Analysis of recurrently protected genomic regions in cell-free DNA found in urine

Cell-free DNA (cfDNA) in urine is a promising analyte for noninvasive diagnostics. However, urine cfDNA is highly fragmented. Whether characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we characterized fragmentation patterns in urine cfDNA using whole-genome sequencing. Size distribution of urine cfDNA fragments showed multiple strong peaks between 40 and 120 base pairs (bp) with a modal size of 81- and sharp 10-bp periodicity, suggesting transient protection from complete degradation. These properties were robust to preanalytical perturbations, such as at-home collection and delay in processing. Genome-wide sequencing coverage of urine cfDNA fragments revealed recurrently protected regions (RPRs) conserved across individuals, with partial overlap with nucleosome positioning maps inferred from plasma cfDNA. The ends of cfDNA fragments clustered upstream and downstream of RPRs, and nucleotide frequencies of fragment ends indicated enzymatic digestion of urine cfDNA. Compared to plasma, fragmentation patterns in urine cfDNA showed greater correlation with gene expression and chromatin accessibility in epithelial cells of the urinary tract. We determined that tumor-derived urine cfDNA exhibits a higher frequency of aberrant fragments that end within RPRs. By comparing the fraction of aberrant fragments and nucleotide frequencies of fragment ends, we identified urine samples from cancer patients with an area under the curve of 0.89. Our results revealed nonrandom genomic positioning of urine cfDNA fragments and suggested that analysis of fragmentation patterns across recurrently protected genomic loci may serve as a cancer diagnostic.

Automated summary

Urinary cfDNA fragments exhibit specific patterns of fragmentation, suggesting temporary protection and robustness against preanalytical perturbations. The genome-wide sequencing coverage revealed protected regions conserved across individuals, with potential implications for cancer diagnostics. Furthermore, the fragmentation patterns of urine cfDNA show correlations with gene expression and chromatin accessibility in epithelial cells of the urinary tract, indicating their potential use in cancer diagnostics.