摘要
目的建立一种适用于不同浓度乙型肝炎病毒(hepatitis B Virus, HBV)的全基因组高通量测序方法。方法分别采用HBV病毒核酸直接建库方法及巢式PCR扩增建库方法,对含高、中、低HBV拷贝的3个血浆样本进行文库构建。通过Illumina Miseq平台对HBV全基因组进行高通量测序。结果基于两种不同建库方法获得数据产量差异巨大。HBV病毒核酸直接建库方法只有很少数据可以匹配到HBV基因组。巢式PCR扩增能够成功扩增出HBV全基因组,文库经高通量测序后,HBV基因组匹配率接近80%,且样本覆盖度和深度不受HBV浓度影响。3个不同浓度HBV样本共发现27个宿主内单核苷酸突变(intra-host single nucleotide variations, iSNVs),其中13个是低频突变。逆转录(reverse transcription, RT)区域的突变出现在低拷贝数和中等拷贝数样品中,而在高拷贝数样品中没有发现。结论本研究建立的巢式PCR扩增结合高通量测序对HBV全基因组测序的方法,不仅可以快速准确的检测HBV感染,还可以发现低拷贝样品中整个HBV基因组的突变。
Objective To establish a high-throughput sequencing method for a whole genome in different hepatitis B virus (HBV) concentrations. Methods Two method of amplieon-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 nueleotide 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.
作者
王贤军
刘云惠
孟飞
杨宁敏
Wang Xianjun;Liu Yunhui;Meng Fei;Yang Ningmin(Clinical Laboratory, Hangzhou First People' s Hospital, Hanghzou 310006, China;Zhiyuan Inspection Medical Institute, Hangzhou 310030, Chin)
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2018年第2期203-207,共5页
Chinese Journal of Experimental and Clinical Virology
基金
浙江省自然科学基金(LY13H190003)
关键词
肝炎病毒
乙型
基因组
聚合酶链式反应
高通量测序
突变
Hepatitis B virus
Genome
Polymerase chain reaction
High-throughput sequencing
Mutation