Division of Infectious Diseases, Stanford University School of Medicine, Stanford, California, USA
Vela Diagnostics, Singapore Science Park II, Singapore.
J Clin Microbiol. 2018 May 25;56(6). doi: 10.1128/JCM.00105-18. Print 2018 Jun.
The ability of next-generation sequencing (NGS) technologies to detect low frequency HIV-1 drug resistance mutations (DRMs) not detected by dideoxynucleotide Sanger sequencing has potential advantages for improved patient outcomes. We compared the performance of an diagnostic (IVD) NGS assay, the Sentosa SQ HIV genotyping assay for HIV-1 genotypic resistance testing, with Sanger sequencing on 138 protease/reverse transcriptase (RT) and 39 integrase sequences. The NGS assay used a 5% threshold for reporting low-frequency variants. The level of complete plus partial nucleotide sequence concordance between Sanger sequencing and NGS was 99.9%. Among the 138 protease/RT sequences, a mean of 6.4 DRMs was identified by both Sanger and NGS, a mean of 0.5 DRM was detected by NGS alone, and a mean of 0.1 DRM was detected by Sanger sequencing alone. Among the 39 integrase sequences, a mean of 1.6 DRMs was detected by both Sanger sequencing and NGS and a mean of 0.15 DRM was detected by NGS alone. Compared with Sanger sequencing, NGS estimated higher levels of resistance to one or more antiretroviral drugs for 18.2% of protease/RT sequences and 5.1% of integrase sequences. There was little evidence for technical artifacts in the NGS sequences, but the G-to-A hypermutation was detected in three samples. In conclusion, the IVD NGS assay evaluated in this study was highly concordant with Sanger sequencing. At the 5% threshold for reporting minority variants, NGS appeared to attain a modestly increased sensitivity for detecting low-frequency DRMs without compromising sequence accuracy.
下一代测序 (NGS) 技术能够检测到传统双脱氧核苷酸 Sanger 测序无法检测到的低频 HIV-1 耐药突变 (DRMs)，这对改善患者预后具有潜在优势。我们比较了一种诊断用（IVD）NGS 检测方法——Sentosa SQ HIV 基因分型检测，用于 HIV-1 基因型耐药检测，与 Sanger 测序在 138 个蛋白酶/逆转录酶（RT）和 39 个整合酶序列上的表现。NGS 检测方法使用 5%的阈值来报告低频变异体。Sanger 测序和 NGS 在完全及部分核苷酸序列上的一致性水平为 99.9%。在 138 个蛋白酶/RT 序列中，Sanger 测序和 NGS 均检测到平均 6.4 个 DRMs，NGS 单独检测到平均 0.5 个 DRM，Sanger 测序单独检测到平均 0.1 个 DRM。在 39 个整合酶序列中，Sanger 测序和 NGS 均检测到平均 1.6 个 DRMs，NGS 单独检测到平均 0.15 个 DRM。与 Sanger 测序相比，NGS 估计 18.2%的蛋白酶/RT 序列和 5.1%的整合酶序列对一种或多种抗逆转录病毒药物的耐药水平更高。NGS 序列中几乎没有技术伪影的证据，但在三个样本中检测到 G 到 A 的超突变。总之，本研究中评估的 IVD NGS 检测方法与 Sanger 测序高度一致。在报告少数变异体的 5%阈值下，NGS 似乎在不影响序列准确性的情况下，略微提高了检测低频 DRM 的灵敏度。
