Fig. 7

Diagram of dual index, four-primer amplicon-bisulfite PCR. The bisulfite converted template DNA (1) is amplified in an initial cycle with primers containing a sequence specific region (green) and overhanging ‘handle’ sequence (blue) (2). After amplification, the handle is incorporated into the amplicon (3). A second amplification cycle is completed with primers complementary to the handle (blue) and an overhang consisting of a multiplex sequencing index and Illumina adapters (red) (4) such that the indexes and adapter are incorporated into the final PCR product (5)