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

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV -1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 36 heavily treated HIV-1-infected individuals in two laboratories using: dideoxynucleotide sequencing. The rates of comp lete sequence concordance between the two laboratories were 99.1% for the p rotease sequence and 99.0% for the RT sequence. Approximately 90% of the di scordances 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 laborato ries, and these were significantly more likely to occur in plasma samples w ith lower plasma HIV-I RNA levels, Nucleotide mixtures were detected at app roximately 1% of the nucleotide positions, and in every case in which one l aboratory detected a mixture, the second laboratory either detected the sam e mixture or detected one of the mixture's components. The high rate of con cordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were cause d by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.