基于多重PCR的HAdV-3全基因组高通量测序方法的建立  

Establishment of a high-throughput sequencing method for the whole genome of human adenovirus 3 based on multiplex PCR

作　　者：林琦[1] 黄枝妙 翁育伟[1] 何文祥[1] 游丽斌 Lin Qi;Huang Zhimiao;Weng Yuwei;He Wenxiang;You Libin(Fujian Provincial Center for Disease Control and Prevention,Fujian Provincial Key Laboratory of Zoonosis Research,Fuzhou 350012,China)

机构地区：[1]福建省疾病预防控制中心福建省人兽共患病研究重点实验室,福州350012

出　　处：《中华实验和临床病毒学杂志》2023年第5期530-536,共7页Chinese Journal of Experimental and Clinical Virology

基　　金：福建省卫生健康科研人才培养项目(2022QNA062);福建省卫生健康中青年科研重大项目(2021ZQNZD006)。

摘　　要：目的建立基于多重PCR的人3型腺病毒(human adenovirus 3,HAdV-3)全基因组高通量测序方法,提高HAdV-3全基因组测序效率和成功率。方法设计适用于HAdV-3全基因组扩增的多重PCR引物,通过多重PCR扩增HAdV-3全基因序列,使用琼脂糖凝胶电泳初步验证扩增产物的特异性。使用Illumina二代测序对多重PCR产物进行高通量测序。获得序列后使用CLC、IGV等软件分析测序有效数据量、平均深度及全基因组覆盖度等质量参数,评估测序质量。基于全基因组测序结果构建系统发育树,确认病毒型别及分析序列的同源性,评价该方法的准确性。结果设计得到HAdV-3全基因组多重PCR扩增引物共计70条(35对),扩增子大小约1200 bp,预期基因组全长约35240 bp、全基因组覆盖度可达99.8%;电泳结果显示多重PCR产物大小与预期相符且扩增特异性高;高通量测序结果显示基于该方法所获病毒全基因组序列完整无缺失,基因组覆盖度达预期范围;序列质量分析显示基于多重PCR的高通量测序方法能获得更多有效数据和更大测序深度,全基因组覆盖更均匀;进化分析显示病毒DNA经多重PCR扩增再测序与病毒DNA直接测序所获序列的进化分型结果一致且同源性高,表明该多重PCR方法的扩增忠实性高,结合高通量测序所获序列结果准确。结论本研究成功建立了一种基于多重PCR的HAdV-3全基因组高通量测序方法,该方法能够提高HAdV-3全基因测序效率和成功率,旨在为基于全基因组测序的HAdV-3病原监测及疫情溯源提供更好的技术支持与参考。Objective To improve the efficiency and success rate of human adenovirus 3(HAdV-3)whole genome sequencing,a high-throughput sequencing method for the whole genome of HAdV-3 based on multiplex PCR was established.Methods Multiplex PCR primers suitable for the whole genome amplification of HAdV-3 were designed.The whole genome sequence of HAdV-3 was amplified by multiplex PCR,and the specificity of the amplification product was preliminarily verified by agarose gel electrophoresis.High-throughput sequencing of the multiplex PCR products was performed using Illumina second-generation sequencing.After obtaining the sequence,software such as CLC and IGV were used to analyze the effective data amount,average sequencing depth,and whole genome coverage obtained by high-throughput sequencing,then the sequencing quality was evaluated.Based on the whole genome sequencing result,a phylogenetic tree was constructed to confirm the virus type and analyze homology of the sequences,and then the accuracy of this method was evaluated.Results A total of 70(35 pairs)multiplex PCR amplification primers for the whole genome of HAdV-3 were designed,with amplicon size of approximately 1200 bp.And the expected whole genome coverage could reach 99.8%(with a total genome length of approximately 35240 bp).Agarose gel electrophoresis analysis showed that the size of the multiplex PCR products was consistent with expectations,and the amplification specificity was high.The high-throughput sequencing result showed that the whole genome sequences obtained by this method were complete and intact,and the genome coverage was consistent with expectations.Sequence quality analysis showed that the high-throughput sequencing method based on multiplex PCR could obtain more effective data and greater sequencing depth,resulting in more uniform whole genome coverage.Phylogenetic analysis showed that the evolutionary typing result of viral DNA sequenced after multiplex PCR amplification were consistent with those of viral DNA sequenced directly and had high

关 键 词：人3型腺病毒 多重PCR 全基因组序列 高通量测序 

分 类 号：R373[医药卫生—病原生物学]