A comprehensive urine workflow enables robust methylation and fragmentation analysis of cell-free DNA

Urine cell-free DNA (cfDNA) holds great promise as a non-invasive biomarker for diagnosis, prognosis and treatment monitoring of diseases. However, its clinical utility is challenged by high nuclease activity and rapid degradation, necessitating an optimized urine cfDNA workflow. We evaluated our workflow for urine cfDNA methylation and fragmentation profiling, comparing a closed, non-crosslinking urine stabilization system (PAXgene Urine Technology), with a crosslinking reagent (Streck Urine Preserve) and unstabilized urine, using enzymatic methyl sequencing of cfDNA from 20 healthy individuals, across three urine storage time points (immediate, 6 h, and 72 h). Urine stabilization significantly improved cfDNA yield and preserved cfDNA fragment sizes, while unstabilized samples exhibited marked degradation and reduced sequencing quality. Methylation analysis revealed high concordance between stabilized samples, with subtle divergence after 72 h. In contrast, unstabilized samples showed widespread methylation changes. Stabilized urine cfDNA retained key biological patterns including donor-, sex-, and tissue- methylation signatures and fragment coverage profiles around transcription start sites and exon-intron junctions, consistent with underlying chromatin features. Our study underscores the importance of well-designed (pre)analytical urine workflows to allow urine cfDNA (epi)genomic analyses. These findings align with emerging standards for pre-examination processes and support clinically compatible urine cfDNA workflows.