Epigenetic heterogeneity after de novo assembly of native full-length Hepatitis B Virus genomesPublication
Date: 3rd December 2020 | Source: bioRxiv
Methylation of Hepatitis B Virus (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 we enriched in HBV DNA from PHH infected with HBV Genotype A, E or D prior to sequencing with Nanopores MinION. In addition, we were also able to enrich and sequence HBV from patient liver tissue achieving coverage of ~2000x.
Moreover, using the developed technique, we have provided the first de novo assembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences.
Furthermore, the developed tools are scalable to the Nanopore Flongle device, providing whole genome length HBV sequences in < 4h. 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.