Fig. 1

Triple indexing design. The triple indexing strategy incorporates two PCR steps. During the first PCR (PCR1), the template sequence of interest is targeted and amplified (green). The primers for this reaction also contain an indexing sequence and a heterogeneity spacer sequence (red), and a partial Illumina adapter (blue). A second PCR (PCR2) allows for the introduction of a third indexing sequence (dark blue) as well as completion of the Illumina sequencing adapter