基于长片段PCR扩增的牡蛎疱疹病毒基因组高通量测序

摘要

为获取2001年低温冻存栉孔扇贝感染牡蛎疱疹病毒(OsHV-1)变异株(ZK2001)基因组序列,并分析ZK2001与其他OsHV-1变异株的序列差异和系统发育关系,利用基于长片段PCR的基因组DNA的扩增和富集技术,获取2001年栉孔扇贝感染OsHV-1变异株的基因组DNA;再使用Illumina Hiseq 2500 PE250高通量测序平台对其测序.最后分析ZK2001与OsHV-1其他变异株基因组的序列差异和系统发育关系.测序数据组装后获得8个Scaffold.基因组变异分析结果显示,ZK2001与参考基因组相比存在328个SNP位点,SNP和序列插入/缺失变异是导致OsHV-1基因组序列变异的主要变异类型.系统发育分析结果显示,ZK2001变异株与分离自我国的OsHV-1变异株亲缘关系最近,与分离自欧洲的OsHV-1μvar及其相关变异株的亲缘关系最远,说明中国和欧洲分布OsHV-1间存在因地理隔离导致的遗传分化.研究表明,基于长片段PCR的DNA富集技术,可以有效地扩增和富集冷冻样本中OsHV-1基因组DNA,并应用于高通量测序.OsHV-1不同变异株基因组序列数据的获取和积累,将为其基因组尺度的基因变异、株系演化和系统发育关系等研究提供重要基础.%To obtain the genome sequence of Ostreid herpesvirus 1 (OsHV-1) infected Zhikong scallop (Chlamys farreri) in 2001 (ZK2001), and its phylogenetic relationship with the other reported variants, the genome DNA of the ZK2001 variant was firstly enriched through long-range PCR, and sequenced with Illumina Hiseq 2500 PE250 platform. Then the variation and phylogenetic relationship between ZK2001 and the other reported variants were analyzed. 8 scaffolds were obtained after assembly. Genome variation analysis indicated that there were 328 SNPs between ZK2001 and the reference genome. SNPs and insert/deletion were the main cause of genome differenti-ation. Phylogenetic inference indicated that ZK2001 variant was more closely related to OsHV-1 variants isolated from China than those from Europe. These results indicated that genetic differentiation had occurred as a result of geographic isolation. This study indicated genomic DNA of OsHV-1, which had been frozen for a long time, could be enriched with long-range PCR and used for high-throughput sequencing. The genomic data of different OsHV-1 variants will be necessary for further study of the genetic variation, evolution and phylogenetic analysis of the virus.