Gs junior sequencing platform

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).

Universal primer PCR targeting the 549–567 bp cpn60 UT region was performed using a mixture of cpn60 primers consisting of a 1:3 M ratio of primers H279/H280:H1612/H1613, as described previously [41 (link)–43 (link)]. 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).
Samples were handled in small batches to avoid cross-contamination, and experimental controls were included at several steps in the study. Regular monitoring of DNA extraction controls in our lab by universal PCR confirms that these procedures are sufficient to eliminate detectable template contamination of study samples. A no template control was also included in each set of PCR reaction as negative controls. Experimental controls were not sequenced as they did not yield any amplification.