Wang Xianjun, Xu Lihui, Chen Yueming, Liu Anbing, Wang Liqian, Xu Peisong, Liu Yunhui, Li Lei, Meng Fei
Clinical Laboratory, Hangzhou First People's Hospital Zhejiang Chinese Medical University Department of Research Service, Zhiyuan Inspection Medical Institute, Hangzhou, Zhejiang, People's Republic of China.
Medicine (Baltimore). 2017 Jul;96(30):e7588. doi: 10.1097/MD.0000000000007588.
Due to the low viral load of hepatitis B virus (HBV) in plasma samples, conventional techniques have limitations to the detection of antiviral resistance mutations. To solve the problem, we developed a fast, highly sensitive, and accurate method to sequence the HBV whole-genome sequencing in plasma samples which had various viral loads from very low to high.Twenty-one plasma samples were collected from patients who were carriers of HBV from the Hangzhou First People's Hospital. Two pairs of conserved, overlapping, nested primers were used to amplify and sequence the whole HBV genome in 8 plasma samples with different viral loads. High-throughput sequencing was performed on Illumina MiSeq platform. Concomitantly, 3 samples were directly sequenced without PCR amplification. We compared amplicon-sequencing with direct sequencing to develop a method for amplifying and characterizing the whole genome of HBV.HBV genome was amplified from all samples and verified by Sanger sequencing, regardless of the viral loads. Sequencing results revealed that only a few reads were mapped to the HBV genome following direct sequencing, while the amplicon-sequencing reads had a good coverage and depth. We identified 50 intrahost single nucleotide variations (iSNVs), 14 of which were low frequency mutations. Interestingly, iSNVs were more common in low viral load samples than in high viral load samples, and mutations in the reverse transcriptase (RT) region were most prevalent.We conclude that amplicon-sequencing is not only a practical method to detect HBV infection with a high sensitivity and accuracy but also enables to detect mutations in the HBV genome in low viral load samples from HBV-infected patients. Thus, our findings provide a new diagnosis method of HBV infection, which is capable of detection of low frequent mutations in low viral load samples.
由于血浆样本中乙型肝炎病毒（HBV）的病毒载量较低，传统技术在检测抗病毒耐药性突变方面存在局限性。为了解决这个问题，我们开发了一种快速、高度灵敏且准确的方法，用于对血浆样本中的HBV全基因组进行测序，这些样本的病毒载量从极低到极高各不相同。从杭州市第一人民医院收集了21份来自HBV携带者患者的血浆样本。使用两对保守、重叠、嵌套引物对8份具有不同病毒载量的血浆样本中的整个HBV基因组进行扩增和测序。在Illumina MiSeq平台上进行高通量测序。同时，对3份样本进行直接测序，无需PCR扩增。我们将扩增子测序与直接测序进行比较，以开发一种扩增和鉴定HBV全基因组的方法。无论病毒载量如何，所有样本中的HBV基因组均被扩增并通过Sanger测序验证。测序结果显示，直接测序后只有少数 reads 映射到HBV基因组，而扩增子测序 reads 具有良好的覆盖度和深度。我们鉴定出50个宿主内单核苷酸变异（iSNV），其中14个为低频突变。有趣的是，iSNV在低病毒载量样本中比在高病毒载量样本中更常见，并且逆转录酶（RT）区域的突变最为普遍。我们得出结论，扩增子测序不仅是一种高灵敏度和准确性检测HBV感染的实用方法，而且能够检测HBV感染患者低病毒载量样本中HBV基因组的突变。因此，我们的研究结果提供了一种新的HBV感染诊断方法，该方法能够检测低病毒载量样本中的低频突变。
