Reconstructing the Gigabase Plant Genome of Solanum pennellii using Nanopore Sequencing, bioRxiv, 2017-04-22

Recent updates in sequencing technology have made it possible to obtain Gigabases of sequence data from one single flowcell. Prior to this update, the nanopore sequencing technology was mainly used to analyze and assemble microbial samples1-3. Here, we describe the generation of a comprehensive nanopore sequencing dataset with a median fragment size of 11,979 bp for the wild tomato species Solanum pennellii featuring an estimated genome size of ca 1.0 to 1.1 Gbases. We describe its genome assembly to a contig N50 of 2.5 MB using a pipeline comprising a Canu4 pre-processing and a subsequent assembly using SMARTdenovo. We show that the obtained nanopore based de novo genome reconstruction is structurally highly similar to that of the reference S. pennellii LA7165 genome but has a high error rate caused mostly by deletions in homopolymers. After polishing the assembly with Illumina short read data we obtained an error rate of &lt;0.02 % when assessed versus the same Illumina data. More importantly however we obtained a gene completeness of 96.53% which even slightly surpasses that of the reference S. pennellii genome5. Taken together our data indicate such long read sequencing data can be used to affordably sequence and assemble Gbase sized diploid plant genomes.Raw data is available at <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpwww.plabipd.deportalsolanum-pennellii>httpwww.plabipd.deportalsolanum-pennellii<jatsext-link> and has been deposited as PRJEB19787.

biorxiv genomics 0-100-users 2017

Nanopore sequencing and assembly of a human genome with ultra-long reads, bioRxiv, 2017-04-21

AbstractNanopore sequencing is a promising technique for genome sequencing due to its portability, ability to sequence long reads from single molecules, and to simultaneously assay DNA methylation. However until recently nanopore sequencing has been mainly applied to small genomes, due to the limited output attainable. We present nanopore sequencing and assembly of the GM12878 UtahCeph human reference genome generated using the Oxford Nanopore MinION and R9.4 version chemistry. We generated 91.2 Gb of sequence data (∼30× theoretical coverage) from 39 flowcells. De novo assembly yielded a highly complete and contiguous assembly (NG50 ∼3Mb). We observed considerable variability in homopolymeric tract resolution between different basecallers. The data permitted sensitive detection of both large structural variants and epigenetic modifications. Further we developed a new approach exploiting the long-read capability of this system and found that adding an additional 5×-coverage of ‘ultra-long’ reads (read N50 of 99.7kb) more than doubled the assembly contiguity. Modelling the repeat structure of the human genome predicts extraordinarily contiguous assemblies may be possible using nanopore reads alone. Portable de novo sequencing of human genomes may be important for rapid point-of-care diagnosis of rare genetic diseases and cancer, and monitoring of cancer progression. The complete dataset including raw signal is available as an Amazon Web Services Open Dataset at <jatsext-link xmlnsxlink=httpwww.w3.org1999xlink ext-link-type=uri xlinkhref=httpsgithub.comnanopore-wgs-consortiumNA12878>httpsgithub.comnanopore-wgs-consortiumNA12878<jatsext-link>.

biorxiv genomics 500+-users 2017

Improved de novo Genome Assembly Linked-Read Sequencing Combined with Optical Mapping Produce a High Quality Mammalian Genome at Relatively Low Cost, bioRxiv, 2017-04-19

AbstractCurrent short-read methods have come to dominate genome sequencing because they are cost-effective, rapid, and accurate. However, short reads are most applicable when data can be aligned to a known reference. Two new methods for de novo assembly are linked-reads and restriction-site labeled optical maps. We combined commercial applications of these technologies for genome assembly of an endangered mammal, the Hawaiian Monk seal.We show that the linked-reads produced with 10X Genomics Chromium chemistry and assembled with Supernova v1.1 software produced scaffolds with an N50 of 22.23 Mbp with the longest individual scaffold of 84.06 Mbp. When combined with Bionano Genomics optical maps using Bionano RefAligner, the scaffold N50 increased to 29.65 Mbp for a total of 170 hybrid scaffolds, the longest of which was 84.78 Mbp. These results were 161X and 215X, respectively, improved over DISCOVAR de novo assemblies. The quality of the scaffolds was assessed using conserved synteny analysis of both the DNA sequence and predicted seal proteins relative to the genomes of humans and other species. We found large blocks of conserved synteny suggesting that the hybrid scaffolds were high quality. An inversion in one scaffold complementary to human chromosome 6 was found and confirmed by optical maps.The complementarity of linked-reads and optical maps is likely to make the production of high quality genomes more routine and economical and, by doing so, significantly improve our understanding of comparative genome biology.

biorxiv genomics 0-100-users 2017

 

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