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

J Clin Microbiol. 2018 May 25;56(6):e00105-18. doi: 10.1128/JCM.00105-18. Print 2018 Jun.


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.

Keywords: antiviral drug resistance; human immunodeficiency virus; integrase; mutations; next-generation sequencing; proteases; reverse transcriptase.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-HIV Agents / therapeutic use
  • Drug Resistance, Viral / genetics*
  • Genotype
  • HIV Infections / drug therapy
  • HIV Infections / virology
  • HIV Integrase / genetics
  • HIV Reverse Transcriptase / genetics
  • HIV-1 / genetics*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Microbial Sensitivity Tests
  • Mutation / genetics*
  • RNA, Viral / genetics
  • Reagent Kits, Diagnostic
  • Viral Load


  • Anti-HIV Agents
  • RNA, Viral
  • Reagent Kits, Diagnostic
  • HIV Integrase
  • HIV Reverse Transcriptase