Output characteristics of the all-in-one multiplex assay versus 4 singleplex assays. (A) Relative copy number changes for the individual assays for paired samples presented as the ratio of the multiplex over the singleplex copy numbers. Dot plots for the paired copy numbers of ACTB (B), APC (C), GSTP1 (D) and RASSF1 (E) for the multiplex assay (y-axis) versus the singleplex assay (x-axis). The gray line represents identity, i.e. equal result for both versions of the assay, while the black, dashed line represents the median signal change obtained from the transition of a singleplex to a multiplex assay. The greater the angle between both lines, the higher the increase. The results from the linear fit are shown with the black, full line. The quality of the fit is represented by the adjusted R2-values. (F) Phi or Matthews correlation coefficient (MCC) in function of the methylation ratio that is used as a cutoff. The maximal value for MCC is shown as a circle, for each individual assay. Shifts due to the transition of singleplex to multiplex, between the optimal cutoffs, identified here through maximal correlation, can be observed for each assay separately.