CHU de Toulouse, Laboratoire de Virologie, Toulouse, France.
INSERM UMR1291-CNRS UMR5051-Université Toulouse III, Toulouse Institute for Infectious and Inflammatory Diseases, Toulouse, France.
J Med Virol. 2024 Oct;96(10):e29951. doi: 10.1002/jmv.29951.
Accurate HIV-1 genome sequencing is necessary to identify drug resistance mutations (DRMs) in people with HIV-1 (PWH). Next-generation-sequencing (NGS) allows the detection of minor variants and is now available in many laboratories. Our study aimed to compare two NGS approaches, a "short read" sequencing protocol using DeepChek® Whole Genome HIV-1 Assay on Illumina, and a "long read" sequencing protocol of HIV-1 pol and env single-molecule real-time sequencing (SMRT) on Pacific Biosciences (PacBio). We analyzed 16 plasma samples and 13 cellular samples from PWH. HIV-1 whole genome was amplified into five amplicons using DeepChek® Whole Genome HIV-1 Assay and sequenced on an iSeq. 100. In parallel, HIV-1 pol and env genes were separately amplified and sequenced using PacBio SMRT system with the circular consensus sequencing mode on a Sequel IIe. Concordance rates for determining DRMs with both approaches varied depending on the HIV-1 region, with higher concordance in the integrase region compared to the reverse transcriptase and protease regions. DeepChek® Whole Genome HIV-1 Assay exhibited better sensitivity in HIV-1 RNA sequencing of plasmas with lower viral loads. In cell HIV-1 DNA sequencing, the DeepChek® Whole Genome HIV-1 Assay performed better in pol and env sequencing but detected more APOBEC-induced DRMs, which can represent defective proviruses. Our findings indicate that both DeepChek® Whole Genome HIV-1 Assay and PacBio SMRT sequencing exhibit good performance for subtype determination, detection, and quantification of DRMs of the HIV-1 genome. However, some discrepancies were found in cellular samples, highlighting the challenges of interpreting HIV-1 DNA DRMs.
准确的 HIV-1 基因组测序对于识别 HIV-1 感染者（PWH）中的耐药突变（DRMs）是必要的。下一代测序（NGS）允许检测次要变异，并且现在许多实验室都可以使用。我们的研究旨在比较两种 NGS 方法，一种是使用 Illumina 上的 DeepChek®全基因组 HIV-1 分析的“短读”测序方案，另一种是使用 Pacific Biosciences（PacBio）上的 HIV-1 pol 和 env 单分子实时测序（SMRT）的“长读”测序方案。我们分析了 16 个血浆样本和 13 个 PWH 的细胞样本。使用 DeepChek®全基因组 HIV-1 分析扩增 HIV-1 全基因组成五个扩增子，并在 iSeq 100 上进行测序。同时，使用 PacBio SMRT 系统以圆形共识测序模式在 Sequel IIe 上分别扩增和测序 HIV-1 pol 和 env 基因。两种方法确定 DRMs 的一致性率因 HIV-1 区域而异，与逆转录酶和蛋白酶区域相比，整合酶区域的一致性更高。DeepChek®全基因组 HIV-1 分析在病毒载量较低的血浆中 HIV-1 RNA 测序中表现出更好的灵敏度。在细胞 HIV-1 DNA 测序中，DeepChek®全基因组 HIV-1 分析在 pol 和 env 测序中表现更好，但检测到更多 APOBEC 诱导的 DRMs，这些 DRMs 可能代表缺陷前病毒。我们的研究结果表明，DeepChek®全基因组 HIV-1 分析和 PacBio SMRT 测序都能很好地用于 HIV-1 基因组的亚型确定、耐药突变的检测和定量。然而，在细胞样本中发现了一些差异，突出了解释 HIV-1 DNA 耐药突变的挑战。
