FIG 3.

Overview of the VISA amplicon sequencing process, from sample isolation to SNP calling and comparison to known VISA mutations. Steps 1 to 5 represent sample isolation, DNA extraction, PCR, nanopore sequencing, and variant calling, respectively. In step 1 bacteria are grown in a blood bottle (left), from which multiple colonies are cultured (right), including both the two individual VISA mutants (red and green) and the VSSA parental strain (blue). Step 2 represents DNA extraction from the pooled colonies cultured at the end of step 1, while step 3 represents PCR amplification of VISA amplicons from the extracted bacterial DNA. Step 4 shows the loading of an amplicon nanopore library on an Oxford Nanopore MinION instrument. Finally, step 5 shows the calling of the two VISA mutations in the sequenced nanopore library against reference gene sequences. Two VISA mutations are illustrated in red and green (steps 2, 3, and 5), while their parental strain is in blue.