Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group

J Virol Methods. 1998 Nov;75(1):93-104. doi: 10.1016/s0166-0934(98)00100-1.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Codon
  • DNA, Viral / analysis*
  • Drug Resistance, Microbial
  • Gene Amplification
  • HIV Reverse Transcriptase / genetics*
  • HIV-1 / drug effects
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • Humans
  • Laboratories / standards*
  • Mutation*
  • Polymerase Chain Reaction
  • Proviruses / genetics
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods*
  • Sequence Analysis, DNA / standards
  • Zidovudine / pharmacology

Substances

  • Codon
  • DNA, Viral
  • Zidovudine
  • HIV Reverse Transcriptase