Fig 4. Day-to-day variation before and after normalization.

(A) Unsupervised PCA analysis of un-normalized log2 transformed raw data. The samples were colored with respect to different rounds (days) of analysis. (B) Random Forest supervised classification differentiates between different days of analysis. (C) Unsupervised PCA analysis of processed data. The data was normalized using the “subtract by group mean” approach. (D) Random Forest classifier applied after normalization step. (E) Systemic technical variation, expressed in terms of CV, with respect to mean (median) inter- and intra-day as well as mean intra-slide variations.