![]() The majority (91%) of drug resistance mutations were detected by both Sanger and NGS, with 1.7% only by Sanger and 7.3% only by NGS. An optimum variant frequency threshold of 10% was determined by comparing Sanger sequences of archived samples from ViroSeq testing, resulting in a sensitivity of 98.2% and specificity of 99.0%. Implemented as a Torrent Suite software plugin, the pipeline runs automatically after sequencing. We developed a complete NGS protocol and companion analysis and reporting pipeline using AmpliSeq multiplex PCR, Ion Torrent S5 XL sequencing, and Stanford’s HIVdb resistance algorithm. Implementation of NGS assays in the clinical laboratory is hindered by complicated assay design, cumbersome wet bench procedures, and the complexity of data analysis and bioinformatics. Next generation sequencing (NGS) platforms enable variant detection at frequencies as low as 1% allowing for earlier detection of resistance and modification of therapy. Most resistance testing is performed with Sanger sequencing, which has limited ability to detect minor variants. ![]() HIV-1 antiretroviral therapy management requires sequencing the protease, reverse transcriptase, and integrase portions of the HIV-1 pol gene.
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