Fig. 1: Overview of the mDETECT assay.

a mDETECT relies on the blood-based detection of large numbers of differentially methylated regions that are specific to tumours represented by the green stripes on the grey chromosome. b To identify these regions with a high density of tumour-specific methylation, those CpG dinucleotides found to show high levels of methylation in the tumour populations but low levels in the normal tissues were identified as being differentially hypermethylated. β-values represent methylation levels at a given probe (CpG dinucleotide). c Bisulfite conversion of the DNA samples of these differentially methylated regions allows the multiple CpG residues in each region to be assessed and subsequently allows for differentiation between tumour and normal samples. d Methylation biased primers, as indicated by the arrows, are designed to preferentially but not exclusively amplify methylated DNA. This ensures that small amounts of methylated tumour DNA are amplified even in a high background of normal DNA. These PCR “probes” are designed to amplify products less than 125 bp (<125 bp) due to the highly fragmented nature of ctDNA (e). PCR is carried out by various types of multiplexing. f Next-generation sequencing (NGS) is carried out to identify multiple CpG dinucleotides that were methylated and to allow for high-level multiplexing.