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Volume 7, Issue 5

Cas9-targeted nanopore sequencing reveals epigenetic heterogeneity after assembly of native full-length hepatitis B virus genomes Open Access

Abstract

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 assembly and detect small fluctuations in HBV methylation, providing the first 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.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): 5mC , cccDNA , cirrhosis , DNA methylation , epigenetics , HBV , HCC , long-read sequencing , nanopore and ONT
Funding
This study was supported by the:
  • Ligue Contre le Cancer (Award AAP 2018)
    • Principle Award Recipient: NotApplicable
  • Agence Nationale de Recherches sur le Sida et les Hépatites Virales (Award ECTZ50137)
    • Principle Award Recipient: NotApplicable
  • Agence Nationale de Recherches sur le Sida et les Hépatites Virales (Award ECTZ47287)
    • Principle Award Recipient: ApplicableNot
  • Agence Nationale de Recherches sur le Sida et les Hépatites Virales (Award CTZ107765)
    • Principle Award Recipient: CheminIsabelle
© 2021 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2021-05-18
2024-04-20
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Cas9-targeted nanopore sequencing reveals epigenetic heterogeneity after de novo assembly of native full-length hepatitis B virus genomes
M Gen 7, 000507 (2021); https://doi.org/10.1099/mgen.0.000507
/content/journal/mgen/10.1099/mgen.0.000507
/content/journal/mgen/10.1099/mgen.0.000507
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