Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Kowloon, Hong Kong SAR, China.
Department of Computer Science, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.
Clin Chem. 2023 Oct 3;69(10):1174-1185. doi: 10.1093/clinchem/hvad108.
HIV infections often develop drug resistance mutations (DRMs), which can increase the risk of virological failure. However, it has been difficult to determine if minor mutations occur in the same genome or in different virions using Sanger sequencing and short-read sequencing methods. Oxford Nanopore Technologies (ONT) sequencing may improve antiretroviral resistance profiling by allowing for long-read clustering.
A new ONT sequencing-based method for profiling DRMs in HIV quasispecies was developed and validated. The method used hierarchical clustering of long amplicons that cover regions associated with different types of antiretroviral drugs. A gradient series of an HIV plasmid and 2 plasma samples was prepared to validate the clustering performance. The ONT results were compared to those obtained with Sanger sequencing and Illumina sequencing in 77 HIV-positive plasma samples to evaluate the diagnostic performance.
In the validation study, the abundance of detected quasispecies was concordant with the predicted result with the R2 of > 0.99. During the diagnostic evaluation, 59/77 samples were successfully sequenced for DRMs. Among 18 failed samples, 17 were below the limit of detection of 303.9 copies/μL. Based on the receiver operating characteristic analysis, the ONT workflow achieved an F1 score of 0.96 with a cutoff of 0.4 variant allele frequency. Four cases were found to have quasispecies with DRMs, in which 2 harbored quasispecies with more than one class of DRMs. Treatment modifications were recommended for these cases.
Long-read sequencing coupled with hierarchical clustering could differentiate the quasispecies resistance profiles in HIV-infected samples, providing a clearer picture for medical care.
HIV 感染常发生耐药突变（DRMs），这会增加病毒学失败的风险。然而，使用桑格测序和短读测序方法，很难确定是否在同一基因组或不同病毒粒子中出现次要突变。牛津纳米孔技术（ONT）测序通过允许长读长聚类，可能改善抗逆转录病毒耐药性分析。
开发并验证了一种新的基于 ONT 测序的 HIV 准种耐药突变分析方法。该方法使用覆盖与不同类型抗逆转录病毒药物相关区域的长扩增子的层次聚类。为了验证聚类性能，制备了 HIV 质粒和 2 个血浆样本的梯度系列。将 ONT 结果与 77 份 HIV 阳性血浆样本中的桑格测序和 Illumina 测序结果进行比较，以评估诊断性能。
在验证研究中，检测到的准种丰度与预测结果一致，R2>0.99。在诊断评估中，59/77 个样本成功进行了 DRM 测序。在 18 个失败样本中，17 个样本的拷贝数低于 303.9 拷贝/μL 的检测下限。基于受试者工作特征分析，ONT 工作流程的截断值为 0.4 变异等位基因频率时，F1 评分为 0.96。有 4 个样本发现存在耐药突变的准种，其中 2 个携带了一种以上耐药类型的准种。建议对这些病例进行治疗调整。
长读测序与层次聚类相结合，可以区分 HIV 感染样本中的准种耐药谱，为医疗提供更清晰的信息。
