Comparative performance of high-density oligonucleotide sequencing and dideoxynucleotide sequencing of HIV type 1 pol from clinical samples.
The performance of the high-density oligonucleotide array methodology (GeneChip) in detecting drug resistance mutations in HIV-1 pol was compared with that of automated dideoxynucleotide sequencing (ABI) of clinical samples, viral stocks, and plasmid-derived NL4-3 clones. Sequences from 29 clinical samples (plasma RNA, n = 17; lymph node RNA, n = 5; lymph node DNA, n = 7) from 12 patients, from 6 viral stock RNA samples, and from 13 NL4-3 clones were generated by both methods. Editing was done independently by a different investigator for each method before comparing the sequences. In addition, NL4-3 wild type (WT) and mutants were mixed in varying concentrations and sequenced by both methods. Overall, a concordance of 99.1% was found for a total of 30,865 bases compared. The comparison of clinical samples (plasma RNA and lymph node RNA and DNA) showed a slightly lower match of base calls, 98.8% for 19,831 nucleotides compared (protease region, 99.5%, n = 8272; RT region, 98.3%, n = 11,316), than for viral stocks and NL4-3 clones (protease region, 99.8%; RT region, 99.5%). Artificial mixing experiments showed a bias toward calling wild-type bases by GeneChip. Discordant base calls are most likely due to differential detection of mixtures. The concordance between GeneChip and ABI was high and appeared dependent on the nature of the templates (directly amplified versus cloned) and the complexity of mixes.
Department of Pathology, School of Medicine, University of California at San Diego, La Jolla 92093-0679, USA.
H F
HF
Günthard
J K
JK
Wong
C C
CC
Ignacio
D V
DV
Havlir
D D
DD
Richman
D019380:Anti-HIV Agents / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D015658:HIV Infections / Q000821:virology*; D016333:HIV Protease / Q000235:genetics*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D019469:Indinavir / Q000494:pharmacology; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000097:blood; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D015215:Zidovudine / Q000494:pharmacology
D019380:Anti-HIV Agents; D017320:HIV Protease Inhibitors; D009841:Oligonucleotides; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D019469:Indinavir; D054303:HIV Reverse Transcriptase; D016333:HIV Protease