Fig. 5

Determination of the limit of detection of methylated cfDNA spiked into plasma from healthy individuals. A serial dilution of Sss1-treated plasma cfDNA, ranging from 40 to 5 copies of methylated cfDNA, was spiked into healthy pooled plasma and analyzed with the TAM-MSP assay. Each dilution point was repeated with 6 replicates. a The Cts for ACTB, cg054 and SCG3 for all dilution replicates showed a strong reproducibility of ACTB at all spike-in dilutions. b Marker cg054 shows no amplification in the healthy plasma (0 copies of spike in DNA) in 4 of 6 replicates and had high ∆Cts. c. Marker SCG3 shows no amplification in the healthy plasma (0 copies of spike in DNA) in 4 of 6 replicates and had high ∆Cts. d The total MAF at all dilutions was significantly different from the healthy plasma without spiked-in of methylated cfDNA. The dots in the gray box represent a failure of the marker to cross the cycle threshold. N number of samples, P Mann–Whitney statistics, MAF methylation allele frequency