title: Reproducibility of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequencing of plasma samples from heavily treated HIV-1-infected individuals.
abstract: The reproducibility of population-based human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT) sequencing was assessed using replicate aliquots of cryopreserved plasma samples obtained from seven heavily treated HIV-1-infected individuals. The sequence of each sample replicate was compared with the consensus sequence for that sample and 99.4% of 35128 amino acids were found to be concordant with the sample consensus. Partial discordances were present at 0.5% of positions and complete discordances were present at <0.1% of positions. To assess the reproducibility at detecting mutations (defined here as differences from the subtype B consensus sequence), the proportion of sequences having a mutation when at least two sequences from that sample had the same mutation were examined. There was a median of 13 protease and 18 RT mutations per sample for a total of 3126 mutations; 95% of these mutations were detected. However, sequencing of multiple clones from two samples demonstrated that those mutations present in a minority of clones were often not detected by population-based sequencing. These results suggest that HIV-1 protease and RT sequencing of circulating plasma virus is highly reproducible but that the sensitivity at detecting mutations may be low if those mutations are present as minor variants.
authors.affiliation: Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. rshafer@cmgm.stanford.edu
authors.forename: R W
authors.initials: RW
authors.lastname: Shafer
authors.forename: A
authors.initials: A
authors.lastname: Warford
authors.forename: M A
authors.initials: MA
authors.lastname: Winters
authors.forename: M J
authors.initials: MJ
authors.lastname: Gonzales
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology / Q000627:therapeutic use*; D001483:Base Sequence; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D004359:Drug Therapy, Combination; D015658:HIV Infections / Q000097:blood / Q000188:drug therapy* / Q000821:virology*; D016333:HIV Protease / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D009154:Mutation; D012367:RNA, Viral / Q000097:blood; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D019380:Anti-HIV Agents; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D016333:HIV Protease