二代测序与分子克隆测序技术在HBV X基因准种变异研究中的应用及差异分析

目的　比较二代测序（next-generation sequencing，NGS）和克隆测序（clone-based sequencing，CBS）两种方法检测乙型肝炎病毒（hepatitis B virus，HBV）X基因准种变异位点的差异。方法 在广西肝癌高危人群队列中选择发展成肝癌的病例（A）和无癌的对照（B）各1例为研究对象。自研究对象进入队列起，每半年采集外周血液5 mL，分离血清备用，直至病例Ａ肝癌发病，共收集研究对象血清12份。提取血清HBV DNA并采用巢式PCR法扩增HBV X基因区。PCR产物同时采用NGS和CBS两种方法检测HBV X基因区核苷酸序列，对两种方法获得的突变位点进行比较分析。结果 NGS共检测到HBV X基因区准种40个变异位点和1段插入突变（nt1649），CBS共检测到41个变异位点和1段插入突变（nt1672~1673）。两种方法同时检测到的变异位点有29个，NGS检测到而CBS未检测到的变异位点有11个，NGS未检测到而CBS检测到的变异位点有12个。NGS和CBS两种方法均发现在肝病的进展中突变位点发生波动。结论 在研究HBV碱基变异方面，CBS和NGS发现变异位点的能力相当，CBS发现长片段插入和缺失的能力优于NGS，在实际应用时应根据研究目的选择相应的检测方法。

Comparison of next-generation sequencing and clone-based sequencing for analysis of X gene mutation sites in hepatitis B virus quasispecies

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.