Fig. 4 Dual barcoding a) workflow for PCR libraries b) test case c) results after splitting barcodes
When looking at PCR amplicons or plasmids, only a small amount of data is needed. Here, it would be helpful to be able to sequence larger numbers of templates on a single flow cell. To accommodate this, we have developed PCR-based (Fig. 4a) and PCR-free dual barcoding protocols. We tested the concept by taking swabs from a variety of environments and growing bacteria from each environment on a separate agar plate (Fig. 4b). We performed 16S PCR on each colony, and barcoded each set of amplicons with an inner, colony-specific, barcode. We then made a pool of amplicons for each plate and labelled each pool with an outer, plate- specific, barcode. Fig. 4c shows the counts of each barcode combination after splitting.