Reproducibility of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequencing of plasma samples from heavily treated HIV-1-infected individuals

The reproducibility of population-based human immunodeficiency virus type 1 (HIV-I) protease and reverse transcriptase (RT) sequencing was assessed us ing replicate aliquots of cryopreserved plasma samples obtained from seven heavily treated HIV-1-infected individuals. The sequence of each sample rep licate was compared with the consensus sequence for that sample and 99.4% o f 35128 amino acids were found to be concordant with the sample consensus. Partial discordances were present at 0.5% of positions and complete discord ances were present at <0.1% of positions. To assess the reproducibility at detecting mutations (defined here as differences from the subtype B consens us sequence), the proportion of sequences having a mutation when at least t wo sequences from that sample had the same mutation were examined. There wa s a median of 13 protease and 18 RT mutations per sample for a total of 312 6 mutations; 95% of these mutations were detected. However, sequencing of m ultiple clones from two samples demonstrated that those mutations present i n a minority of clones were often not detected by population-based sequenci ng. These results suggest that HIV-I protease and RT sequencing of circulat ing plasma virus is highly reproducible but that the sensitivity at detecti ng mutations may be low if those mutations are present as minor variants. ( C) 2000 Elsevier Science B.V. All rights reserved.