Design of genotyping-by-sequencing adapters for use with Ion Torrent sequencing chemistry.
1) Genomic DNA (black) is digested with a combination of PstI and MspI producing fragments with corresponding 3’ TGCA (PstI) and 5’ CG (MspI) overhangs. The barcoded forward Ion Adapter 1 (blue) is ligated to the PstI generated overhang and the common Ion Adapter 2 (green) is ligated to the MspI generated overhang. The variable bar code is in bold and the unpaired tail of the Y-adapter is underlined. 2) During the first PCR cycle, the forward primer (orange) binds to the corresponding Adapter 1 site and proceeds to synthesize the complementary strand (grey) to the genomic sequence tag and then the unpaired tail of the Y-adapter. The common MspI-MspI fragments (not shown) have a Y-adapter on both ends and lack a complementary binding site to initialize PCR amplification. 3) During the second and subsequent rounds of PCR, the reverse primer (purple) can bind to the newly synthesized complement and initialize synthesis on the reverse strand. These PCR reactions continue until completion of the fully synthesized fragments. 4) The final fragment is ready to sequence and consists of the Ion Torrent forward priming site (orange) with a bar code (blue) followed by the genomic sequence fragment (black) and the Ion Torrent reverse priming site (purple).