Department of Ophthalmology and Visual Sciences, University of Wisconsin Biotechnology Center,University of Wisconsin-Madison, Madison, WI 53706, USA.
Invest Ophthalmol Vis Sci. 2011 Nov 25;52(12):9061-73. doi: 10.1167/iovs.11-7812.
Little is known about the role of sequence variation in the pathology of HSV-1 keratitis virus. The goal was to show that a multiplex, high-throughput genome-sequencing approach is feasible for simultaneously sequencing seven HSV-1 ocular strains.
A genome sequencer was used to sequence the HSV-1 ocular isolates TFT401, 134, CJ311, CJ360, CJ394, CJ970, and OD4, in a single lane. Reads were mapped to the HSV-1 strain 17 reference genome by high-speed sequencing. ClustalW was used for alignment, and the Mega 4 package was used for phylogenetic analysis (www.megasoftware.net). Simplot was used to compare genetic variability and high-speed sequencing was used to identify SNPs (developed by Stuart Ray, Johns Hopkins University School of Medicine, Baltimore, MD, http://sray.med.som.jhml.edu/SCRoftware/simplot).
Approximately 95% to 99% of the seven genomes were sequenced in a single lane with average coverage ranging from 224 to 1345. Phylogenetic analysis of the sequenced genome regions revealed at least three clades. Each strain had approximately 200 coding SNPs compared to strain 17, and these were evenly spaced along the genomes. Four genes were highly conserved, and six were more variable. Reduced coverage was obtained in the highly GC-rich terminal repeat regions.
Multiplex sequencing is a cost-effective way to obtain the genomic sequences of ocular HSV-1 isolates with sufficient coverage of the unique regions for genomic analysis. The number of SNPs and their distribution will be useful for analyzing the genetics of virulence, and the sequence data will be useful for studying HSV-1 evolution and for the design of structure-function studies.
关于单纯疱疹病毒 1 角膜炎病毒的序列变异在病理学中的作用，人们知之甚少。本研究旨在展示一种多重、高通量的基因组测序方法，用于同时对 7 株单纯疱疹病毒 1 眼部分离株进行测序。
在单道测序中，使用基因组测序仪对 HSV-1 眼部分离株 TFT401、134、CJ311、CJ360、CJ394、CJ970 和 OD4 进行测序。通过高速测序将读取序列映射到 HSV-1 株 17 参考基因组上。使用 ClustalW 进行比对，使用 Mega 4 软件包进行系统发育分析（www.megasoftware.net）。Simplot 用于比较遗传变异性，高速测序用于鉴定单核苷酸多态性（由马里兰州巴尔的摩市约翰霍普金斯大学医学院的 Stuart Ray 开发，http://sray.med.som.jhml.edu/SCRoftware/simplot）。
大约 95%到 99%的七个基因组在单道测序中完成，平均覆盖率范围为 224 到 1345。对测序基因组区域的系统发育分析显示，至少存在三个分支。与 17 株相比，每个分离株大约有 200 个编码 SNP，这些 SNP 均匀分布在基因组上。有 4 个基因高度保守，6 个基因更具变异性。在高度 GC 丰富的末端重复区获得的覆盖度较低。
多重测序是一种具有成本效益的方法，可以获得具有足够基因组分析独特区域覆盖度的眼部单纯疱疹病毒 1 分离株的基因组序列。SNP 的数量及其分布将有助于分析毒力的遗传基础，序列数据将有助于研究单纯疱疹病毒 1 的进化，并为结构-功能研究的设计提供参考。
