深度测序法在检测HCV不同基因型/亚型混合感染中的应用  

Mixed hepatitis C virus( HCV) genotypes / subtypes infection identified by deep sequencing

作　　者：许茹[1] 黄杰庭[1] 王敏[1] 梁明月[1] 廖峭[1] 黄珂[1] 熊华平[1] 戎霞[1] 付涌水[1] 

机构地区：[1]广州血液中心输血研究所,广东广州510095

出　　处：《中国输血杂志》2015年第10期1214-1218,共5页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金(81273145);国家科技重大专项子项目(2012ZX10004702);广州市医药卫生科技重大项目(20141A031001);广州市医药卫生科技项目(20131A011121)

摘　　要：目的将深度测序法用于HCV感染者中不同基因型/亚型混合感染的检测。方法 2例HCV RNA阳性血浆标本(标记为1和2号标本),以NS5B和E1编码区为目的基因,经RT-PCR扩增后做sanger核苷酸序列测定,对其做基因分型发现2个编码区的分型结果后,重复3次NS5B基因PCR扩增;合并3次PCR产物用Ion TorrentTM半导体测序仪做深度测序。获得的序列用Geneious软件的"The map to reference algorithm"和"De novo assembly"2种方法做序列分析和比对,并用Mega 5构建进化树。结果 sanger测序:1、2号标本NS5B基因分型均为1b,E1基因分型均为2a;深度测序:2个标本均为HCV 1b与2a混合感染,其中The map to reference algorithm(Geneious)方法显示,1号标本中1b占92.7%、2a占3.8%、没有比对到的序列(unused seq)占3.5%,2号标本相应为84.3%、5.4%,没有比对到的序列占10.3%;De novo assembly(Geneious)方法显示,1号标本中1b占96.3%、2a占3.7%,2号标本相应为94.1%、5.9%。结论深度测序技术可以精确地获得HCV混合感染的不同基因型/亚型信息,De novo assembly(Geneious)分析HCV的深度测序数据更为精确和直观。Objective To identify infection of mixed hepatitis C virus （HCV） genotype/subtype by deep sequencing. Methods Plasma samples from two individuals infected with HCV were collected. Disparate HCV genotypes were found in both persons between NS5B and E1 gene by RT-PCR and then Sanger sequencing. NS5B was amplified for three times and pooled together, which were subsequently applied to deep sequencing by the Ion Torrent system. Sequencing results were an-alyzed and aligned by two methods in Genious software： ＂map to reference algorithm＂ and ＂de novo assembly＂. Mega5 was used to construct phylogenetic tree. Results Sanger sequencing revealed subtype lb on NS5B and 2a on E1 for both sam- pies. Deep sequencing on NS5B also revealed a mixed infection with HCV lb and 2a. From the analysis in ＂the map of ref- erence algorithm＂, subtypes lb and 2a accounted for 92.7% and 3.7% of the total reads （ unused reads accounted for 3.5% ） for sample 1 , while lb and 2a accounted for 84. 3% , and 5.4% for sample 2 （ 10. 3% unused reads） , respectively. ＂De novo assembly＂ analysis revealed that the percentage for lb and 2a were 96. 3% and 3.7% for sample 1, and 94. 1% and 5.9% for sample 2. Conclusion Deep sequencing is a powerful tool that can precisely identify mixed infection with multiple HCV genotypes/subtypes. De novo assembly performs a better analysis on deep sequencing data.

关 键 词：丙型肝炎病毒 HCV混合型感染 深度测序 HCV基因型 亚型 

分 类 号：R446.1[医药卫生—诊断学] Q503[医药卫生—临床医学]