To find the predictors of early HCC based on the dynamic changes of HBV quasispecies, this study utilizing the second-generation sequencing (NGS) and high-order multiplex droplet digital PCR (ddPCR) technology to examine the HBV quasispecies in serum of total 247 subjects recruited from high-incidence area of HCC. In the discovery stage, 15 non-synonymous Single Nucleotide Polymorphisms (SNPs) with higher variant proportion in HCC case group were founded (all P<0.05). Furthermore, the variant proportions in some of these SNPs were observed changing regularly within 5 years before the onset of HCC, and 5 of them located in HBX, 2 in HBS and 2 in HBC. The HBV predominant quasispecies and their consensus sequences were identified by genetic evolution analysis, in which the high HBS and HBC quasispecies heterogeneity were found associated with the forming of multifarious quasispecies clones, and the HBX gene had the highest proportion of predominant quasispecies (46.7 % in HBX vs 12.7 % and 13.8 % in HBS and HBC respectively) with the key variations (G1512A, A1630G, T1753C/G/A, A1762T and G1764A) determined. In the validation stage, we confirmed that the combined double mutations of G1512A+A1630G, A1762T+G1764A, and the combined triple mutations of T1753C/G/A + A1762T+G1764A, all expressed higher in early HCC cases when comparing with control group (all P<0.05). We also demonstrated the advantages of ddPCR using in multi-variations detection in large-sample for early HCC surveillance and screening. So we think that the dynamic of key HBV variation positions and their different combinations determined by quasispecies anlysis in this study can act as the novel predictors of early hepatocarcinoma and suitable to popularize and apply in HCC screening.

为了根据HBV准种的动态变化寻找早期HCC的预测因子，本研究利用二代测序（NGS）和高阶多重液滴数字PCR（ddPCR）技术检测了总共247名受试者血清中的HBV准种招募自肝癌高发区。在发现阶段，发现HCC病例组中变异比例较高的15个非同义单核苷酸多态性（SNP）（均P <0.05）。此外，在HCC发病前5年内，观察到其中一些SNP的变异比例有规律地变化，其中5个位于HBX，2个位于HBS，2个位于HBC。通过遗传进化分析鉴定了HBV优势准种及其共有序列，其中HBS和HBC准种异质性较高，与多种准种克隆的形成有关，其中HBX基因在优势准种中所占比例最高（46.7%）。 HBX 与 HBS 和 HBC 分别为 12.7% 和 13.8%），并确定了关键变异（G1512A、A1630G、T1753C/G/A、A1762T 和 G1764A）。在验证阶段，我们证实与对照组相比，G1512A+A1630G、A1762T+G1764A的组合双突变以及T1753C/G/ A  +A1762T+G1764A的组合三突变在早期HCC病例中的表达均较高（所有P <0.05）。我们还展示了 ddPCR 在大样本多变异检测中用于早期 HCC 监测和筛查的优势。因此我们认为本研究通过准种分析确定的HBV关键变异位点及其不同组合的动态可以作为早期肝癌的新预测因子，适合在HCC筛查中推广应用。