Next-generation sequencing (NGS) is a powerful and high-throughput method for the detection of viral mutations. This article provides a brief overview about optimization of NGS analysis for hepatocellular carcinoma (HCC)-associated hepatitis B virus (HBV) mutations, and hepatocarcinogenesis of relevant mutations. For the application of NGS analysis in the genome of HBV, four noteworthy steps were discovered in testing. First, a sample-specific reference sequence was the most effective mapping reference for NGS. Second, elongating the end of reference sequence improved mapping performance at the end of the genome. Third, resetting the origin of mapping reference sequence could probed deletion mutations and variants at a certain location with common mutations. Fourth, using a platform-specific cut-off value to distinguish authentic minority variants from technical artifacts was found to be highly effective. One hundred and sixty-seven HBV single nucleotide variants (SNVs) were found to be studied previously through a systematic literature review, and 12 SNVs were determined to be associated with HCC by meta-analysis. From comprehensive research using a HBV genome-wide NGS analysis, 60 NGS-defined HCC-associated SNVs with their pathogenic frequencies were identified, with 19 reported previously. All the 12 HCC-associated SNVs proved by meta-analysis were confirmed by NGS analysis, except for C1766T and T1768A which were mainly expressed in genotypes A and D, but including the subgroup analysis of A1762T. In the 41 novel NGS-defined HCC-associated SNVs, 31.7% (13/41) had cut-off values of SNV frequency lower than 20%. This showed that NGS could be used to detect HCC-associated SNVs with low SNV frequency. Most SNV II (the minor strains in the majority of non-HCC patients) had either low (< 20%) or high (> 80%) SNV frequencies in HCC patients, a characteristic U-shaped distribution pattern. The cut-off values of SNV frequency for HCC-associated SNVs represent their pathogenic frequencies. The pathogenic frequencies of HCC-associated SNV II also showed a U-shaped distribution. Hepatocarcinogenesis induced by HBV mutated proteins through cellular pathways was reviewed. NGS analysis is useful to discover novel HCC-associated HBV SNVs, especially those with low SNV frequency. The hepatocarcinogenetic mechanisms of novel HCC-associated HBV SNVs defined by NGS analysis deserve further investigation.

Background Hepatitis B virus (HBV) quasispecies are crucial in the pathogenesis of chronic liver disease. Next-generation sequencing (NGS) is powerful for identifying viral quasispecies. To improve mapping quality and single nucleotide variant (SNV) calling accuracy in the NGS analysis of HBV, we compared different mapping references, including the sample-specific reference sequence, same genotype sequences and different genotype sequences, according to the sample. Methods Real Illumina HBV datasets from 86 patients, and simulated datasets from 158 HBV strains in the GenBank database, were used to assess mapping quality. SNV calling accuracy was evaluated using different mapping references to align Real Illumina datasets from a single HBV clone. Results Using the sample-specific reference sequence as a mapping reference produced the largest number of mappable reads and coverages. With a different genotype mapping reference, the consensus sequence derived from the Real Illumina datasets of the single HBV clone showed 21 false SNV callings in polymerase and surface genes, the regions most divergent between the mapping reference and this HBV clone. A ~6 % coverage of most of these false SNVs was yielded even with a same genotype mapping reference, but none with the sample-specific reference sequence. Conclusions Using sample-specific reference sequences as a mapping reference in NGS analysis optimized mapping quality and the SNV calling accuracy for HBV quasispecies.

To investigate the utility of hepatitis B surface antigen (HBsAg) kinetics in chronic hepatitis B patients during long-term entecavir treatment. This retrospective study included treatment-naïve chronic hepatitis B patients who received at least 2 years of consecutive entecavir treatment. Patients were followed up at three to six month intervals with liver biochemistry, hepatitis B virus DNA, and abdominal sonography. In hepatitis B e antigen (HBeAg)-positive patients, HBeAg levels were assessed every three to six month until results became negative. Serum HBsAg levels were determined at the baseline, one-year and five-year time points. Liver cirrhosis was diagnosed through liver biopsy, imaging examinations, or clinical findings of portal hypertension. Hepatocellular carcinoma was diagnosed by histological examination or dynamic image studies. A total of 211 patients were enrolled. The median treatment time was 5.24 (2.00-9.62) years. Multivariate analysis showed that lower baseline HBsAg levels were associated with an earlier virological response, earlier hepatitis B e antigen (HBeAg) seroconversion, and earlier biochemical response in HBeAg-positive patients (cut-off value: 4 log IU/mL) and an earlier virological response in HBeAg-negative non-cirrhotic patients (cut-off value: 2.4 log IU/mL). Although HBsAg levels decreased slowly during long-term entecavir treatment, higher HBsAg decrease rates were found in the first year for HBeAg-positive non-cirrhotic patients, and patients with higher baseline HBsAg levels. More favorable clinical outcomes were not observed by a rapid HBsAg decline per se, but depended on lower baseline HBsAg levels. Baseline HBsAg can be used to predict treatment responses. HBsAg levels and decrease rates should be considered together according to disease status while interpreting HBsAg changes.

Serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) is correlated with covalently closed circular DNA. We aimed to investigate the utility of serum HBV pgRNA in chronic hepatitis B patients receiving nucleos(t)ide analogue treatment and those achieving HBsAg loss. One hundred and eighty-five patients were enrolled for studying long-term HBV pgRNA kinetics during treatment. Twenty patients achieving HBsAg loss after treatment were enrolled for examining HBV pgRNA kinetics around HBsAg loss. HBV pgRNA significantly decreased in the high baseline HBV pgRNA (≥6 log copies/mL) group but significantly increased in the low baseline HBV pgRNA (<4 log copies/mL) group after 3-month entecavir treatment. Among the 20 patients achieving HBsAg loss, 13 (65%) patients had serum HBV pgRNA higher than the limit of detection (LOD, 1466 copies/mL) when they achieved HBsAg loss. Finally, all 20 patients had HBV pgRNA going below the LOD within 3 years after achieving HBsAg loss. In conclusion, baseline serum HBV pgRNA alone is insufficient for predicting the trajectory of HBV pgRNA. Most patients still had HBV pgRNA higher than the LOD when they achieved HBsAg loss. Further studies on HBV pgRNA kinetics around HBsAg loss would provide an enhanced basis for further applications of HBV pgRNA.