Establishment and application of whole genome high-throughput sequencing of hepatitis B virus with different concentrations / 中华实验和临床病毒学杂志

ABSTRACT

Objective@#To establish a high-throughput sequencing method for a whole genome in different hepatitis B virus (HBV) concentrations.@*Methods@#Two method of amplicon-sequencing and direct sequencing without PCR amplification were used for library construction in the three plasmids, including the low HBV load sample, the moderate HBV load sample and the high HBV load sample. Whole genome sequencing was performed on Illumina MiSeq platform.@*Results@#There are significant differences in data yield between the two different library construction method. Only a few reads could be mapped to the HBV genome for direct sequencing. However, three samples were successfully amplified by the nested PCR amplification and amplicon-sequencing showed that all HBV samples had a good coverage and depth, which was not affected by HBV concentration. The alignment rate of HBV genome approached 80%. A total of 27 intra-host single nucleotide variations (iSNVs) were identified and 13 iSNVs were low-frequency mutation in three samples. Compared with the high HBV load sample, mutations in the reverse transcription (RT) region was more easily appeared in the low HBV sample and the moderate HBV load sample.@*Conclusions@#Integrating nested PCR with high-throughput sequencing to the HBV whole genome sequencing is not only a practical method to detect the infection of HBV with a high-sensitivity and accuracy, but also enables to detect the mutation in the infected patient with low HBV copy number.