Analysis of hepatitis B virus genotyping and drug resistance gene mutations based on massively parallel sequencing

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• 作者：Yingxin Han ; Yinxin Zhang ; Yanhua Mei ; Yuqi Wang ; Tao Liu ; Yanfang Guan ; Deming Tan ; Yu Liang ; Ling Yang ; Xin Yi
• 关键词：Drug resistance gene ; Genetic variation ; Genotyping ; Hepatitis B virus ; HiSeq 2000 sequencing ; Reverse-transcriptase (RT) district
• 刊名：Journal of Virological Methods
• 出版年：2013
• 出版时间：November, 2013
• 年：2013
• 卷：193
• 期：2
• 页码：341-347
• 全文大小：1924 K

文摘

Drug resistance to nucleoside analogs is a serious problem worldwide. Both drug resistance gene mutation detection and HBV genotyping are helpful for guiding clinical treatment. Total HBV DNA from 395 patients who were treated with single or multiple drugs including Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir and Emtricitabine were sequenced using the HiSeq 2000 sequencing system and validated using the 3730 sequencing system. In addition, a mixed sample of HBV plasmid DNA was used to determine the cutoff value for HiSeq-sequencing, and 52 of the 395 samples were sequenced three times to evaluate the repeatability and stability of this technology. Of the 395 samples sequenced using both HiSeq and 3730 sequencing, the results from 346 were consistent, and the results from 49 were inconsistent. Among the 49 inconsistent results, 13 samples were detected as drug-resistance-positive using HiSeq but negative using 3730, and the other 36 samples showed a higher number of drug-resistance-positive gene mutations using HiSeq 2000 than using 3730. Gene mutations had an apparent frequency of 1 % as assessed by the plasmid testing. Therefore, a 1 % cutoff value was adopted. Furthermore, the experiment was repeated three times, and the same results were obtained in 49/52 samples using the HiSeq sequencing system. HiSeq sequencing can be used to analyze HBV gene mutations with high sensitivity, high fidelity, high throughput and automation and is a potential method for hepatitis B virus gene mutation detection and genotyping.