Next generation sequencing (NGS) parallelizes the sequencing process, empowering scientists the dexterity to study HIV genome for drug resistance, tropism, and virus evolution. The full genome data can also be used to develop better clinical decision support systems. We developed and validated an NGS setup for HIV full genome deep sequencing on the MiSeq (Illumina), and applied this technique for gag-pol drug resistance mutation analysis.
Near full length HIV genome was amplified using extracted nucleic acid from 10 patient samples (Fig 1). The NGS workflow is described in Fig 2. The accuracy of the in-house alignment pipeline was validated by pairwise sequence comparison of the gag-pol sequences generated from the in-house pipeline and a Sanger sequencing method. The accuracy of the in-house variant calling pipeline was assessed with a CE-marked NGS analysis software (DeepChek).
The viral load of the 10 samples ranged between 20400 and 7690000 copies/mL. Seven of them were from treatment-na´ve patients. CRF01_AE was identified as the main subtype (70%), followed by subtype B (30%). Pairwise sequence comparison of the NGS and Sanger sequences showed 100% concordance, except for low abundant variants (<25%) that were undetectable by Sanger sequencing. For variant calling accuracy, DeepChek identified 935 gag-pol mutations and 879 of them were also detected by the in-house pipeline (Fig 3). Of the 56 missed mutations, all were non-drug resistance minor variants (<1.5%), except for a 230I non-nucleoside reverse transcriptase inhibitor-resistance mutation found in a treatment-na´ve patient. Among the 879 mutations found by both pipelines, 9 drug resistance mutations (reverse transcriptase: 74V, 98G, 103N, 138K, 219R; integrase: 66I, 153Y, 163R) were present in low abundance (<5%). They were found in 2 treatment-na´ve patients and all antiretroviral-experienced patients.
Our validated bioinformatics pipeline was able to assemble a reliable consensus sequence from the MiSeq sequencing data. We found several minor drug resistance mutations in both treatment-na´ve and antiretroviral-experienced patients. However, the therapeutic consequences of these minor variants, except for 103N, remain unclear.
C. K. Lee,
T. P. Loh, None
J. W. Tang, None
L. L. Chiu, None
S. Archuleta, None
E. S. C. Koay, None
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