Fig 6. 2-step PCR method for deep sequencing preparation of libraries.

PCR reactions are shown for two separate gene tiles containing single mutations (orange and green). Primers are designed to be complementary to flanking regions (grey) of each tile, with encoded single mutations. The first set of primers includes the flanking regions and Illumina sequencing primers (purple). In the next step, outer primers add the Illumina adaptor (pink) and multiplexing index (teal) sequences to the gene. The PCR reaction is performed in a single tube using a 1:2 molar ratio of inner to outer primers and bead purified to remove primer dimer products. The purified library is ready for sequencing without further modifications. While the first set of primers is specific to a single gene tile, the outer primer set is universal.