Thys Kim, Verhasselt Peter, Reumers Joke, Verbist Bie M P, Maes Bart, Aerssens Jeroen
Discovery Sciences, Janssen Research & Development, Turnhoutseweg 30, B-2340 Beerse, Belgium.
Department of Mathematical Modeling, Statistics and Bioinformatics, Ghent University, Coupure Links 653, 9000 Gent, Belgium.
J Virol Methods. 2015 Sep 1;221:29-38. doi: 10.1016/j.jviromet.2015.04.022. Epub 2015 Apr 25.
Massively parallel sequencing (MPS) technology has opened new avenues to study viral dynamics and treatment-induced resistance mechanisms of infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Whereas the Roche/454 platform has been used widely for the detection of low-frequent drug resistant variants, more recently developed short-read MPS technologies have the advantage of delivering a higher sequencing depth at a lower cost per sequenced base. This study assesses the performance characteristics of Illumina MPS technology for the characterization of genetic variability in viral populations by deep sequencing. The reported results from MPS experiments comprising HIV and HCV plasmids demonstrate that a 0.5-1% lower limit of detection can be achieved readily with Illumina MPS while retaining good accuracy also at low frequencies. Deep sequencing of a set of clinical samples (12 HIV and 9 HCV patients), designed at a similar budget for both MPS platforms, reveals a comparable lower limit of detection for Illumina and Roche/454. Finally, this study shows the possibility to apply Illumina's paired-end sequencing as a strategy to assess linkage between different mutations identified in individual viral subspecies. These results support the use of Illumina as another MPS platform of choice for deep sequencing of viral minority species.
大规模平行测序（MPS）技术为研究病毒动力学以及诸如人类免疫缺陷病毒（HIV）和丙型肝炎病毒（HCV）等感染的治疗诱导耐药机制开辟了新途径。虽然罗氏/454平台已被广泛用于检测低频耐药变异体，但最近开发的短读长MPS技术具有以较低的每测序碱基成本提供更高测序深度的优势。本研究评估了Illumina MPS技术通过深度测序表征病毒群体遗传变异性的性能特征。来自包含HIV和HCV质粒的MPS实验的报告结果表明，使用Illumina MPS可以轻松实现0.5-1%的检测下限，同时在低频时也能保持良好的准确性。对一组临床样本（12名HIV患者和9名HCV患者）进行深度测序，两个MPS平台的预算相似，结果显示Illumina和罗氏/454的检测下限相当。最后，本研究表明应用Illumina的双末端测序作为一种策略来评估在单个病毒亚群中鉴定出的不同突变之间的连锁关系是可行的。这些结果支持将Illumina用作病毒少数群体深度测序的另一个首选MPS平台。
