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Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes.

ABSTRACT: De novo assembly of a human genome using nanopore long-read sequences has been reported, but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly, we present Shasta, a de novo long-read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled 11 highly contiguous human genomes de novo in 9 d. We achieved roughly 63× coverage, 42-kb read N50 values and 6.5× coverage in reads >100 kb using three flow cells per sample. Shasta produced a complete haploid human genome assembly in under 6 h on a single commercial compute node. MarginPolish and HELEN polished haploid assemblies to more than 99.9% identity (Phred quality score QV = 30) with nanopore reads alone. Addition of proximity-ligation sequencing enabled near chromosome-level scaffolds for all 11 genomes. We compare our assembly performance to existing methods for diploid, haploid and trio-binned human samples and report superior accuracy and speed.

SUBMITTER: Shafin K

PROVIDER: S-EPMC7483855 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes.

Shafin Kishwar K Pesout Trevor T Lorig-Roach Ryan R Haukness Marina M Olsen Hugh E HE Bosworth Colleen C Armstrong Joel J Tigyi Kristof K Maurer Nicholas N Koren Sergey S Sedlazeck Fritz J FJ Marschall Tobias T Mayes Simon S Costa Vania V Zook Justin M JM Liu Kelvin J KJ Kilburn Duncan D Sorensen Melanie M Munson Katy M KM Vollger Mitchell R MR Monlong Jean J Garrison Erik E Eichler Evan E EE Salama Sofie S Haussler David D Green Richard E RE Akeson Mark M Phillippy Adam A Miga Karen H KH Carnevali Paolo P Jain Miten M Paten Benedict B

Nature biotechnology 20200504 9

De novo assembly of a human genome using nanopore long-read sequences has been reported, but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly, we present Shasta, a de novo long-read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled 11 highly contiguous human genomes de novo in 9 d. We achieved roughly 63× coverage, 42-kb read N50 values and 6.5× covera ...[more]

PMID: 32686750

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Project description:Hepatitis B virus (HBV) contains a 3.2 kb DNA genome and causes acute and chronic hepatitis. HBV infection is a global health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). Methylation of HBV DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues as to why certain infections are cleared or persist with or without progression to cancer. The detection of 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. With Cas9-guided RNPs to specifically target the HBV genome, we enriched in HBV DNA from primary human hepatocytes (PHHs) infected with different HBV genotypes, as well as enriching in HBV from infected patient liver tissue, followed by sequencing with Oxford Nanopore Technologies MinION. Detection of 5mCpG by nanopore sequencing was benchmarked with bisulfite-quantitative methyl-specific qPCR (BS-qMSP). The 5mCpG levels in HBV determined by BS-qMSP and nanopore sequencing were highly correlated. Our nanopore sequencing approach achieved a coverage of ~2000× of HBV depending on infection efficiency, sufficient coverage to perform a de novo assembly and detect small fluctuations in HBV methylation, providing the first de novo assembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences. Moreover, by capturing entire HBV genomes, we explored the epigenetic heterogeneity of HBV in infected patients and identified four epigenetically distinct clusters based on methylation profiles. This method is a novel approach that enables the enrichment of viral DNA in a mixture of nucleic acid material from different species and will serve as a valuable tool for infectious disease monitoring.

Dataset Information

Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes.

Publications

Nanopore sequencing and the Shasta toolkit enable efficient de novo assembly of eleven human genomes.

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