Assembling large genomes with single-molecule sequencing and locality-sensitive hashing

Berlin, Konstantin; Koren, Sergey; Chin, Chen-Shan; Drake, James; Landolin, Jane M.; Phillippy, Adam M.

doi:10.1038/nbt.3238

Cited by 884 publications

(740 citation statements)

References 83 publications

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“…Read clustering approaches are beneficial for assembling highly similar sequences with PacBio data (11,1416). To integrate this with our assembly pipeline, we used the PacBio SMRTanalysis HGAP3 algorithm (11) with empirically determined custom parameters (see github repository: https://github.com/paajanen/RenSeq).…”

Section: Resultsmentioning

confidence: 99%

Targeted Capture and Sequencing of Gene-Sized DNA Molecules

et al. 2016

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Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family-key genes in plant immune systems.

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Section: Resultsmentioning

confidence: 99%

Targeted Capture and Sequencing of Gene-Sized DNA Molecules

et al. 2016

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“…The last step called the consensus contigs from output files from two previous steps and raw PacBio reads. In PacBio‐only assembly, first, the raw reads from the PacBio platform were corrected using the PBcR pipeline (Berlin et al ., 2015) with the self‐correction feature enabled and the minimum length of PacBio fragment to keep set to 500. The assembly was done using The Celera Assembler (CA) version 8.3rc2 (Myers et al ., 2000) leaving the parameters as default.…”

Section: Methodsmentioning

confidence: 99%

Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice

Brożyńska

Copetti

Furtado

et al. 2017

Plant Biotechnology Journal

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SummaryThe related A genome species of the Oryza genus are the effective gene pool for rice. Here, we report draft genomes for two Australian wild A genome taxa: O. rufipogon‐like population, referred to as Taxon A, and O. meridionalis‐like population, referred to as Taxon B. These two taxa were sequenced and assembled by integration of short‐ and long‐read next‐generation sequencing (NGS) data to create a genomic platform for a wider rice gene pool. Here, we report that, despite the distinct chloroplast genome, the nuclear genome of the Australian Taxon A has a sequence that is much closer to that of domesticated rice (O. sativa) than to the other Australian wild populations. Analysis of 4643 genes in the A genome clade showed that the Australian annual, O. meridionalis, and related perennial taxa have the most divergent (around 3 million years) genome sequences relative to domesticated rice. A test for admixture showed possible introgression into the Australian Taxon A (diverged around 1.6 million years ago) especially from the wild indica/O. nivara clade in Asia. These results demonstrate that northern Australia may be the centre of diversity of the A genome Oryza and suggest the possibility that this might also be the centre of origin of this group and represent an important resource for rice improvement.

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“…An essential component of SMRT sequencing is the zero-mode waveguide (ZMW) 4 , a zeptolitre-volume cylindrical cavity (~100 nm diameter and height) in which the DNA/polymerase complex is immobilised 4 . Major advantages of SMRT sequencing over second-generation sequencing methods include long average read lengths of more than 10,000 bases and lack of GC% bias 3, 5, 6 , critical for gap-free sequencing, and the ability to directly detect DNA base modifications by monitoring polymerase kinetics 2 . Apart from DNA sequencing, ZMWs have been exploited for single molecule RNA sequencing/epigenetics 7 and a variety of other single-molecule studies 8–13 .…”

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confidence: 99%

Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing

et al. 2017

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Compared to conventional methods, single molecule, real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC per cent bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-ng quantities is impractical due to inefficient delivery of DNA molecules into the confines of zero-mode waveguides, zeptolitre optical cavities in which DNA sequencing proceeds. Here we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000 bp long DNA template within seconds from a sub-ng input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.

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Assembling large genomes with single-molecule sequencing and locality-sensitive hashing

Cited by 884 publications

References 83 publications

Targeted Capture and Sequencing of Gene-Sized DNA Molecules

Targeted Capture and Sequencing of Gene-Sized DNA Molecules

Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice

Length-independent DNA packing into nanopore zero-mode waveguides for low-input DNA sequencing

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