De novo assembly of haplotype-resolved genomes with trio binning

Koren, Sergey; Rhie, Arang; Walenz, Brian P.; Dilthey, Alexander; Bickhart, Derek M.; Kingan, Sarah B.; Hiendleder, Stefan; Williams, J. L.; Smith, Timothy P. L.; Phillippy, Adam M.

doi:10.1038/nbt.4277

Cited by 435 publications

(568 citation statements)

References 58 publications

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“…While the haploid source material has been extremely useful for benchmarking, the ultimate challenge is the accurate assembly of human diploid genomes where both chromosomal haplotypes are fully resolved. Incorporation of linking-read technologies, such as Strand-seq, Hi-C, and 10X Genomics, or trio-binning approaches have been shown to significantly improve phasing and SV sequence and assembly Koren et al, 2018;Kronenberg et al, 2019) . It is likely that such approaches 6 could be combined with HiFi datasets to enhance telomere-to-telomere phasing and improve the accuracy of more complex repeats.…”

Section: Discussionmentioning

confidence: 99%

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

Logsdon

Audano

Sulovari

et al. 2019

Preprint

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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.

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Section: Discussionmentioning

confidence: 99%

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

Logsdon

Audano

Sulovari

et al. 2019

Preprint

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“…The ability to generate genomes de novo from field-collected arthropods makes high-quality genomes accessible for many more species. This approach also enables comprehensive comparisons of genetic diversity within and between populations without the bias from single reference-based studies [15] and allows generation of a diploid genome assembly that more closely captures the organism's biology [22]. Library preparation and sequencing.…”

Section: Discussionmentioning

confidence: 99%

“…An additional advantage to single-insect assemblies is that genome assembly for a diploid sample is algorithmically simpler than for a sample of many pooled individuals, each of which may contribute up to two unique haplotypes. Several de novo assembly methods are available for diploid samples [21,22,33], and have been broadly applied taxonomically [5,34]. Recent work indicates that assembly of high-heterozygosity samples is more accurate than for inbred samples when parental data can be used to partition long-read sequence data by haplotype, an approach called trio-binning [22,35].…”

Section: Nilaparvata Lugensmentioning

confidence: 99%

“…Pooling introduces multiple haplotypes into the sample and complicates the assembly and curation process [19], and while this issue may be ameliorated by inbreeding it is not always an option for organisms that cannot be cultured in the laboratory. Moreover, genomic regions with high heterozygosity tend to be assembled into more fragmented contigs [20], so computational methods specifically developed for heterozygous samples are needed [21][22][23]. Recently, high-quality long-read assemblies have been published for a single diploid mosquito (Anopheles coluzzii) [24] and a single haploid honeybee (Apis mellifera) [7].…”

Section: Introductionmentioning

confidence: 99%

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A High-Quality Genome Assembly from a Single, Field-collected Spotted Lanternfly (Lycorma delicatula) using the PacBio Sequel II System

Kingan

Urban

Lambert

et al. 2019

Preprint

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A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies, however, long-read methods have historically had greater input DNA requirements and higher costs than next generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female Spotted Lanternfly (Lycorma delicatula) using a single PacBio SMRT Cell. The Spotted Lanternfly is an invasive species recently discovered in the northeastern United States, threatening to damage economically important crop plants in the region. The DNA from one individual was used to make one standard, size-selected library with an average DNA fragment size of ~20 kb. The library was run on one Sequel II SMRT Cell 8M, generating a total of 132 Gb of long-read sequences, of which 82 Gb were from

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“…Global haplotype partitioning of reads prior to assembly was shown to be possible by using trio data in conjunction with long reads. An approach that leverages parent-specific k-mers for this has been pioneered by Koren et al 10 However, such parental sequencing data are not always available, especially in clinical settings. Combining Hi-C data with long reads towards single-individual phased assembly has shown considerable promise 11,12 , but reliable scaffolding and phasing across the entire chromosomes still remains challenging.…”

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confidence: 99%

A fully phased accurate assembly of an individual human genome

Porubský

Ebert

Audano

et al. 2019

Preprint

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Figure 1: Conceptual overview of the complete genome assembly pipeline. a) In a single Strand-seq library only the template DNA strand (solid line) is sequenced for each parental homologous chromosome. b) Template strands of each homologue in a given diploid cell are randomly inherited by daughter cells ('+' positive strand, teal -Crick and '-' negative strand, orange -Watson), resulting in three possible template-strand states for homologous chromosomes (height of bars plotted along each chromosome represents the number of '+' and '-' reads mapped in each genomic bin): WC -one Crick and one Watson strand represented for given homologues; WW -only Watson template strands represented; or CC -only Crick template strands represented. c) Unassigned contigs follow the same pattern of template-strand-state inheritance based on the homologue they belong to. d) Contig order can be inferred based on low frequency changes in a templatestrand state resulting from sister chromatid exchange events (SCEs) in the parental cell: Contigs that are closer to each other tend to share the same template-strand state more often than more distant contigs. e) Regions with WC strand-state are haplotype informative and can be assembled into continuous haplotypes. f) Haplotypes can then be used to split long reads into their respective homologues. g) Generation of long-read (HiFi/CLR) based assemblies: i) Producing collapsed assemblies; ii) Assigning contigs to clusters using Strand-seq (StrandS); iii) Phasing clustered assemblies using the combination of Strand-seq and long PacBio reads; iv) Partitioning and reassembling of haplotype-specific PacBio reads and polishing of the final diploid assemblies.

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De novo assembly of haplotype-resolved genomes with trio binning

Cited by 435 publications

References 58 publications

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

A High-Quality Genome Assembly from a Single, Field-collected Spotted Lanternfly (Lycorma delicatula) using the PacBio Sequel II System

A fully phased accurate assembly of an individual human genome

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