Unraveling cfDNA Fragmentation Patterns: Linking Chromatin Architecture to Cancer Diagnostic Sensitivity

Summary Circulating cell-free DNA (cfDNA) liquid biopsies offer a powerful, non-invasive approach for cancer diagnostics, leveraging DNA fragments primarily released during apoptosis. Using droplet digital PCR on cfDNA samples from 88 breast and colorectal cancer patients, we uncovered highly consistent nucleosome breakpoints across individuals. Proximity to these cfDNA breakpoints diminishes the sensitivity of somatic mutation assays, including those targeting IDH1 and NRAS hotspot mutations, underscoring the importance of precise nucleosome positioning data. We introduce a probabilistic nucleosome protection scoring method that enables high-resolution mapping of nucleosome positions and cfDNA breakpoints. These maps reveal distinct nucleosome spacing patterns between heterochromatin and euchromatin, corresponding to different chromatin fiber topologies. Analysis of X-chromosome inactivation further supports the topoisomer model: heterochromatin’s 187 bp nucleosome repeat length differs from euchromatin’s 182 bp, generating wave interference patterns in nucleosome signals. Our findings suggest that loop-mediated transitions from T2 to T1 topoisomers shape chromatin accessibility and cfDNA fragmentation, advancing the diagnostic potential of liquid biopsy assays. Highlights Nucleosome breakpoint conservation impacts somatic mutation assay sensitivity Probabilistic protection scoring enhances cfDNA nucleosome peak resolution Chromatin states exhibit unique nucleosome spacing linked to distinct topologies, influencing cfDNA fragmentation Chromosome X-inactivation phasing analyses reveal waveform interference in nucleosome positioning signals Graphical Abstract