title: Factors influencing the sensitivity and specificity of conventional sequencing in human immunodeficiency virus type 1 tropism testing.
abstract: Human immunodeficiency virus type 1 (HIV-1) V3 loop sequence can be used to infer viral coreceptor use. The effect of input copy number on population-based sequencing of the V3 loop of HIV-1 was examined through replicate deep and population-based sequencing of samples with known tropism, a heterogeneous clinical sample (624 population-based sequences and 47 deep-sequencing replicates), and a large cohort of clinical samples from phase III clinical trials of maraviroc including the MOTIVATE/A4001029 studies (n = 1,521). Proviral DNA from two independent samples from each of 101 patients from the MOTIVATE/A4001029 studies was also analyzed. Cumulative technical error occurred at a rate of 3 × 10(-4) mismatches/bp, without observed effect on inferred tropism. Increasing PCR replication increased minority species detection with an ~10% minority population detected in 18% of cases using a single replicate at a viral load of 1,072 copies/ml and in 44% of cases using three replicates. The nucleotide prevalence detected by population-based and deep sequencing were highly correlated (Spearman's ρ, 0.73), and the accuracy increased with increasing input copy number (P < 0.001). Triplicate sequencing was able to predict tropism changes in the MOTIVATE/A4001029 studies for both low (P = 0.05) and high (P = 0.02) viral loads. Sequences derived from independently extracted and processed samples of proviral DNA for the same patient were equivalent to replicates from the same extraction (P = 0.45) and had correlated position-specific scoring matrix scores (Spearman's ρ, 0.75; P << 0.001); however, concordance in tropism inference was only 83%. Input copy number and PCR replication are important factors in minority species detection in samples with significant heterogeneity.
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, British Columbia, Canada.
authors.forename: David J H F
authors.initials: DJ
authors.lastname: Knapp
authors.forename: Rachel A
authors.initials: RA
authors.lastname: McGovern
authors.forename: Winnie
authors.initials: W
authors.lastname: Dong
authors.forename: Art F Y
authors.initials: AF
authors.lastname: Poon
authors.forename: Luke C
authors.initials: LC
authors.lastname: Swenson
authors.forename: Xiaoyin
authors.initials: X
authors.lastname: Zhong
authors.forename: Conan K
authors.initials: CK
authors.lastname: Woods
authors.forename: P Richard
authors.initials: PR
authors.lastname: Harrigan
mesh_terms: D001483:Base Sequence; D056726:Genetic Association Studies; D016679:Genome, Viral; D005838:Genotype; D060005:Genotyping Techniques; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000254:growth & development; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D010641:Phenotype; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load; D056189:Viral Tropism / Q000235:genetics*; D014779:Virus Replication