Abstract
Single-stranded ends of double-stranded DNA (jagged ends) are more abundant in urinary DNA than in plasma DNA. However, the lengths of jagged ends in urinary DNA remained undetermined, as a previous method used for urinary DNA jagged end sequencing analysis (Jag-seq) relied on unmethylation at CpG sites, limiting the resolution. Here, we performed high-resolution Jag-seq analysis using methylation at non-CpG cytosine sites, allowing determination of exact length of jagged ends. The urinary DNA bore longer jagged ends (~26-nt) than plasma DNA (~17-nt). The jagged end length distribution displayed 10-nt periodicities in urinary DNA, which were much less observable in plasma DNA. Amplitude of the 10-nt periodicities increased in patients with renal cell carcinoma. Heparin treatment of urine diminished the 10-nt periodicities. The urinary DNA jagged ends often extended into nucleosomal cores, suggesting potential interactions with histones. This study has thus advanced our knowledge of jagged ends in urine DNA.
Highlights
A number of recent efforts have been taken to explore the biological properties and clinical utilities of urinary cell-free DNA molecules[1,2]
It was reported that the use of methylation signal at CH (H: A, C, or T) sites allowed the determination of exact length of jagged ends, on the basis of enhanced jagged end sequencing analysis (Jag-seq)[4]
For data generated by an experimental protocol without exonuclease T (Exo T) (Fig. 2a), we observed that 99.9% of nucleotides at the first base relative to the 3′ end were determined to be Cs, whereas only 0.2% of nucleotides at the second base relative to the 3′ end were determined to be Cs
Summary
A number of recent efforts have been taken to explore the biological properties and clinical utilities of urinary cell-free DNA molecules[1,2]. The previous study regarding urinary DNA jagged ends was based on the detection of unmethylated CpG sites introduced during the DNA end-repair process[1]. It was reported that the use of methylation signal at CH (H: A, C, or T) sites allowed the determination of exact length of jagged ends, on the basis of enhanced jagged end sequencing analysis (Jag-seq)[4]. Such methylation signals at CH sites were introduced during the DNA end-repair process with the use of methylated cytosines instead of unmethylated cytosines[4]
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