(A) Schematics of the sample preparation protocol, see text and Materials and methods for details. (B) Read coverage for a representative sample. Coverage of separate PCR amplicons is shown in different hues, the black line is the total coverage. The coverage of PCR fragment F5 is lower than the other amplicons, but it is still larger than number of input HIV-1 RNA molecules; this situation is typical in our samples. (C) Each blue circle corresponds to a SNP frequency in amplicon F1 of a late sample of patient 11, while red squares are SNP frequencies in the sequence data generated from 10,000 copies of plasmid NL4-3. The histogram on the right shows the distribution of SNP frequencies in the patient sample and the control. Minor SNPs observed in reads generated from the plasmid, which represent PCR and sequencing errors, did not exceed 0.3%. SNP, single nucleotide polymorphism; RT-PCR, reverse transcriptase polychromase chain reaction.