An international multicenter study on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing

•First multicenter study on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing to evaluate reproducibility and concordance of this new protocol.•All HIV-1 drug resistance mutations identified by population sequencing were also identified by 454 ultra-deep pyrosequencing.•Minority HIV-1 drug resistance mutations were only detected by 454 ultra-deep pyrosequencing.•454 ultra-deep pyrosequencing provided highly concordant results together with excellent cross-site correlation. The detection of mutant spectra within the viral quasispecies is critical for therapeutic management of HIV-1 infections. Routine clinical application of ultrasensitive genotyping requires reproducibility and concordance within and between laboratories. The goal of the study was to evaluate a new protocol on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing (454-UDS) in an international multicenter study. Sixteen blinded HIV-1 subtype B samples were provided for 454-UDS as both RNA and cDNA with viral titers of 88,600–573,000 HIV-1 RNA copies/ml. Eight overlapping amplicons spanning protease (PR) codons 10–99 and reverse transcriptase (RT) codons 1–251 were generated using molecular barcoded primers. 454-UDS was performed using the 454 Life Sciences/Roche GS FLX platform. PR and RT sequences were analyzed using 454 Life Sciences Amplicon Variant Analyzer (AVA) software. Quantified variation data were analyzed for intra-laboratory reproducibility and inter-laboratory concordance. Routine population sequencing was performed using the ViroSeq HIV-1 genotyping system. Eleven laboratories and the reference laboratory 454 Life Sciences sequenced the HIV-1 sample set. Data presented are derived from seven laboratories and the reference laboratory since severe study protocol execution errors occurred in four laboratories leading to exclusion. The median sequencing depth across all sites was 1364 reads per position (IQR=809–2065). 100% of the ViroSeq-reported mutations were also detected by 454-UDS. Minority HIV-1 drug resistance mutations, defined as HIV-1 drug resistance mutations identified at frequencies of 1–25%, were only detected by 454-UDS. Analysis of 10 preselected majority and minority mutations were consistently found across sites. The analysis of drug-resistance mutations detected between 1 and 10% demonstrated high intra- and inter-laboratory consistency in frequency estimates for both RNA and prepared cDNA samples, indicating robustness of the method. HIV-