Ho Cynthia K Y, Welkers Matthijs R A, Thomas Xiomara V, Sullivan James C, Kieffer Tara L, Reesink Henk W, Rebers Sjoerd P H, de Jong Menno D, Schinkel Janke, Molenkamp Richard
Department of Medical Microbiology, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands.
Department of Infectious Diseases, Vertex Pharmaceuticals Incorporated, Cambridge, MA 02139, USA.
J Virol Methods. 2015 Jul;219:28-37. doi: 10.1016/j.jviromet.2015.03.018. Epub 2015 Mar 26.
We compared 454 amplicon sequencing with clonal sequencing for the characterization of intra-host hepatitis C virus (HCV) NS3 variants. Clonal and 454 sequences were obtained from 12 patients enrolled in a clinical phase I study for telaprevir, an NS3-4a protease inhibitor. Thirty-nine datasets were used to compare the consensus sequence, average pairwise distance, normalized Shannon entropy, phylogenetic tree topology and the number and frequency of variants derived from both sequencing techniques. In general, a good concordance was observed between both techniques for the majority of datasets. Discordant results were observed for 5 out of 39 clonal and 454 datasets, which could be attributed to primer-related selective amplification used for clonal sequencing. Both 454 and clonal datasets consisted of a few major variants and a large number of low-frequency variants. Telaprevir resistance-associated variants were observed in low frequencies and were detected more often by 454. We conclude that performance of 454 and clonal sequencing is comparable for the characterization of intra-host virus populations. Not surprisingly, 454 is superior for the detection of low frequency resistance-associated variants. However, despite the greater coverage, 454 failed to detect some low frequency variants detected by clonal sequencing.
我们比较了454扩增子测序和克隆测序在宿主内丙型肝炎病毒（HCV）NS3变异体特征分析中的应用。克隆序列和454序列来自12名参加替拉瑞韦（一种NS3 - 4a蛋白酶抑制剂）临床I期研究的患者。使用39个数据集比较了两种测序技术得出的共有序列、平均成对距离、标准化香农熵、系统发育树拓扑结构以及变异体的数量和频率。总体而言，大多数数据集的两种技术之间观察到良好的一致性。在39个克隆和454数据集中，有5个出现了不一致的结果，这可能归因于克隆测序中使用的引物相关选择性扩增。454数据集和克隆数据集均由少数主要变异体和大量低频变异体组成。替拉瑞韦耐药相关变异体的出现频率较低，454检测到的频率更高。我们得出结论，在宿主内病毒群体特征分析方面，454测序和克隆测序的性能相当。不出所料，454在检测低频耐药相关变异体方面更具优势。然而，尽管覆盖范围更大，454仍未能检测到克隆测序检测到的一些低频变异体。
