Hepatitis B Virus (HBV) is a 3.2KB DNA virus that causes acute and chronic hepatitis. HBV infection is a world 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 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 (PHH) 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 PCR (BS-qMSP). 5mCpG levels in HBV determined by BS-qMSP and Nanopore sequencing were highly correlated. Our Nanopore sequencing approach achieved a coverage of ~2000x of HBV depending on infection efficacy, 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 4 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.

中文翻译：

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从头开始组装天然全长乙型肝炎病毒基因组后的表观遗传异质性

乙型肝炎病毒（HBV）是一种3.2KB的DNA病毒，可引起急性和慢性肝炎。HBV感染是世界卫生问题，3.5亿慢性感染者罹患肝病和肝细胞癌（HCC）的风险增加。在CpG环境中（5mCpG）HBV DNA的甲基化可以改变与感染和细胞转化有关的病毒基因的表达方式。此外，它还可以提供线索来说明为什么某些感染会被清除，或者无论是否发展为癌症都持续存在。5mCpG的检测通常需要通过无数限制来破坏DNA或引入偏倚的技术。因此，我们开发了一种不依赖于亚硫酸氢盐转化或PCR的HBV基因组中5mCpG的检测方法。利用cas9指导的RNP特异性靶向HBV基因组，我们从感染了不同HBV基因型的原代人肝细胞（PHH）中富集了HBV DNA，并从受感染的患者肝脏组织中富集了HBV；然后使用牛津纳米孔技术MinION进行测序。用亚硫酸氢盐定量甲基特异性PCR（BS-qMSP）对通过纳米孔测序检测到的5mCpG进行基准测试。通过BS-qMSP和Nanopore测序确定的HBV中5mCpG水平高度相关。我们的Nanopore测序方法根据感染的功效实现了约2000倍的HBV覆盖率，足以进行从头组装并检测HBV甲基化的细微波动，从而提供了天然HBV DNA的第一个从头组装以及第一个概况来自天然HBV序列的5mCpG。此外，通过捕获整个HBV基因组，我们探讨了感染患者中乙肝病毒的表观遗传异质性，并基于甲基化图谱确定了4个表观遗传上不同的簇。该方法是一种新颖的方法，能够在不同物种的核酸材料混合物中富集病毒DNA，并将成为监测传染病的宝贵工具。