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

## TL;DR

A new sequencing method reveals that most HBV RNA comes from integrated viral DNA, not circular DNA, and supports HDV production without circular DNA.

## Contribution

A novel method combining semi-nested PCR and Nanopore sequencing to analyze HBV transcripts and their integration into human DNA.

## Key findings

- Over 90% of HBV preS2 RNA reads were found to be derived from integrated DNA.
- Chimeric HBV-human preS1 transcripts were detected, supporting cccDNA-independent HDV production.
- Integration-derived RNA accounted for up to 100% of HBV RNA in some patients.

## Abstract

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.

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.

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.

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.

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.

Nanopore long‐read sequencing of HBV RNA provides information about the entire transcripts including HBV–human fusion sites and splicing.Over 90% of HBV preS2 reads, but no pregenomic or core reads, were found to be integration‐derived.Chimeric HBV–human preS1 transcripts were detected, supporting the presence of cccDNA‐independent hepatitis D virus production.

Nanopore long‐read sequencing of HBV RNA provides information about the entire transcripts including HBV–human fusion sites and splicing.

Over 90% of HBV preS2 reads, but no pregenomic or core reads, were found to be integration‐derived.

Chimeric HBV–human preS1 transcripts were detected, supporting the presence of cccDNA‐independent hepatitis D virus production.