National HIV and Retrovirology Laboratories, National Microbiology Laboratory at JC Wilt Infectious Diseases Research Centre, Public Health Agency of Canada, Winnipeg, Canada.
Bioinformatics Core, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada.
Sci Rep. 2019 Jun 20;9(1):8970. doi: 10.1038/s41598-019-45328-3.
Conventional HIV drug resistance (HIVDR) genotyping utilizes Sanger sequencing (SS) methods, which are limited by low data throughput and the inability of detecting low abundant drug resistant variants (LADRVs). Here we present a next generation sequencing (NGS)-based HIVDR typing platform that leverages the advantages of Illumina MiSeq and HyDRA Web. The platform consists of a fully validated sample processing protocol and HyDRA web, an open web portal that allows automated customizable NGS-based HIVDR data processing. This platform was characterized and validated using a panel of HIV-spiked plasma representing all major HIV-1 subtypes, pedigreed plasmids, HIVDR proficiency specimens and clinical specimens. All examined major HIV-1 subtypes were consistently amplified at viral loads of ≥1,000 copies/ml. The gross error rate of this platform was determined at 0.21%, and minor variations were reliably detected down to 0.50% in plasmid mixtures. All HIVDR mutations identifiable by SS were detected by the MiSeq-HyDRA protocol, while LADRVs at frequencies of 1~15% were detected by MiSeq-HyDRA only. As compared to SS approaches, the MiSeq-HyDRA platform has several notable advantages including reduced cost and labour, and increased sensitivity for LADRVs, making it suitable for routine HIVDR monitoring for both patient care and surveillance purposes.
传统的 HIV 耐药性（HIVDR）基因分型利用 Sanger 测序（SS）方法，这些方法受到数据吞吐量低和无法检测低丰度耐药变体（LADRVs）的限制。在这里，我们提出了一种基于下一代测序（NGS）的 HIVDR 分型平台，利用了 Illumina MiSeq 和 HyDRA Web 的优势。该平台由一个经过全面验证的样本处理方案和 HyDRA Web 组成，HyDRA Web 是一个开放的网络门户，允许基于 NGS 的 HIVDR 数据处理的自动化定制。该平台使用代表所有主要 HIV-1 亚型的 HIV 加标血浆、系谱质粒、HIVDR 能力验证样本和临床样本进行了表征和验证。所有检查的主要 HIV-1 亚型在病毒载量≥1000 拷贝/ml 时均得到一致扩增。该平台的总错误率为 0.21%，在质粒混合物中可靠地检测到 0.50%的微小变化。MiSeq-HyDRA 方案可检测到所有可通过 SS 识别的 HIVDR 突变，而 MiSeq-HyDRA 仅可检测到频率为 1%至 15%的 LADRVs。与 SS 方法相比，MiSeq-HyDRA 平台具有几个显著的优势，包括降低成本和劳动力，以及提高对 LADRVs 的敏感性，使其适合于患者护理和监测目的的常规 HIVDR 监测。
