Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing
Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing
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DOI：
10.1128/jcm.00105-18
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发表时间：
2018-06-01
影响因子：
9.4
通讯作者：
Shafer, Robert W.
中科院分区：
文献类型：
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作者：
Tzou, Philip L.;Ariyaratne, Pramila;Shafer, Robert W.
下一代测序 (NGS) 技术能够检测双脱氧核苷酸桑格测序无法检测到的低频 HIV-1 耐药突变 (DRM)，这对于改善患者治疗结果具有潜在优势。我们比较了体外诊断 (IVD) NGS 测定、用于 HIV-1 基因型耐药性测试的 Sentosa SQ HIV 基因分型测定以及对 138 种蛋白酶/逆转录酶 (RT) 和 39 种整合酶序列进行桑格测序的性能。 NGS 检测使用 5% 的阈值来报告低频变异。桑格测序和NGS之间的完整加部分核苷酸序列一致性水平为99.9%。在 138 个蛋白酶/RT 序列中，Sanger 和 NGS 均检测到平均 6.4 个 DRM，单独 NGS 检测到平均 0.5 个 DRM，单独 Sanger 测序检测到平均 0.1 个 DRM。在 39 个整合酶序列中，桑格测序和 NGS 均检测到平均 1.6 个 DRM，单独 NGS 检测到平均 0.15 个 DRM。与桑格测序相比，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性较高。 NGS 序列中几乎没有技术假象的证据，但在三个样本中检测到了 G 到 A 的超突变。总之，本研究评估的 IVD NGS 检测与 Sanger 测序高度一致。在报告少数变异的 5% 阈值下，NGS 似乎在不影响序列准确性的情况下检测低频 DRM 的灵敏度略有提高。
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 in vitro 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.