent with individual haplotypes obtained from diploid HPRC samples produced by trio-hifiasm (Cheng et al 2021; Rautiainen et al 2022) (Figs. S3-S4).…”

Section: Resultsmentioning

confidence: 94%

Increased mutation rate and interlocus gene conversion within human segmental duplications

DeWitt

Dishuck

Harvey

et al. 2022

Preprint

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Single-nucleotide variants (SNVs) within segmental duplications (SDs) have not been systematically assessed because of the difficulty in mapping short-read sequence data to virtually identical repetitive sequences. Using 102 phased human haplotypes, we constructed 1:1 unambiguous alignments spanning high-identity SDs and compared the pattern of SNVs between unique and SD regions. We find that human SNVs are elevated 60% in SDs compared to unique regions. We estimate that at least 23% of this increase is due to interlocus gene conversion (IGC) with >7 Mbp of SD sequence converted on average per human haplotype. We develop a genome-wide map of IGC donors and acceptors, including 498 acceptor and 454 donor hotspots affecting the exons of ~800 protein-coding genes. The latter includes 171 genes that have "relocated" on average 1.61 Mbp in a subset of human haplotypes. Using a coalescent framework, we show that SD regions are evolutionarily older when compared to unique sequences with most of this signal originating from putative IGC loci. SNVs within SDs, however, also exhibit a distinct mutational spectrum where there is a 27.1% increase in transversions that convert cytosine to guanine or the reverse across all triplet contexts. In addition, we observe a 7.6% reduction in the frequency of CpG associated mutations when compared to unique DNA. We hypothesize that these distinct mutational properties help to maintain an overall higher GC content of SD DNA when compared to unique DNA, and we show that these GC-favoring mutational events are likely driven by GC-biased conversion between paralogous sequences.

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Section: Resultsmentioning

confidence: 94%

Increased mutation rate and interlocus gene conversion within human segmental duplications

DeWitt

Dishuck

Harvey

et al. 2022

Preprint

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“…S26, Methods ). Approaches and assembly algorithms that couple both ONT and HiFi data (e.g., Verkko (Rautiainen et al 2022)) may be necessary to close the remaining gaps and to achieve routine T2T assemblies of human genomes. The costs of generating deep long-read sequence coverage from two platforms to achieve a T2T human genome remain a significant cost (>$10,000) and throughput consideration.…”

Section: Discussionmentioning

confidence: 99%

Gaps and complex structurally variant loci in phased genome assemblies

Porubský

Harvey

Rozanski

et al. 2022

Preprint

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There has been tremendous progress in the production of phased genome assemblies by combining long-read data with parental information or linking read data. Nevertheless, a typical phased genome assembly generated by trio-hifiasm still generates more than ~140 gaps. We perform a detailed analysis of gaps, assembly breaks, and misorientations from 77 phased and assembled human genomes (154 unique haplotypes). We find that trio-based approaches using HiFi are the current gold standard although chromosome-wide phasing accuracy is comparable when using Strand-seq instead of parental data. We find two-thirds of defined contig ends cluster near the largest and most identical repeats [including segmental duplications (35.4%) or satellite DNA (22.3%) or to regions enriched in GA/AT rich DNA (27.4%)]. As a result, 1513 protein-coding genes overlap assembly gaps in at least one haplotype and 231 are recurrently disrupted or missing from five or more haplotypes. In addition, we estimate that 6-7 Mbp of DNA are incorrectly orientated per haplotype irrespective of whether trio-free or trio-based approaches are employed. 81% of such misorientations correspond to bona fide large inversion polymorphisms in the human species, most of which are flanked by large identical segmental duplications. In addition, we also identify large-scale alignment discontinuities consistent with an 11.9 Mbp deletion and 161.4 Mbp of insertion per human haploid genome. While 99% of this variation corresponds to satellite DNA, we identify 230 regions of the euchromatic DNA with frequent expansions and contractions, nearly half of which overlap with 197 protein-coding genes. Although not completely resolved, these regions include copy number polymorphic and biomedically relevant genic regions where complete resolution and a pangenome representation will be most useful, yet most challenging, to realize.

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“…A variation graph (5) constructed from centromere-spanning acrocentric contigs indicates the presence of pseudo-homologous regions where most contigs appear as recombinants of heterologous CHM13 acrocentrics. A diploid T2T assembly of a target sample cross-validates these patterns (6). On chromosomes 13, 14, 21, and 22, we observe lower levels of linkage disequilibrium in pseudo-homologous regions than in their short and long arms, indicating higher rates of recombination and/or a larger effective population size (7).…”

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confidence: 86%

Recombination between heterologous human acrocentric chromosomes

Buonaiuto¹,

Potapova²,

Rhie³

et al. 2022

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The short arms of the human acrocentric chromosomes 13, 14, 15, 21, and 22 share large homologous regions, including the ribosomal DNA repeats and extended segmental duplications. While the complete assembly of these regions in the Telomere-to-Telomere consortium's (T2T) CHM13 provided a model of their homology, it remained unclear if these patterns were ancestral or maintained by ongoing recombination exchange. Here, we use pangenomic resources and methods to show that specific pseudo-homologous regions of the acrocentrics recombine. Considering an all-to-all comparison of the high-quality human pangenome from the Human Pangenome Reference Consortium (HPRC), we find that contigs from all of the acrocentric short arms form a community similar to those formed by single chromosomes or the sex chromosome pair. A variation graph constructed from centromere-spanning acrocentric contigs indicates the presence of pseudo-homologous regions where most contigs appear as recombinants of heterologous CHM13 acrocentrics. A diploid T2T assembly of a target sample cross-validates these patterns. On chromosomes 13, 14, 21, and 22, we observe lower levels of linkage disequilibrium in pseudo-homologous regions than in their short and long arms, indicating higher rates of recombination and/or a larger effective population size. The ubiquity of signals of heterologous recombination seen in the HPRC draft pangenome's acrocentric assemblies suggests that this phenomenon is a basic feature of human cellular biology, providing sequence and population-based confirmation of hypotheses first developed cytogenetically fifty years ago.

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Verkko: telomere-to-telomere assembly of diploid chromosomes

Cited by 45 publications

References 82 publications

Increased mutation rate and interlocus gene conversion within human segmental duplications

Increased mutation rate and interlocus gene conversion within human segmental duplications

Gaps and complex structurally variant loci in phased genome assemblies

Recombination between heterologous human acrocentric chromosomes

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