Frequency matters: comparison of drug resistance mutation detection by Sanger and next-generation sequencing in HIV-1. Journal of Antimicrobial Chemotherapy
Balakrishna et al. aimed to compare the HIV drug resistance mutations (DRMs) detected by Sanger sequencing (SS) and next-generation sequencing (NGS) at different thresholds and explored the dependency of the frequency and spectrum of low-abundance HIV-DRMs on the variant-calling thresholds. SS can detect HIV-DRMs that appear in more than 15%–25% of variants, whereas NGS detect low-abundance mutations.
To compare the HIV-DRMs detected by SS and NGS, the authors included participants enrolled in the Swiss HIV Cohort Study (SHCS) with SS and NGS sequences available with sample collection dates ≤7 days apart. They tested for the presence of HIV-DRMs and compared the agreement between SS and NGS at different variant-calling thresholds.
The authors included 594 pairs of SS and NGS from 527 SHCS participants. Males accounted for 80.5% of the participants, 76.3% were ART naive at sample collection and 78.1% of the sequences were subtype B. Overall, the study observed a good agreement (Cohen’s kappa >0.80) for HIV-DRMs for variant-calling thresholds ≥5%. The authors observed an increase in low-abundance HIV-DRMs detected at lower thresholds [28/417 (6.7%) at 10%–25% to 293/812 (36.1%) at 1%–2% threshold]. However, such low-abundance HIV-DRMs were overrepresented in ART-naive participants and were in most cases not detected in previously sampled sequences suggesting high sequencing error for thresholds <3%.
In conclusion, the study observed, for different variant-calling thresholds above 5%, a good concordance (Cohen’s kappa and Light’s kappa >0.8) between SS and NGS in detecting HIV-DRMs as well as for the resulting resistance levels to ARV drugs. The authors observed a substantial number of low-abundance HIV-DRMs detected only by NGS at lower variant-calling thresholds. These findings suggest that a substantial fraction of the low-abundance HIV-DRMs detected at thresholds <3% may represent sequencing errors and hence should not be over interpreted in clinical practice.