Comparison of sequencing by hybridization and cycle sequencing for genotyping of human immunodeficiency virus type 1 reverse transcriptase

The performances of two methods of nucleotide sequencing were compared for the detection of drug resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) in viruses isolated from highly RT inhibi tor-experienced individuals. Of 11,677 amino acids deduced from population PCR products by both cycle sequencing and sequencing by hybridization to hi gh-density arrays of oligonucleotide probes, 97.4% were concordant by both methods, 0.8% were discordant, and 1.7% had an ambiguous determination by a t least one method. A higher rate of discordance (3.9%) was observed among RT inhibitor resistance-associated codons, In 45% of the isolates, RT codon 67 was deduced as the wild-type Asp by hybridization sequencing but as the zidovudine resistance-associated Asn by cycle sequencing. In other resista nce-associated codon discordances, cycle sequencing also more commonly call ed a known resistance-associated amino acid than hybridization sequencing d id. The nucleotide sequence in the vicinity of several codons with discorda nt calls influenced population-based hybridization sequencing. For isolates evaluated by additional sequencing of molecular clones of PCR products by both methods, the discordance between methods was less frequent (0.4% of al l 5,994 amino acids and 0 of 494 drug resistance-associated codons), At pos itions which were discordant or ambiguous in the population sequences, the results of sequencing of clones by both methods were usually in agreement w ith the population cycle sequencing result, In summary, most RT codons were highly concordant by both methods of population-based sequencing, with dis cordances due in large part to genetic mixtures within or adjacent to disco rdant codons.