Phased Diploid Genome Assembly with Single Molecule Real-Time Sequencing

Chin, Chen-Shan; Peluso, Paul; Sedlazeck, Fritz J.; Nattestad, Maria; Concepcion, Gregory T.; Clum, Alicia; Dunn, Christopher; O’Malley, Ronan; Figueroa‐Balderas, Rosa; Morales‐Cruz, Abraham; Cramer, Grant R.; Delledonne, Massimo; Luo, Chongyuan; Ecker, Joseph R.; Cantu, Dario; Rank, David R.; Schatz, Michael C.

doi:10.1101/056887

Cited by 157 publications

(213 citation statements)

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“…Additionally, ARS1 is a haplotype-mixed representation of a diploid animal. Haplotype phasing is possible using single-molecule 55 and Hi-C 56 technologies, so a future aim is to generate a phased reference assembly.…”

Section: Discussionmentioning

confidence: 99%

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

Bickhart¹,

et al. 2017

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The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus), based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ~400-fold improvement in continuity due to properly assembled gaps compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex ever produced for an individual of a ruminant species.

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Section: Discussionmentioning

confidence: 99%

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

Bickhart¹,

et al. 2017

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“…Recently, single-molecule sequencing has revolutionized assembly by producing reads longer than 10 kbp (Gordon et al 2016), which has significantly reduced the number of unresolvable repeats ) and enabled the complete assembly of microbial genomes (Chin et al 2013;Koren et al 2013;Koren and Phillippy 2014). These long reads also aid assembly phasing (Chin et al 2016), where the conserved alleles in a diploid, polyploid, or meta-genome can be thought of as a special kind of repeat. However, in contrast to improved read length, single-molecule sequencing is less accurate than past technologies (Eid et al 2009;Schneider and Dekker 2012), requiring sensitive alignment methods and limiting the discrimination of divergent alleles and non-exact repeats.…”

Section: Introductionmentioning

confidence: 99%

Canu: scalable and accurate long-read assembly via adaptivek-mer weighting and repeat separation

Koren

Walenz

Berlin³

et al. 2016

Preprint

1,136

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Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either PacBio or Oxford Nanopore technologies, and achieves a contig NG50 of greater than 21 Mbp on both human and Drosophila melanogaster PacBio datasets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes.. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a

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“…Xu et al, 2011) and from sequencing data performed on an RSII (Pacific Biosciences) using a total of 197 SMRT cells. PacBio raw subreads were extracted in FASTA format using DEXTRACTOR (Gene Myers; https://github.com/thegenemyers/DEXTRACTOR) and assembled in a diploid-aware fashion with FALCON (Chin et al, 2016). Residual errors in the assembly were corrected using quiver and pbalign (smrtpipe v. 2.3).…”

Section: Characterization Of the Cho Genome Ervsmentioning

confidence: 99%

Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

Duroy

Bosshard

Schmid‐Siegert

et al. 2019

Biotech & Bioengineering

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The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type‐C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type‐C ERV sequences clustering into three functionally conserved groups. Transcripts from one type‐C ERV group were full‐length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral‐like particles, suggesting that this ERV group may produce functional viruses. CRISPR‐Cas9 genome editing was used to disrupt the gag gene of the expressed type‐C ERV group. Comparison of CRISPR‐derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA‐loaded viral particles. Clones bearing a Gag loss‐of‐function mutation in this ERV showed a reduction of RNA‐containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.

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Phased Diploid Genome Assembly with Single Molecule Real-Time Sequencing

Cited by 157 publications

References 50 publications

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

Canu: scalable and accurate long-read assembly via adaptivek-mer weighting and repeat separation

Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

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