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

Abstract

Current evidence suggests that plasma cell-free DNA (cfDNA) is fragmented around a mode of 166 bp. Data supporting this view has been mainly acquired through the analysis of double-stranded cfDNA. The characteristics and diagnostic potential of single-stranded and damaged double-stranded cfDNA in healthy individuals and cancer patients remain unclear. Here, through a combination of high-affinity magnetic bead-based DNA extraction and single-stranded DNA sequencing library preparation (MB-ssDNA), we report the discovery of a large proportion of cfDNA fragments centred at ~50 bp. We show that these 'ultrashort' cfDNA fragments have a greater relative abundance in plasma of healthy individuals (median = 19.1% of all sequenced cfDNA fragments, n = 28) than in plasma of patients with cancer (median = 14.2%, n = 21, P < 0.0001). The ultrashort cfDNA fragments map to accessible chromatin regions of blood cells, particularly in promoter regions with the potential to adopt G-quadruplex (G4) DNA secondary structures. G4-positive promoter chromatin accessibility is significantly enriched in ultrashort plasma cfDNA fragments from healthy individuals relative to patients with cancers (P < 0.0001), in whom G4-cfDNA enrichment is inversely associated with copy number aberration-inferred tumor fractions. Our findings redraw the landscape of cfDNA fragmentation by identifying and characterizing a novel population of ultrashort plasma cfDNA fragments. Sequencing of MB-ssDNA libraries could facilitate the characterization of gene regulatory regions and DNA secondary structures via liquid biopsy. Our data underline the diagnostic potential of ultrashort cfDNA through classification for cancer patients.

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 During our manuscript revisions we discovered that three other research groups (one unpublished) also isolated ultrashort single-strand fragments in plasma using distinctly different methods ( Cheng et al., 2021 ; Hisano et al., 2021 ; Hudecova et al.,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.…”

Real-time analysis of the cancer genome and fragmentome from plasma and urine short and long cell-free DNA using Nanopore sequencing

“…Current strategies based on either tumor-naïve or tumor-guided sequencing are reaching high level of sensitivity for detecting tumor-derived signal in plasma of cancer patients (Chabon et al, 2020; Shen et al, 2018; Wan et al, 2020; Zviran et al, 2020). They can also determine and leverage the biological properties of cfDNA (Hudecova et al, 2022; Lo et al, 2021; Mouliere et al, 2018).…”

New Perspectives on the Importance of Cell-Free DNA Biology

“…While more research is needed, this represents an alternative view to the view in which the presence of mono-nucleosomes is ascribed mainly to apoptosis. In addition to HMW cfDNA and mono-nucleosomes, a growing number of studies are demonstrating the presence of sub-nucleosomal and ultrashort, single-stranded cfDNA fragments in body fluids [ 106 , 184 , 252 , 460 , 461 , 462 , 463 , 464 ]. Whether these shortened cfDNA fragments represent short fragments released from cells or extracellularly digested products of longer fragments is not yet clear.…”