FIGURE 1.

Modified Circle‐Seq method for mapping ucf‐eccDNAs. (A) Workflow of cell‐free eccDNA purification and identification from urine samples. Total cell‐free DNAs consisting of both circular and linear DNAs were isolated from the urine samples. To purify circular DNAs, linear DNA molecules were removed by exonuclease digestion. The enriched circular DNAs were then amplified by rolling circle amplification (RCA). The RCA‐amplified products were finally subjected to library construction and sequencing. Circle‐Map software was used to detect eccDNAs from sequencing data based on plit read and discordant read pairs. (B‐E) GC content and length distributions of ucf‐eccDNAs from healthy individuals (pooled data from 28 cases). (B) GC content distribution of ucf‐eccDNAs, in silico eccDNAs and their downstream and upstream regions of equivalent length. (C) Heatmap showing the GC content of 4000 randomly selected ucf‐eccDNAs and their downstream and upstream regions of equivalent length. (D) Length distribution of ucf‐eccDNAs. (E) Cumulative frequency plot of ucf‐eccDNAs. Ucf‐eccDNAs: urinary cell‐free extrachromosomal circular deoxyribonucleic acid