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Nanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations.

ABSTRACT: BACKGROUND:Human cytomegalovirus (HCMV) has a double-stranded DNA genome of approximately 235 Kbp that is structurally complex including extended GC-rich repeated regions. Genomic recombination events are frequent in HCMV cultures but have also been observed in vivo. Thus, the assembly of HCMV whole genomes from technologies producing shorter than 500 bp sequences is technically challenging. Here we improved the reconstruction of HCMV full genomes by means of a hybrid, de novo genome-assembly bioinformatics pipeline upon data generated from the recently released MinION MkI B sequencer from Oxford Nanopore Technologies. RESULTS:The MinION run of the HCMV (strain TB40/E) library resulted in ~?47,000 reads from a single R9 flowcell and in ~?100× average read depth across the virus genome. We developed a novel, self-correcting bioinformatics algorithm to assemble the pooled HCMV genomes in three stages. In the first stage of the bioinformatics algorithm, long contigs (N50?=?21,892) of lower accuracy were reconstructed. In the second stage, short contigs (N50?=?5686) of higher accuracy were assembled, while in the final stage the high quality contigs served as template for the correction of the longer contigs resulting in a high-accuracy, full genome assembly (N50?=?41,056). We were able to reconstruct a single representative haplotype without employing any scaffolding steps. The majority (98.8%) of the genomic features from the reference strain were accurately annotated on this full genome construct. Our method also allowed the detection of multiple alternative sub-genomic fragments and non-canonical structures suggesting rearrangement events between the unique (UL /US) and the repeated (T/IRL/S) genomic regions. CONCLUSIONS:Third generation high-throughput sequencing technologies can accurately reconstruct full-length HCMV genomes including their low-complexity and highly repetitive regions. Full-length HCMV genomes could prove crucial in understanding the genetic determinants and viral evolution underpinning drug resistance, virulence and pathogenesis.

SUBMITTER: Karamitros T

PROVIDER: S-EPMC6090854 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Nanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations.

Karamitros Timokratis T van Wilgenburg Bonnie B Wills Mark M Klenerman Paul P Magiorkinis Gkikas G

BMC genomics 20180802 1

<h4>Background</h4>Human cytomegalovirus (HCMV) has a double-stranded DNA genome of approximately 235 Kbp that is structurally complex including extended GC-rich repeated regions. Genomic recombination events are frequent in HCMV cultures but have also been observed in vivo. Thus, the assembly of HCMV whole genomes from technologies producing shorter than 500 bp sequences is technically challenging. Here we improved the reconstruction of HCMV full genomes by means of a hybrid, de novo genome-ass ...[more]

PMID: 30068288

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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 full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations.

Publications

Nanopore sequencing and full genome de novo assembly of human cytomegalovirus TB40/E reveals clonal diversity and structural variations.

Similar Datasets

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