Figure 2. Reference normalization factor (RN-factor) based color channel normalization for the 450K methylation array.

(A) The 450K chip includes n=93 normalization control probes in both assay colors (red and green). The mean values of these sites are used to create RN-factors for normalizing both color channels over all samples (i.e. an experiment). The Illumina first sample normalization (IFSN) method uses the first sample’s mean red and green control probes as RN-factors ($\overline{R}$._,1 and $\overline{G}$._,1). The all sample mean normalization (ASMN) method instead uses the mean read and green control probes taken across all control sites and all samples in a given experiment ($\overline{R}$._,. and $\overline{G}$._,.) as RN-factors. (B) A set of sample-wise normalization values, taken as the ratio of the RN-factor to each sample’s mean control probe values, is then computed. This results in a vector of length n normalization values for each color channel (R-RNV and G-RNV). (C) Color channel normalization of sample data occurs by multiplying the each of the j^th sample’s red and green signals by the j^th normalization value from the corresponding RN-vector (where j=1,2,…, n).