High degree of interlaboratory reproducibility of human immunodeficiency virus type 1 protease and reverse transcriptase sequencing of plasma samples from heavily treated patients.
We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.
Division of Infectious Diseases and Center for AIDS Research, Stanford University Medical Center, Stanford, California 94305, USA. rshafer@cmgm.stanford.edu
R W
RW
Shafer
K
K
Hertogs
A R
AR
Zolopa
A
A
Warford
S
S
Bloor
B J
BJ
Betts
T C
TC
Merigan
R
R
Harrigan
B A
BA
Larder
D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology / Q000627:therapeutic use; D001483:Base Sequence; D004352:Drug Resistance, Microbial / Q000235:genetics; D004359:Drug Therapy, Combination; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D016333:HIV Protease / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007753:Laboratories / Q000592:standards*; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D017422:Sequence Analysis, DNA
D019380:Anti-HIV Agents; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D016333:HIV Protease