Schematic illustration for CpG-associated nucleosomal pattern analysis of cfDNA molecules. cfDNA molecules were aligned to the human reference genome and analyzed according to the genomic positions relative to the CpG sites, spanning multinucleosomal distance. To dissect the relationship between the nucleosomal patterns and DNA methylation, we determined DMSs between blood cells that are major contributors to plasma DNA and a targeted tissue of interest and deduced the nucleosomal patterns associated with genomic positions surrounding DMSs. Two types of DMSs are involved in this study. Type-A DMSs are CpG sites that exhibit hypomethylation in blood cells but are hypermethylated in a specific tissue of interest. Conversely, type-B DMSs are hypermethylated in blood cells and hypomethylated in the specific tissue. The nucleosomal pattern is defined as the proportion of cfDNA molecules fully spanning a window (e.g., 140 bp) centered at each queried genomic position. Making use of such nucleosomal patterns allows cancer detection and tissue-of-origin analysis for different pathophysiological states (e.g., pregnancy and cancer).