HIVdb-NGS (beta) accepts user-submitted protease, RT, and/or integrase codon frequency tables (CodFreq files) or AAVF files generated by the HYDRA pipeline. Results are returned at 8 mutation detection thresholds. At each threshold the program quantifies the number of unusual mutations and the number of signature APOBEC mutations. Thresholds with large numbers of unusual or signature APOBEC mutations are likely too low and pose unacceptable risks of identifying artifactual mutations caused either by machine error, PCR error, or G-to-A hypermutation.
By default, genotypic resistance interpretations are returned for the 20% threshold. A drop-down menu allows users to view results at lower thresholds. A multi-threshold mutation summary table is provided to help users identify thresholds to reduce the risk of identifying artifactual mutations. Genotypic resistance interpretations are suppressed at thresholds for which >1% of positions have an unusual mutation. The appropriate mutation detection threshold, however, cannot be identified with certainty in the absence of a sequencing protocol that uses unique molecular identifiers (UMIs) for each virus template.
Mutations detected at low thresholds are difficult to interpret because they are at increased risk of being artifactual and because few data have linked such mutations with an increased risk of virological failure to contemporary ART regimens. Nonetheless, the genotypic resistance interpretation of HIVdb-NGS beta version currently does not consider the threshold at which a mutation is detected. Like the main HIVdb program, the purpose of HIVdb-NGS beta is educational with regard to its genotypic resistance interpretations and quality control analysis.