The circulating cell-free DNA landscape in sepsis is dominated by impaired liver clearance.

Circulating cell-free DNA (cfDNA) is a promising molecular biomarker, but its role in severe infection is unclear. Here, we profile cfDNA from sepsis patients and controls, demonstrating a 41-fold increase during disease. Methylation-based deconvolution revealed similar cfDNA compositions in the two groups, suggesting that cfDNA accumulation during disease is due not to excess cell death but to impaired hepatic clearance. Fragmentation and end-motif patterns both support this hypothesis, suggesting prolonged exposure of cfDNA to circulating nucleases. In addition, we show that cfDNA retains nucleosome footprints informative of gene activity. By developing a novel method to quantify these footprints and integrate them with single-cell data, we report an increase in cfDNA from Kupffer cells and liver parenchyma in patients with liver dysfunction. Finally, we show that cfDNA contains pathogen-derived material, highlighting its diagnostic potential. This high-throughput, multimodal study provides a reference for understanding cfDNA's role in sepsis and critical illness.