Superscribe gainsville ga address3/29/2023 After sequencing, datasets are clustered according to the unique Primer-ID, and a consensus sequence for each template is generated. 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. 31 developed a Primer-ID approach which solved many of the technical artifacts and biases described above. Thus, quantification of minority variants using standard deep sequencing approaches may not be accurate. However, even with the next generation sequencing (NGS) technology, the original quasispecies structure may be distorted by technical artifacts during sample preparation, which may incorporate errors during reverse transcription and PCR, resampling errors from low copy number of viral templates, PCR amplification bias, and sequencing errors from Roche/454 or Illumina sequencing. Numerous studies have used deep sequencing methods such as Roche/454 pyrosequencing and Illumina sequencing to detect minority HIV variants 23– 30. However, the level at which minority variants become clinically relevant remains unclear. Many studies have shown that minority drug resistant variants can compromise response to therapy 5, 6, 9– 22. 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. In contrast, genotypic testing by Sanger Sequencing is the preferred approach for most patients beginning or switching ART. However, because phenotypic assays are time-consuming and expensive, they are generally reserved for patients with known or suspected complex drug-resistance patterns. Currently, genotypic and phenotypic assays are used to identify HIV drug resistance in clinical settings. The presence of drug resistant HIV in the viral population is known to compromise virologic response to ART 4– 6. As antiretroviral drug coverage increases and patients become more treatment experienced, the prevalence of HIV drug resistance is expected to increase 3. There are approximately 37.9 million people living with HIV 1, 2. 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. In addition, the measured frequencies were comparable to the expected frequencies. Our results showed that minority variants present at 1% of quasispecies were detected reproducibly with minimal variations between technical replicates. 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. Thus, its sensitivity and quantitative accuracy have not been adequately validated using known controls. However, most studies that used the Primer-ID method have analyzed clinical samples directly. A combination of Primer-ID and deep sequencing is a promising approach that may quantify minority variants more accurately compared to standard deep sequencing. Many studies have investigated the clinical relevance of drug resistant minority variants, but the level at which minority variants become clinically relevant remains unclear. Drug resistant HIV variants, including minority variants, can compromise response to antiretroviral therapy. HIV drug resistance is a major threat to achieving long-term viral suppression in HIV-positive individuals.
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