Novel Long-Read Sequencing Method for Characterisation of Hepatitis B Transcripts Show High Expression of Chimeric...

Area of Science:

• Virology
• Genomics
• Hepatology

Background:

• Hepatitis B virus (HBV) integration into human DNA contributes to HBsAg production and hepatocellular carcinoma (HCC).
• Long-read Nanopore sequencing offers advantages for analyzing whole transcripts compared to short-read NGS.
• Current applications of Nanopore sequencing for HBV integration analysis are limited.

Purpose of the Study:

• To develop and apply a method combining semi-nested PCR with Nanopore sequencing.
• To analyze HBV transcripts, including canonical RNA, HBV-human fusion transcripts, and spliced forms.
• To study patients with HBV- or hepatitis D virus (HDV)-induced liver disease.

Main Methods:

• Studied nine liver-transplanted patients with HBV- or HDV-related cirrhosis or HCC.
• Used semi-nested PCR to amplify all HBV transcripts, followed by Nanopore sequencing.
• Differentiated canonical (cccDNA-derived) and fusion transcripts by quantifying reads with 3' redundancy beyond nucleotide 1826.

Main Results:

• Unique and total HBV-human fusion RNA reads correlated with serum HBV DNA and HBsAg levels.
• Integration-derived RNA constituted a median of 97% of total HBV RNA.
• PreS1 RNA levels were sufficient for HDV particle production even without cccDNA-derived transcripts.

Conclusions:

• The developed method simplifies and comprehensively analyzes HBV transcripts.
• Results show the predominance of integration-derived RNA and support cccDNA-independent HDV production.
• Nanopore sequencing provides valuable insights into HBV and HDV biology for therapeutic targeting.