基于二代测序技术建立覆盖我国主要流行亚型的HIV-1近全长基因组测序方法  

Establishing a HIV-1 near full-length genome amplification and sequencing method suitable for the major subtype epidemic in China using next-generation sequencing technology

作　　者：张瑞凤 刘静 刘佩 张鑫 王宇 丁梦君 丁海锋 姚均[1] 金聪[1] 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京102206 [2]河北医科大学公共卫生学院,石家庄050017

出　　处：《中国艾滋病性病》2023年第5期509-516,共8页Chinese Journal of Aids & STD

基　　金：国家病原微生物资源库-NPRC-32;北京科委课题(D171100006717001)。

摘　　要：目的 基于二代测序技术建立一种适合我国主要流行亚型的HIV-1近全长基因组扩增和测序方法,并优化建立标准检测流程,为HIV-1的全基因组序列鉴定和相关系统进化分析等应用提供重要技术手段。方法 基于我国HIV-1主要流行亚型设计扩增引物,通过扩增两段交叉重叠的基因序列获得HIV-1近全长基因组。并通过使用不同反转录试剂与扩增试剂以及尝试不同反应体系和反应条件,分析对HIV-1近全长基因组扩增的效果以及进行二代测序所获序列质量的影响。结果 本研究基于二代测序技术建立的HIV-1近全长基因组扩增和测序方法可成功扩增我国主要HIV-1流行亚型CRF01_AE、CRF07_BC、CRF08_BC、B、CRF55_01B,进行二代测序所获基因组序列的平均比对率为95.18%(89.74%~99.16%),平均覆盖率为94.96%(83.58%~99.98%),平均测序深度为5323.10×(3026.68×~6790.57×)。进一步分析不同亚型毒株每个基因位点的测序深度覆盖图,发现CRF08_BC亚型与CRF105_0108亚型的毒株测序深度分布均匀,均高于6000×,其他亚型的毒株样本在6672~8196 bp之间有部分基因位点的测序深度较低。优化和建立的标准检测流程可成功扩增病毒载量大于104copies/mL的样本并获得准确的近全长基因组序列。结论 本研究基于二代测序技术建立了一种HIV-1近全长基因组扩增与测序方法,该方法可成功扩增我国主要流行亚型毒株,为我国应用HIV-1基因组序列开展艾滋病精准防控工作提供了重要技术支撑。Objective To establish a HIV-1 near full-length genome amplification and sequencing method suitable for the major subtypes epidemic in China using next-generation sequencing technology,and to optimize a standard detection process as an important technical method for identifying the full-length genome sequence of HIV-1 and related phylogenetic analysis.Methods Based on the major subtypes of HIV-1 epidemic in China,amplification primers were designed to obtain the near full-length genome of HIV-1 by amplifying two overlapping genome sequences.Different reverse transcription and amplification reagents,as well as various reaction systems and conditions,were used to analyze the effect of near full-length genome amplification of HIV-1 and the impact on sequence quality obtained by next generation sequencing.Results The HIV-1 near full-length genome amplification and sequencing method established in this study based on next-generation sequencing technology could successfully amplify the major HIV-1 subtypes epidemic in China,including CRF01_AE,CRF07_BC,CRF 08_BC,B,and CRF55_01B.The average HIV-1 mapping rate of the obtained genome sequences by next-generation sequencing was 95.18%(89.74%-99.16%),the average coverage rate was 94.96%(83.58%~99.98%),and the meandepthwas 5323.10x(3026.68x-6790.57x).Further analysis of the sequencing depth coverage map of each gene locus of different subtypes showed that the sequencing depth of CRF08_BC subtype and CRF105_0108 subtype was evenly distributed,both higher than 6000x,while the sequencing depth of some gene loci from the samples of other subtypes between 6672 bp and 8196 bp,were lower.The optimized and established standard detection process could successfully amplify samples with viral loads greater than 10*copies/mL and obtain accurate near full-length genome sequences.Conclusions This study established a HIV-1 near fll-length genome amplification and sequencing method based on next-generation sequencing technology,which can successfully amplify the major epidemic subtype strain

关 键 词：艾滋病病毒 近全长基因组扩增 二代测序 

分 类 号：R373.9[医药卫生—病原生物学]