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Fig A: Overview of amplicon locations. Reference amplicon sizes (incl. adapters and MIDs) are as follows: (A) RTP1 = 419 bp, RTP2 = 510 bp, RTP3 = 400 bp, RTP4 = 599 bp, RTP5 = 558 bp, RTP6 = 434 bp; (B) V3A = 465 bp, V3B = 423 bp. Fig B: HQ reads and aligned reads distribution. I) The number of HQ reads, aligned and not aligned, per sequencing region of the PTP for each site II) The percent composition of total sequencing reads (in brackets below the site name) obtained from each amplicon at each site using region 3 as a representative example. Fig C: AVA software screen shot of the global alignment of sample 3 from site 31 of low frequency mutation RT215. Population sequencing indicated the possibility of amino acids Threonine (ACT), Tyrosine (TAT), Serine (TCT), and Asparagine (AAT) at codon 215 in the reverse transcriptase. These could not be further resolved, as the codon nucleotides cannot be phased. The 454 pyrosequencing reads resolved the actual variant codon compositions: 52.0% TAT (Tyr), and 46.9% ACT (Thr) (only mutations present at >1% included). The variants TCT (Ser) and AAT (Asn) could not be detected. Table A: Overview of sample viral titers and clades used for this study. 30 HIV-1 subtype B and 6 subtype non-B samples were used for RTP and V3 sequencing, three HIV-1 subtype B samples were run in triplicate (samples 37, 38 and 39) and two HIV-1 subtype B samples were used for the dilution series (samples 40–44 and 45–49). The RTP, dilution samples and triplicate samples are cultured recombinant virus, whereas V3 samples are cultured samples from patient isolates. The viral titer for each is shown. Table B: Primers. Table C: Details of amplicon drop out across sites per amplicon. Lack of amplicon production was reported by each site as any amplicon measuring less than 1 ng/μL or that was comprised of primer dimer alone. All amplicon concentration measurements were taken following PCR and purification. Table D: Drug resistance mutations found only by UDS. Details on reports of each site. The summary of the data are given in table 4. Hits, number of reads reporting the variant; denom, number of total reads at this position.
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