Validation of multiplex PCR sequencing assay of SIV

Abstract Background The generation of accurate and reproducible viral sequence data is necessary to understand the diversity present in populations RNA viruses isolated from clinical samples. While various sequencing methods are available, they often require high quality templates titer ensure reliable data. Methods We modified a multiplex PCR approach characterize simian immunodeficiency virus (SIV) nonhuman primates. chose this with aim reducing number required input while maintaining fidelity sensitivity. conducted replicate experiments using different numbers quantified (vRNA) or cDNA as material. performed assays clonal SIVmac239 detect false positives, we mixed variant 24 point mutations (SIVmac239-24X) measure detection Results found that utilizing starting material had lower rate positives increased reproducibility when compared vRNA templates. This study identifies importance rigorously validating deep including samples new method low frequency variants population small Conclusions Because need generate diverse samples, SIV non-human increasing template decreased identified, was further seen used Ultimately, highlight vigorously prevent overinterpretation sample.