Fig. 2

Schematic representation of biomarker discovery and validation pipeline. Infinium HumanMethylation450K Beadchip® array data were used to evaluate the methylation status of CpG sites in CRC, PBL, normal mucosa, and other cancer types (left panel). We excluded CpG sites that were methylated in blood cells and CpG sites with low methylation in CRC. The remaining 6700 CpG sites were ranked according to CRC sensitivity and specificity against other common cancers. To confirm uniform methylation in genomic regions of candidate CpG sites, bisulfite Sanger sequencing was performed on paired samples of CRC, PBL, and normal colorectal mucosa. Twenty-nine of the top 50 CpG sites were located in regions compatible with successful methylation-specific ddPCR assay design. A total of 58 methylation-specific ddPCR assays were designed for the 29 CpG sites (markers) and tested in a sequence of validation steps (right panel). Assays were excluded if their performance was suboptimal in methylated and unmethylated control DNA, PBLs, and CRC tissue or in plasma from CRC patients. Three markers passed all selection criteria. CRC colorectal cancer, PBL peripheral blood leukocytes, ddPCR droplet digital PCR