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

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.