A flexible and efficient template format for circular consensus sequencing and SNP detection

Travers, Kevin; Chin, Chen-Shan; Rank, David R.; Eid, John; Turner, Stephen W.

doi:10.1093/nar/gkq543

Cited by 382 publications

(324 citation statements)

References 18 publications

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“…SMRTbell template libraries were prepared as previously described (53,54). SMRT sequencing was carried out on the PacBio RSII instrument using standard protocols for large insert SMRTbell libraries.…”

Section: Methodsmentioning

confidence: 99%

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk

O’Connor

Fung

Sharp

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

130

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Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.Teredinidae | endosymbionts | symbiosis | xylotrophy | carbohydrate-active enzymes

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Section: Methodsmentioning

confidence: 99%

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk

O’Connor

Fung

Sharp

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

130

View full text Add to dashboard Cite

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“…The "1D" mode sequences only the template strand, whereas the "2D" mode sequences both the template and complement strands via a hairpin adapter. This technique is similar to PacBio circular consensus sequencing (CCS) (Travers et al 2010). Because the 2D mode provides two independent observations of each base, the per-read accuracy is improved (e.g., from 70% to 86% for R7.3 chemistry) (Fig.…”

Section: Nanopore Sequence Assemblymentioning

confidence: 99%

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

Koren

Walenz

Berlin³

et al. 2017

Genome Res.

5,881

4,185

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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 Pacific Biosciences (PacBio) or Oxford Nanopore technologies and achieves a contig NG50 of >21 Mbp on both human and Drosophila melanogaster PacBio data sets. 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.

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“…SMRTbell libraries were prepared for each sample by ligation of hairpin adaptors at both the ends (Travers et al 2010), using PacBio DNA Template Prep Kit 2.0 (3-10 kbp) for SMRT sequencing with C2 chemistry on the PacBio RS according to manufacturer instructions. Libraries were purified using (0.453) Agencourt AMPure beads to remove short sheared inserts <1.5 kbp.…”

Section: Pacbio Sequencingmentioning

confidence: 99%

Reconstructing complex regions of genomes using long-read sequencing technology

et al. 2014

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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.

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A flexible and efficient template format for circular consensus sequencing and SNP detection

Cited by 382 publications

References 18 publications

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk

Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk

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

Reconstructing complex regions of genomes using long-read sequencing technology

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