Example of the effect of abnormally low fluorescence readouts. a. Upper panel: Individual β values for 36 cell lines examined using the type I probe (cg10428733) interrogating CpG site with a SNP (C>T) in the first position. Only high and intermediate β values are observed, in cell lines with CpG (blue), TpG (yellow) and CpG/TpG (red) genotypes. Lower panel: Fluorescence readouts for the probe cg10428733. Signals are detected as a result of the extension of M (green) and U (red) probes. All lines with homozygous CpG genotypes display strong green and no red signal, indicating methylated status. In lines with homozygous TpG genotypes fluorescence signals from both probes are extremely low (only one colour is visible, due to the overlapping red and green signals). In heterozygotes the only readable signal comes from M probes. b. Sequences of the cg10428733 probes M and U, aligned with the genomic sequence (shown in opposed orientation) with the C>T substitution in the first position of the interrogated dinucleotide (bold and underlined). Three additional CpG sites under the probes (marked in red) appear to have the same methylation status as the CpG allele in the interrogated site. Neither U nor M probe would hybridize efficiently to the TpG allele: the M probe due to the mismatch at the interrogated TpG, and the U probe in addition, due to the mismatch with methylated under-a-probe CpGs, localized close to the 3′ end of the probe.