Fig. 2: DNA methylation and cfDNA-fragmentation.

a The frequency of observed CpG’s at different positions in cfDNA fragments, counted from the cfDNA break, differed between methylated and unmethylated CpG sites per healthy individual (n = 543). The bar represents the mean value and the whiskers correspond to the standard deviation. While unmethylated CpG’s showed a more equal distribution over the cfDNA fragments, methylated CpG’s showed enrichment at the beginning of cfDNA-fragments. b The preference of fragments to start at a CpG increases with higher levels of methylation of that CpG. The preference for cfDNA-fragments to start at a CpG is measured in aggregate in 543 healthy individuals by the ratio of cfDNA-fragments starting with a CpG, over the total amount of cfDNA fragments overlapping a window of ±50 bases around the first base of that CpG. The degree of methylation at each CpG is indicated as the beta value in the horizontal axis using methylation data from cfDNA of healthy individuals at ~850 K CpG positions¹¹. c The opposite relationship is seen when a CpG is preceded by a cytosine: there is a preference for cfDNA fragments to start with CCG, when the CpG in this motif is not methylated. d CpG’s on chromosome X are known to be differently methylated in male (n = 264) and female individuals (n = 279). Due to X-inactivation by methylation of CpG-islands, these CpG’s show a difference in the preference of cfDNA-fragment end-positions. With increased methylation, more fragments start with a CpG, while less fragments start with a CCG. P-values were calculated using a two-sample t-test, without correction for multiple testing. e On autosomes, we do not observe a difference in methylation between male (n = 264) and female individuals (n = 279). P-values were calculated using a two-sample t-test, without correction for multiple testing. The middle hinge in the boxplots corresponds to the median, while the lower and upper hinges correspond to the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 × interquartile range from the hinge.