Genotypic HIV-1 drug resistance testing with standard Sanger sequencing is limited to the detection of mutations with >20% prevalence. A new protocol for variant detection of protease and reverse transcriptase genes of HIV-1 genotype B samples with ultra-deep sequencing on the GS-FLX sequencer (Roche 454 Life Sciences, Branford, CT) was evaluated. The new technology was compared with the standard Sanger sequencing method. For accuracy testing, genotype B samples obtained from proficiency panels were examined with ultra-deep sequencing. Reproducibility was determined by repeat GS-FLX sequencing of 21 clinical samples. Clinical performance was evaluated with 44 samples and the results were compared to the TRUGENE HIV-1 Genotyping Kit (Siemens Healthcare Diagnostics, Tarrytown, NY). Sequences generated with both protocols were analyzed using the Stanford University HIV drug resistance database. When accuracy was tested, 316 of 317 mutation codons included in the analysis of proficiency panels could be identified correctly with ultra-deep sequencing. Reproducibility testing resulted in a correlation value of R(2)=0.969. Analysis of 44 routine clinical samples with the Stanford University HIV drug resistance database revealed a total number of 269 and 171 mutations by the ultra-deep and standard Sanger sequencing, respectively. Drug resistance interpretations showed differences for 11 samples. With ultra-deep sequencing, total time to result was four times longer in comparison to standard Sanger sequencing. Manual work was increased significantly using the new protocol. The ultra-deep sequencing protocol showed good accuracy and reproducibility. However, automation and shorter time to obtain results are essential for use in the routine diagnostic laboratory.
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