Cell-free DNA in plasma has been used for noninvasive prenatal testing and cancer liquid biopsy. The physical properties of cell-free DNA fragments in plasma, such as fragment sizes and ends, have attracted much recent interest, leading to the emerging field of cell-free DNA fragmentomics. However, one aspect of plasma DNA fragmentomics as to whether double-stranded plasma molecules might carry single-stranded ends, termed a jagged end in this study, remains underexplored. We have developed two approaches for investigating the presence of jagged ends in a plasma DNA pool. These approaches utilized DNA end repair to introduce differential methylation signals between the original sequence and the jagged ends, depending on whether unmethylated or methylated cytosines were used in the DNA end-repair procedure. The majority of plasma DNA molecules (87.8%) were found to bear jagged ends. The jaggedness varied according to plasma DNA fragment sizes and appeared to be in association with nucleosomal patterns. In the plasma of pregnant women, the jaggedness of fetal DNA molecules was higher than that of the maternal counterparts. The jaggedness of plasma DNA correlated with the fetal DNA fraction. Similarly, in the plasma of cancer patients, tumor-derived DNA molecules in patients with hepatocellular carcinoma showed an elevated jaggedness compared with nontumoral DNA. In mouse models, knocking out of the Dnase1 gene reduced jaggedness, whereas knocking out of the Dnase1l3 gene enhanced jaggedness. Hence, plasma DNA jagged ends represent an intrinsic property of plasma DNA and provide a link between nuclease activities and the fragmentation of plasma DNA.

Cell-free DNA (cfDNA) in bodily fluids has rapidly transformed the development of noninvasive prenatal testing, cancer liquid biopsy, and transplantation monitoring. Plasma cfDNA consists of a mixture of molecules originating from various bodily tissues. The study of the fragmentation patterns of cfDNA, also referred to as ‘fragmentomics’, is now an actively pursued area of biomarker research. Clues that cfDNA fragmentation patterns might carry information concerning the tissue of origin of cfDNA molecules have come from works demonstrating that circulating fetal, tumor-derived, and transplanted liver-derived cfDNA molecules have a shorter size distribution than the background mainly of hematopoietic origin. More recently, an improved understanding of cfDNA fragmentation has provided many emerging fragmentomic markers, including fragment sizes, preferred ends, end motifs, single-stranded jagged ends, and nucleosomal footprints. The intrinsic biological link between activities of various DNA nucleases and characteristic fragmentations has been demonstrated. In this review, we focus on the biological properties of cell-free DNA unveiled recently and their potential clinical applications.

Background Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends. Methods Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing. Results The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83. Conclusions Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.