10.1371/journal.pone.0076925.g001
Martin Mascher
Martin
Mascher
Shuangye Wu
Shuangye
Wu
Paul St. Amand
Paul
St. Amand
Nils Stein
Nils
Stein
Jesse Poland
Jesse
Poland
Design of genotyping-by-sequencing adapters for use with Ion Torrent sequencing chemistry.
Public Library of Science
2013
genotyping-by-sequencing
adapters
ion
torrent
sequencing
2013-10-03 01:53:06
Figure
https://plos.figshare.com/articles/figure/_Design_of_genotyping_by_sequencing_adapters_for_use_with_Ion_Torrent_sequencing_chemistry_/813551
<p>1) Genomic DNA (black) is digested with a combination of <i>Pst</i>I and <i>Msp</i>I producing fragments with corresponding 3’ TGCA (<i>Pst</i>I) and 5’ CG (<i>Msp</i>I) overhangs. The barcoded forward Ion Adapter 1 (blue) is ligated to the <i>Pst</i>I generated overhang and the common Ion Adapter 2 (green) is ligated to the <i>Msp</i>I 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 <i>Msp</i>I-<i>Msp</i>I 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).</p>