We evaluated the performance of the Applied Biosystems Viroseq HIV-1 Genotyping System for analysis of Ugandan plasma samples. Applied Biosystems recommends the use of 0.5-ml plasma samples with viral loads that were >2,000 copies/ml for analysis in the ViroSeq system. In our sample set, the volume of plasma available for analysis was limited. The samples used for analysis were <0.5 ml for 6 of 105 women and all 25 infants (Table ). The viral loads of these samples were relatively high, although some samples had viral loads <10,000 copies/ml (Table ). In the present study, PCR provided sufficient DNA for sequencing for all samples tested (Table ).
Summary of assay performance
The locations and positions of the sequencing primers in the ViroSeq system are shown in Fig. . Primers A, B, C, and D sequence the sense strand of the PCR product. Primers A and D are alternate primers that bind to the heterogeneous gag region. Primers F, G, and H sequence the antisense strand of the PCR product. In this analysis, a total of 848 primer reactions were performed. This included analysis of all 130 samples with primers A, B, C, F, G, and H and analysis of the first 68 samples with the alternate primer, primer D (Table ). Only 24 (35%) of the 68 reactions with primer D were successful. In contrast, the reactions with primer A were successful for most of those samples. Therefore, primer D was not included for routine analysis of the remaining 62 samples. An advantage of including only six primers for each sample (either primer A or D, but not both) is that 16 samples can be analyzed with a single 96-well plate and a single 96-lane sequencing gel. For primers A, B, C, F, G, and H, 96% of the reactions were successful on the first sequencing attempt. Sequencing reactions were repeated for 33 primers that initially failed. Eight (24%) of the repeat sequencing reactions were successful (Table ). Note that, for one sample, reactions with all primers failed, accounting for 6 of 25 (24%) of the primer failures. Also, 2 of 7 primer A failures were for samples from a mother-infant pair, and 3 of 13 primer F failures were for samples from a mother and her twins. The alternate primer, primer D, was tested with four of the seven samples for which reactions with primer A failed, and reactions with primer D were successful for three of the four samples. Full-length sequences were obtained for 102 of 105 (97%) of the samples from women tested and 22 of 25 (88%) of the samples from infants tested. Complete double-stranded sequences were obtained for 112 of 130 (86%) of the samples tested (Table ).
PCR and sequencing primers. The orientation and position of the PCR primers (PCR-F and PCR-R) and the seven sequencing primers in the ViroSeq HIV-1 Genotyping System are shown with respect to the protease and RT coding regions.
Performance of sequencing primers
To confirm the absence of sample cross-contamination, protease and RT nucleotide sequences from each sample were aligned and phylogenetic reconstructions were performed. No two sequences in the data set were identical, and the sequence obtained from each infant most closely resembled the sequence obtained from the corresponding mother.
HIV-1 subtyping was performed for the 102 women and 22 infants whose samples generated full-length sequences. Subtyping methods and the subtype analysis of the samples from the women are described in a separate report (11
); those subtypes were 50A, 35D, 12 A/D recombinant, and 4C; the subtype of one sequence could not be determined. The subtypes identified in the infants were 10A, 9D, 2 A/D recombinant, and 1C. We compared the subtypes of samples for which reactions with one or more of the sequencing primers failed. No subtype was identified for the sample for which reactions with all seven primers failed. Among the remaining 6 samples for which reactions with primer A failed, 3 had subtype D and 3 had subtype A; and among the remaining 12 samples for which reactions with primer F failed, 4 had subtype A, 6 had subtype D, and 2 had A/D recombinant HIV-1. Samples for which reactions with primer D failed included those with subtype A, C, D, and A/D recombinant HIV-1. We noted no association between subtype and sequencing primer performance among the subtypes tested.