Fig. 5.

a. Panels 1 and 2 refer to the resulting Ct plots (from ViiA7 run) for multiplex detection of the CpG markers m_ncr1, m_NR5A2, and m_PRKCB using as initial template fragmented and bisulfite-converted mixture of 30, and 3000 genomic copies of DNA from breast cancer cell line (MCF7 or MDA-MB-134-VI) and normal human blood (Roche human genomic DNA) respectively. The DNA fragment mixture simulates the likely constitution of patient cfDNA (i.e. majority of which are released by peripheral blood cells). Panel 3 serves as a negative control (3000 copies of genomic DNA from normal human blood). b. The Ct values for the plots depicted in A. Also shown are results from no template controls (NTCs) in various steps of the assay (PCR, LDR, qPCR). “No Ct” means no amplification was detected after 45 cycles of real time PCR. c. The fraction of methylation at a specific CpG site for the 3 CpG sites in the genomes of MCF7 and MDA-MB-134-VI cell lines, as extracted from Illumina 450 K array-generated datasets deposited in Gene Expression Omnibus (GEO). * Average values extracted from datasets GSE57342, GSE68379, GSE78875, and GSE94943. **Value extracted from dataset GSE68379