Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group.
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.
Demeter
L M
LM
University of Rochester School of Medicine and Dentistry, NY, USA. lisa_demeter@urmc.rochester.edu
D'Aquila
R
R
Weislow
O
O
Lorenzo
E
E
Erice
A
A
Fitzgibbon
J
J
Shafer
R
R
Richman
D
D
Howard
T M
TM
Zhao
Y
Y
Fisher
E
E
Huang
D
D
Mayers
D
D
Sylvester
S
S
Arens
M
M
Sannerud
K
K
Rasheed
S
S
Johnson
V
V
Kuritzkes
D
D
Reichelderfer
P
P
Japour
A
A
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
D003062:Codon; D004279:DNA, Viral; D015215:Zidovudine; D054303:HIV Reverse Transcriptase