Low-Pass Nanopore Sequencing of Plasma cfDNA Reveals Fragmentomic, Epigenomic, and Age-Associated Signatures Under Ultra-Low-Coverage Conditions

Circulating cell-free DNA (cfDNA) enables minimally invasive assessment of chromatin organization and DNA modifications. Whether such information can be reliably recovered under conditions of limited plasma input (below 1 mL) and ultra-low sequencing depth remains unclear. We performed low-pass whole-genome Oxford Nanopore sequencing (down to 0.01× coverage) of plasma cfDNA from young and elderly donors and jointly analyzed fragment length distributions and base modifications (5mC, 5hmC, 6mA). In parallel, we analyzed an enzymatically fragmented model DNA system to assess whether controlled in vitro fragmentation can reproduce cfDNA-like nucleosomal profiles and associated modification patterns. Despite shallow coverage, cfDNA samples displayed reproducible mono-, di-, tri-, and tetra-nucleosomal peaks, indicating that major fragmentomic features can be retained under ultra-low-coverage conditions. Modification-aware basecalling enabled exploratory quantification of global modification fractions across nucleosomal size classes and nomination of candidate group-specific modification loci. Overall, these results support the feasibility of low-pass nanopore sequencing as an exploratory framework for simultaneous cfDNA fragmentomic and epigenomic profiling in low-input studies.