Abstract
HIV drug resistance is a major threat to achieving long-term viral suppression in HIV-positive individuals. Drug resistant HIV variants, including minority variants, can compromise response to antiretroviral therapy. Many studies have investigated the clinical relevance of drug resistant minority variants, but the level at which minority variants become clinically relevant remains unclear. A combination of Primer-ID and deep sequencing is a promising approach that may quantify minority variants more accurately compared to standard deep sequencing. However, most studies that used the Primer-ID method have analyzed clinical samples directly. Thus, its sensitivity and quantitative accuracy have not been adequately validated using known controls. Here, we constructed defined proportions of artificial RNA and virus quasispecies and measured their relative proportions using the Primer-ID based, quantitative single-variant sequencing (qSVS) assay. Our results showed that minority variants present at 1% of quasispecies were detected reproducibly with minimal variations between technical replicates. In addition, the measured frequencies were comparable to the expected frequencies. These data validate the accuracy and reproducibility of the qSVS assay in quantifying authentic HIV minority variants, and support the use of this approach to examine the impacts of minority HIV variants on virologic response and clinical outcome.
Highlights
HIV drug resistance is a major threat to achieving long-term viral suppression in HIV-positive individuals
While the Sanger Sequencing method is informative in many settings, it is insensitive in detecting minority variants circulating in less than 20% of the viral quasispecies[7,8]
Since each starting RNA template is tagged with a unique Primer-ID barcode, errors originating from subsequent steps such as nucleotide misincorporation and deep sequencing, allelic skewing from differential amplification, and template resampling, are corrected using this approach
Summary
HIV drug resistance is a major threat to achieving long-term viral suppression in HIV-positive individuals. The measured frequencies were comparable to the expected frequencies These data validate the accuracy and reproducibility of the qSVS assay in quantifying authentic HIV minority variants, and support the use of this approach to examine the impacts of minority HIV variants on virologic response and clinical outcome. Jabara et al.[31] developed a Primer-ID approach which solved many of the technical artifacts and biases described above This approach relies on the use of random sequence tags in the reverse transcription primer such that each RNA template from the original quasispecies population receives a unique Primer-ID. The resulting population of consensus sequences should accurately represent the original quasispecies population, and the approach holds great promise for quantifying minority variants in clinical samples. We constructed artificial RNA pools and artificial viral quasispecies and determined the frequency of minority HIV variants using a Primer-ID, quantitative single variant sequencing assay. The goal was to validate using known controls that the assay can detect minority variants reproducibly at or above 1% of the HIV-1 quasispecies with high precision
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