Novel Long‐Read Sequencing Method for Characterisation of Hepatitis B Transcripts Show High Expression of Chimeric HBV /Human RNA

Background Hepatitis B virus (HBV) genomes integrated into human DNA significantly contribute to surface antigen (HBsAg) production and may drive hepatocellular carcinoma (HCC). Long‐read sequencing methods like Nanopore offer advantages over short‐read next‐generation sequencing (NGS) by providing continuous reads of whole transcripts, but their application to HBV integration analysis remains limited. Objective To develop and apply a method combining semi‐nested PCR with Nanopore sequencing to analyse HBV transcripts, including canonical RNA, HBV–human fusion transcripts, and spliced forms in patients with HBV‐ or hepatitis D virus (HDV)‐induced liver disease. Methods Nine liver‐transplanted patients with HBV‐ or HDV‐related cirrhosis or HCC were studied. Semi‐nested PCR was used to amplify all HBV transcripts, followed by Nanopore sequencing. The approach allowed differentiation between canonical (cccDNA‐derived) and fusion transcripts. Reads containing the 3′ redundancy beyond nucleotide 1826, exclusive to cccDNA‐derived RNA, were quantified to determine the source of HBV RNA. Results Unique and total HBV‐human fusion RNA reads correlated with serum levels of HBV DNA and HBsAg. Integration‐derived RNA accounted for a median of 97% (range: 16%–100%) of HBV RNA. PreS1 RNA levels were much lower than preS2 but sufficient for HDV particle production in an HDV patient without cccDNA‐derived transcripts. Conclusion This method enables a simplified and comprehensive analysis of HBV transcripts. The results highlight the predominance of integration‐derived RNA and support the presence of cccDNA‐independent hepatitis D virus production. Nanopore sequencing offers valuable insights into HBV and HDV biology, supporting its role in understanding viral pathogenesis and therapeutic targeting.