Background Extrachromosomal circular deoxyribonucleic acid (eccDNA) is evolving as a valuable biomarker, while little is known about its presence in urine. Methods Here, we report the discovery and analysis of urinary cell‐free eccDNAs (ucf‐eccDNAs) in healthy controls and patients with advanced chronic kidney disease (CKD) by Circle‐Seq. Results Millions of unique ucf‐eccDNAs were identified and comprehensively characterised. The ucf‐eccDNAs are GC‐rich. Most ucf‐eccDNAs are less than 1000 bp and are enriched in four pronounced peaks at 207, 358, 553 and 732 bp. Analysis of the genomic distribution of ucf‐eccDNAs shows that eccDNAs are found on all chromosomes but enriched on chromosomes 17, 19 and 20 with a high density of protein‐coding genes, CpG islands, short interspersed transposable elements (SINEs) and simple repeat elements. Analysis of eccDNA junction sequences further suggests that microhomology and palindromic repeats might be involved in eccDNA formation. The ucf‐eccDNAs in CKD patients are significantly higher than those in healthy controls. Moreover, eccDNA with miRNA genes is highly enriched in CKD ucf‐eccDNA. Conclusions This work discovers and provides the first deep characterisation of ucf‐eccDNAs and suggests ucf‐eccDNA as a valuable noninnvasive biomarker for urogenital disorder diagnosis and monitoring.

Urine‐based liquid biopsy has emerged as a non‐invasive and effective tool for early screening and diagnosis of bladder cancer. This review provides a comprehensive overview of the current urine‐based biomarkers and methods for the detection and monitoring of bladder cancer. We focus on biomarkers including tumour DNAs, proteins, microbiome, tumour RNAs, long non‐coding RNAs, transfer RNA‐derived fragments, messenger RNAs, microRNAs, circular RNAs, exosomes and extrachromosomal circular DNA.