Fig. 1

Infinium methylation probe design. a The difference in DNA methylation measurement process used by Illumina Infinium Type I and II probes is demonstrated with two probes targeting adjacent CpG sites in the BRCA1 promoter. Both probes are present on EPIC and HM450 platforms. b Infinium I (cg21253966) and Infinium II (cg04110421) probes targeting two adjacent CpG sites in the BRCA1 promoter region; the targeted CpG sites are highlighted in green. Each probe is designed to hybridise a 50 bp DNA sequence, underlined in blue, downstream of the targeted CpG site. c DNA methylation measurement with Infinium I probes is carried out by two beads – the unmethylated (U) bead measures the unmethylated signal and methylated (M) bead measures the methylated signal. The unmethylated signal detection for the cg21253966 probe is schematically represented on the left panel. Briefly, the unmethylated bead probe (U) sequence is designed to match bisulphite converted DNA sequence of the unmethylated locus. (Note that cytosines in both the target CpG site and all other CpG sites bound by the 50 bp probe are assumed to be unmethylated and therefore converted to Ts during bisulphite reaction.) The hybridisation of a bisulphite converted unmethylated DNA fragment to the bead enables single base extension and incorporation of a ddNTP labelled nucleotide matching the nucleotide immediately upstream of the target CpG site; in this case incorporation of an A nucleotide and signal detection in the RED channel. Hybridisation of the methylated bead probe (M), on the other hand, results in mismatch at the 3′ end of the probe and inhibition of single base extension. Detection of the methylated signal, shown on the right panel, follows similar steps. d For Infinium II probes the unmethylated and methylated signals are measured by the same bead (U/M). The bead probe sequence is designed to match bisulphite converted DNA of both the methylated and unmethylated locus. This is achieved by making the cytosine of the target CpG site the single base extension locus and replacing cytosines of all other CpG sites within the probe sequence with degenerate R bases that hybridises to both T (representing unmethylated and converted cytosine) and C (representing methylated and protected cytosine) bases. The unmethylated signal detection for the cg04110421 probe is schematically represented on the left panel. The hybridisation of the bisulphite converted unmethylated DNA fragment enables single base extension and incorporation of ddNTP labelled A nucleotide matching the unmethylated and converted cytosine at the target CpG site and signal detection on the RED channel. The detection of the methylation signal, shown on the right panel, is the same except that in this case single base extension results in incorporation of ddNTP labelled G nucleotide matching the methylated and protected cytosine at the target CpG site and signal detection on the GREEN channel