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Factors Influencing the Sensitivity and Specificity of Conventional Sequencing in Human Immunodeficiency Virus Type 1 Tropism Testing

Authors :
Xiaoyin Zhong
Winnie Dong
Rachel A. McGovern
P. R. Harrigan
David J.H.F. Knapp
Art F. Y. Poon
Luke C. Swenson
Conan K. Woods
Source :
Journal of Clinical Microbiology. 51:444-451
Publication Year :
2013
Publisher :
American Society for Microbiology, 2013.

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.

Details

ISSN :
1098660X and 00951137
Volume :
51
Database :
OpenAIRE
Journal :
Journal of Clinical Microbiology
Accession number :
edsair.doi.dedup.....48b6b4c70b566a6ec1378e80f85a6235
Full Text :
https://doi.org/10.1128/jcm.00739-12