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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 perform deep sequencing for the detection of mixed genotypes / subtypes in HCV-infected individuals. Methods Two HCV RNA positive plasma samples (labeled as Nos. 1 and 2) were obtained. The NS5B and E1 coding regions were used as the target gene. The nucleotide sequence of Sanger was amplified by RT-PCR and genotyped After genotyping of the two coding regions, PCR amplification of NS5B gene was repeated three times. The PCR products were pooled three times and sequenced in depth using an Ion Torrent (TM) semiconductor sequencer. The obtained sequences were sequenced and aligned using Geneious software’s “The map to reference algorithm” and “De novo assembly” methods, and the phylogenetic tree was constructed using Mega 5 software. Results Sanger sequencing showed that the NS5B genotypes of 1b and 1b were both 1b and E1 genotypes were both 2a. Deep sequencing: The two samples were all mixed infection with HCV 1b and 2a, among which The map to reference algorithm (Geneious) Showed that 1b accounted for 92.7%, 2a accounted for 3.8%, no unused seq accounted for 3.5%, No. 2 corresponding to 84.3%, 5.4%, no sequence to 10.3% ; De novo assembly (Geneious) method showed that 1b in specimen 1 accounted for 96.3%, 2a accounted for 3.7%, specimen 2 corresponding 94.1% and 5.9% respectively. Conclusion Deep sequencing technology can obtain different genotypes / subtypes of HCV infection accurately. The De novo assembly (Geneious) analysis of HCV deep sequencing data is more accurate and intuitive.