Deep sequencing of hepatitis B virus using Ion Torrent fusion primer method

Johan Ringlander; Maria E Andersson; Kasthuri Prakash; Simon B Larsson; Magnus Lindh

doi:10.1016/j.jviromet.2021.114315

Deep sequencing of hepatitis B virus using Ion Torrent fusion primer method

J Virol Methods. 2022 Jan:299:114315. doi: 10.1016/j.jviromet.2021.114315. Epub 2021 Oct 11.

Authors

Johan Ringlander¹, Maria E Andersson¹, Kasthuri Prakash², Simon B Larsson², Magnus Lindh³

Affiliations

¹ Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden; Department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden.
² Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden.
³ Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden; Department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden. Electronic address: magnus.lindh@microbio.gu.se.

PMID: 34648822
DOI: 10.1016/j.jviromet.2021.114315

Abstract

Background: Hepatitis B virus (HBV) infection is worldwide a major cause of liver cirrhosis and hepatocellular carcinoma. Thousands of years ago, several HBV genotypes (A-I) evolved and have, as a result of human migration, become globally disseminated. Sequencing of HBV is used for genotyping, and investigation of outbreaks or of antiviral resistance. The present study describes a simplified deep sequencing of the whole HBV genome.

Methods: Sequencing by Ion Torrent was evaluated and its performance compared with Sanger sequencing on clinical samples.

Results: Amplification of overlapping segments spanning the entire HBV genome was successful at HBV DNA levels in serum as low as 100 IU/mL. The use of primers carrying adapter tags generated libraries without the need for fragmentation and ligation steps, and inclusion of barcode sequences allowed parallel analysis of multiple samples. A streamlined bioinformatic platform generated consensus sequences and superior mutation assessment as compared with Sanger sequencing, with which there was a 99.8 % average agreement.

Conclusion: Deep sequencing of the whole HBV genome by using PCR primers tagged with adapters that prepare overlapping amplicons for Ion Torrent analysis was efficient and accurate.

Keywords: Deep sequencing; HBV DNA; Hepatitis B virus; Ion Torrent; Whole genome.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA, Viral / genetics
Drug Resistance, Viral / genetics
Genotype
Hepatitis B virus / genetics
Hepatitis B*
Hepatitis B, Chronic*
High-Throughput Nucleotide Sequencing / methods
Humans
Liver Neoplasms*

Substances

DNA, Viral