Short single-stranded DNAs with putative non-canonical structures comprise a new class of plasma cell-free DNA

Abstract

Background Cell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations. Results We aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments. Conclusions We propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

Characteristics, origin, and potential for cancer diagnostics of ultrashort plasma cell-free DNA

“…Using MB-ssDNA, we observed an average 35-fold higher recovery of US fragments compared to the other methods (86-, 13-, and fivefold higher as compared to SC-dsDNA, MB-dsDNA, SC-ssDNA, respectively). A report released during the revision of this manuscript using alternative chemistry for DNA extraction also suggested a better recovery of US fragments (Hisano et al 2021). The US fragments observed in our study were relatively enriched in plasma from healthy individuals as compared to cancer patients (Fig.…”

Plasma contains ultrashort single-stranded DNA in addition to nucleosomal cell-free DNA

“…However, sequences shorter than 50bp may have greater intergenic proportion which would result in the dilution of sequences in coding regions for SPRI extracted samples ( Figure 4 C). During our manuscript revisions we discovered that three other research groups (one unpublished) also isolated ultrashort single-strand fragments in plasma using distinctly different methods ( Cheng et al., 2021 ; Hisano et al., 2021 ; Hudecova et al., 2021 ). There would be interest to cross-compare these methods in future studies.…”

Tracing the Origin of Cell-Free DNA Molecules through Tissue-Specific Epigenetic Signatures

“…It has become increasingly clear during the last years that another cfDNA population exists in plasma that might exhibit diagnostic potential: ultrashort single-stranded cfDNA fragments up to 100 bp [ 20 , 167 , 168 , 169 , 170 ]. These fragments are often omitted by conventional library preparation due to the adapter ligation process that needs double strands and/or applied size selection procedures.…”

“…An early study employing single-stranded library preparation and subsequent sequencing demonstrated that short cfDNA molecules directly footprint TFBS occupancy [ 20 ]. Recent efforts optimizing different DNA extraction and library preparation procedures independently demonstrated the presence of this novel population of cfDNA molecules that might inform on gene regulatory regions and DNA secondary structures [ 168 , 169 , 170 ].…”