Abstract:

Objective To compare the sensitivity and accuracy of next-generation sequencing （NGS） and clone-based sequencing （CBS） for detection of X gene mutation sites in hepatitis B virus （HBV） quasispecies. Methods  Two individuals，one with hepatocellular carcinoma and one without it，were selected from a prospective cohort of high-risk individuals in Guangxi，China. Peripheral blood samples （5 mL） were collected every six months before diagnosis of hepatocellular carcinoma，giving a total of 12 samples. HBV DNA was extracted from serum，the HBV X gene was amplified using nested PCR，and PCR products were sequenced using NGS or CBS. Results NGS detected 40 mutation sites and 1 insertion in the X gene，while CBS detected 41 mutation sites and 1 insertion；29 mutation sites were detected by both NGS and CBS，11 sites only by NGS and 12 sites only by CBS. Mutations identified by either technique varied as hepatocellular carcinoma progressed. Conclusions NGS and CBS showed similar ability to discover mutation sites，though CBS was better at detecting fragment insertions and deletions. The choice of sequencing method will depend on research purposes.

Key words: Hepatitis B virus, Quasispecies, Next-generation sequencing, Clone-based sequencing, Hepatocellular carcinoma