Fig. 3

Predicted type I probe signal for individuals with a SBE site-associated SNP. For Type I probes, the SBE is located 1 bp upstream of the C site for interrogations on the forward strand, and 2 bp downstream of the C site for interrogations on the reverse strand (defining the C site location using the forward strand). Enumerating signal expectations requires consideration of bisulfite conversion, complementary bases, the expected color channel for fluorescence, and if those latter two factors change in the presence of SNP. Of note is that C and G bases are labeled to fluoresce green signal, while A and T bases are labeled to fluoresce red signal (hence the existence of “Type I Red” and “Type I Green” probes). For example, consider a forward strand type I probe with a C nucleotide at the SBE position, based on a reference genome sequence (top row). After bisulfite conversion, this base will change to a T, the complementary SBE base is an A, which fluoresces in the red channel. If instead of a C there is a G at the SBE due to a C/G SNP, the SBE-incorporated nucleotide would be a C and fluoresce in the green channel. Because the software is programmed to read only the red channel, no fluorescent signal will be detected when a G SNP is present. Inferring the scenarios for interrogating a CpG site on the reverse strand requires similar reasoning but with the added consideration of complementary bases. N/A not applicable (that SNP cannot exist there), S signal, NS no signal