Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing

Abstract

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.

Characterization of minority HIV-1 drug resistant variants in the United Kingdom following the verification of a deep sequencing-based HIV-1 genotyping and tropism assay

“…These variants, usually present as minority members of the virus population, can be selected and become predominant under the appropriate pressure by antiretroviral drugs [ 20 – 22 ]. With the advent of deep (next-generation) sequencing, several new HIV-1 genotyping approaches based on this ultrasensitive methodology have been developed with the goal of detecting drug resistant HIV-1 variants at low frequencies, i.e., below 20% of the viral population [ 19 , 23 – 26 ], with only a few assays being used in the clinical setting [ 19 , 27 , 28 ]. Although the clinical significance of these minority drug resistant HIV-1 variants is still on discussion [ 29 – 33 ], numerous groups are now using these assays not only to monitor HIV-1 drug resistance but also to better understand the role of low-level HIV-1 variants on transmission, disease progression, and HIV-1 cure strategies [reviewed on [ 10 , 11 ]].…”

Analysis of unusual and signature APOBEC-mutations in HIV-1 pol next-generation sequences

“…Second, the analyzed data was generated by research laboratories that adopted measures to improve the detection of low frequency variants, avoided samples with very low virus loads, and made their data publicly available. Indeed, previous studies have suggested that a five percent threshold is likely to be more reproducible in clinical settings [7,24,32,33].…”

“…In such scenarios, much of the observed variability in an NGS sequence may reflect PCR error rather than authentic viral mutations [2][3][4]. Since PCR errors are not subject to selective forces exerted during virus evolution, we have hypothesized that the presence of large numbers of unusual and likely deleterious mutations at an NGS mutation detection threshold suggests the threshold is too low [5][6][7][8].…”

Transmitted and Acquired HIV-1 Drug Resistance from a Family: A Case Study

“… 18 , 19 Sanger-based sequencing (SBS), the most commonly used method to diagnose HIV drug resistance, 20 was reported to have high repeatability and interpretability. 21 However, only virus strains with mutation frequency more than 10% to 20% can be detected by direct sequencing, 22 and the minority variants missed by standard genotyping may be lead to viral failure. 23 – 25 Thus, more sensitive methods of drug resistance testing are necessary to detect minority variants, so as to better guide clinical decision-making.…”