cpn60 UT Region Universal Primer PCR

Universal primer PCR targeting the 552–558 bp cpn60 UT region was performed using a mixture of cpn60 primers consisting of a 1:3 molar ratio of primers H279/H280:H1612/H1613, as described previously^{47 (link), 48 (link), 81 (link)}. To avoid cross-contamination, samples were handled in small batches, and a no template control was included with each set of PCR reactions. To allow multiplexing of samples in a single sequencing run, primers were modified at the 5′ end with one of 24 unique decamer multiplexing identification (MID) sequences, as per the manufacturer’s recommendations (Roche, Brandford, CT, USA). Amplicons were pooled in equimolar amounts for sequencing on the Roche GS Junior sequencing platform. The sequencing libraries were prepared using the GS DNA library preparation kit and emulsion PCR (emPCR) was performed with a GS emPCR kit (Roche Diagnostics, Laval, Canada).