Figure 1

Schematic representation of EDSB-detection using Ion Torrent sequencing. (A) The red lines and parallel vertical bars represent genomic DNA and EDSB ends, respectively. The green line is the First linker sequence, where the darker shade represents the primer sequence. The blue line is the second linker sequence. The green and blue arrows represent the forward and reverse primers, respectively. In the first step, the First linker is ligated to one of the EDSB ends. Next, the DNA ligated to the first linker is digested with a restriction enzyme, RsaI, which does not cut between the linkers. After digestion, the second linker is ligated to the first linker-ligated DNA. Finally, the EDSBs were amplified with primers that bind to the first and second linkers. (B) The five PCR product patterns are shown. The red vertical bar represents the RIND-EDSBs whereas the blue vertical bar represents an RsaI break. (1) An intact sequence with both linkers. The RIND-EDSBs are between the First linker and the DNA sequence. (2) An intact sequence with Second linkers on both sides. There were no RIND-EDSBs here. (3) A sequence derived from multiple ligations with both of the linkers. (4) Multiple ligations with the Second linker only. (5) PCR products generated from nonspecific amplification due to the direct binding of the primers to the genome. These amplicons did not contain the 3’ sequence of the First linker and also lacked RIND-EDSBs.