title: Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group.
abstract: Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.
authors.affiliation: University of Rochester School of Medicine and Dentistry, NY, USA. lisa_demeter@urmc.rochester.edu
authors.forename: L M
authors.initials: LM
authors.lastname: Demeter
authors.forename: R
authors.initials: R
authors.lastname: D'Aquila
authors.forename: O
authors.initials: O
authors.lastname: Weislow
authors.forename: E
authors.initials: E
authors.lastname: Lorenzo
authors.forename: A
authors.initials: A
authors.lastname: Erice
authors.forename: J
authors.initials: J
authors.lastname: Fitzgibbon
authors.forename: R
authors.initials: R
authors.lastname: Shafer
authors.forename: D
authors.initials: D
authors.lastname: Richman
authors.forename: T M
authors.initials: TM
authors.lastname: Howard
authors.forename: Y
authors.initials: Y
authors.lastname: Zhao
authors.forename: E
authors.initials: E
authors.lastname: Fisher
authors.forename: D
authors.initials: D
authors.lastname: Huang
authors.forename: D
authors.initials: D
authors.lastname: Mayers
authors.forename: S
authors.initials: S
authors.lastname: Sylvester
authors.forename: M
authors.initials: M
authors.lastname: Arens
authors.forename: K
authors.initials: K
authors.lastname: Sannerud
authors.forename: S
authors.initials: S
authors.lastname: Rasheed
authors.forename: V
authors.initials: V
authors.lastname: Johnson
authors.forename: D
authors.initials: D
authors.lastname: Kuritzkes
authors.forename: P
authors.initials: P
authors.lastname: Reichelderfer
authors.forename: A
authors.initials: A
authors.lastname: Japour
mesh_terms: D003062:Codon; D004279:DNA, Viral / Q000032:analysis*; D004352:Drug Resistance, Microbial; D005784:Gene Amplification; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007753:Laboratories / Q000592:standards*; D009154:Mutation; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D003062:Codon; D004279:DNA, Viral; D015215:Zidovudine; D054303:HIV Reverse Transcriptase