Nanopore sequencing of amplicons to detect vancomycin-intermediate Staphylococcus aureus

In this study we performed pilot experiments to develop a combined PCR/long-read sequencing-based method for detection previously known VISA-causing mutations. We amplified 16 genes (walR, walK, rpoB, graR, graS, vraF, vraG, stpI, vraR, vraS, agrA, sarA, clpP, ccpA, prsA, and yvqF) found in prior studies to be associated with mutations responsible for VISA as 10 amplicons and sequenced amplicon pools as long reads with Oxford Nanopore adapter ligation on Flongle flow cells. We then detected mutations by mapping reads against a parental consensus or known reference sequence and comparing called variants against a database of known VISA mutations from laboratory selection.