High degree of interlaboratory reproducibility of human immunodeficiency virus type 1 protease and reverse transcriptase sequencing of plasma samples from heavily treated patients

Abstract

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.

FIG. 1

Unrooted neighbor-joining trees of the 46 protease (A) and RT (B) sequence pairs. The sequence name consists of a letter indicating the laboratory (S, Stanford [laboratory A]; V, VIRCO [laboratory B]). In all cases the paired sequences were closer to one another than to any other sequence in the data set.

FIG. 1

Unrooted neighbor-joining trees of the 46 protease (A) and RT (B) sequence pairs. The sequence name consists of a letter indicating the laboratory (S, Stanford [laboratory A]; V, VIRCO [laboratory B]). In all cases the paired sequences were closer to one another than to any other sequence in the data set.

FIG. 2

(A) Twelve samples with one or more complete nucleotide discordances had significantly lower mean plasma HIV-1 RNA levels than the 34 samples without a complete nucleotide mismatch (3.9 versus 4.9 log copies/ml; P < 0.001). Panel A appears to have less than 46 points because 34 of the samples had no complete mismatches and these overlap with one another along the x axis. (B)There was no statistically significant relationship between the number of partial nucleotide mismatches and plasma HIV-1 RNA levels in the 46 samples. Each panel has 46 points, one for each plasma sample. The position along the x axis is based on the HIV-1 RNA level of the plasma sample. The position along the y axis is the number of nucleotide mismatches between laboratories A and B upon testing of the plasma sample.

FIG. 3

Matrices showing the exact numbers of nucleotide concordances and discordances between laboratories A (vertical, left) and B (horizontal, top). Exact matches are shown along the diagonal. The numbers of partial discordances are written in black on a grey background, and the numbers of complete discordances are written in red on a white background. R (A/G) and Y (C/T) represent transitions. M (A/C), W (A/T), K (G/T), and S (C/G) represent transversions. Data from the protease sequencing are shown at the top, and data from the RT sequencing are shown below. One RT sequence had a B and another had an H (data not shown). There were no N's or other highly ambiguous nucleotides.

FIG. 4

Each panel shows the mutations detected in common between the laboratories (A & B) as well as those mutations detected by laboratory A but not laboratory B (A) and those detected by laboratory B but not laboratory A (B). The panels on the left show the total number of mutations (differences from consensus B) for protease and RT. The panels on the right show the number of drug resistance mutations for protease and RT.

FIG. 5

Chromatographic tracings showing the automated DNA sequence analysis of the 10 instances in which a primary resistance mutation was detected by just one laboratory (the PCC numbers at the tops of the tracings indicate patient code-codon number). In seven instances, the laboratory detecting the mutation detected it as part of a mixture, and in three instances (for patient PCC53) there was a complete mismatch between the two laboratories. The tracings for the forward and reverse sequences of each laboratory are shown (the reverse sequence at RT codon 106 for patient PCC45 at laboratory A is missing). Nucleotides with mixtures are shown in bold for laboratory A and in red for laboratory B. Laboratory A reported protease codon 90 for patient PCC25 and protease codon 48 for patient PCC26 as being of the wild type (WT) because the mutant (Mut) peak was detected in only one of the sequencing reactions. Laboratory A's reverse sequence of RT codon 181 for patient PCC45 shows a mixture of TAT and TGT. However, the minor G peak was not flagged by the ABI FACTURA program at the then-recommended cutoff of 30%. For three of the mixtures (for patient PCC25, codon 90; patient PCC45, codon 106; and patient PCC45, codon 181), the predominant nucleotide (having the larger peak) was different between the two laboratories.