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 populationbased 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. Copyright © 2013, American Society for Microbiology.