Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics

Abstract

To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole-genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP, n = 7) and using quantitative PCR (Q-PCR, n = 116). The size profile in healthy individuals was remarkably homogenous when using DSP sequencing or SSP sequencing. CfDNA size profile had a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes, and chromatin of higher molecular size (>1000 bp) can be estimated as 67.5% to 80%, 9.4% to 11.5%, and 8.5% to 21.0%, respectively. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97%–98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between metastatic colorectal cancer patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content.

Plasma derived cell-free mitochondrial DNA originates mainly from circulating cell-free mitochondria

“…This process has also provided the following observations: (i), jagged dsDNA appeared by far the most present cirDNA structural forms; (ii), the lowest cirDNA fragment size is about 70 bp; and (iii), this might be due to the low hybridization forces of the lower-sized jagged fragments leading to their peeling off from histone/nucleosome particles (29). Altogether, our data suggest that the proportion of cir-nDNA inserted in mono-nucleosomes, di-nucleosomes, and chromatin of higher molecular size (<1000 bp) in healthy individuals ranges from 67 to 80%, from 9 to 12%, and from 8 to 21%, respectively (29).…”

“…Subsequent works using WGS analysis confirmed this in greater detail (25–27,50,51). In addition, WGS-assisted fragmentomics revealed nucleosome footprints, which indicated that detected short fragments (30 to 400 bp) derive from the nucleosome around which cirDNA is packed and relatively stabilized in the blood compartment (25,27,29). Furthermore, Shendure’s team (25) inferred tissue of origins from nucleosome occupancy as determined by WGS.…”

“…Holdenrieder et al suggested that cirDNA were mainly packed in nucleosomes (24), and several reports have shown that cirDNA associated structures have nucleosome footprints (25–27). We recently demonstrated that cirDNA are mainly compacted within mono-nucleosomes, which apparently constitutes their most stable form, while di- or oligo-nucleosomes or larger pieces of cirDNA constitute a very minor fraction of its population (28,29). In addition, we demonstrated that blunted and jagged double-stranded DNA (dsDNA) of size up to 220 bp and down to 70 bp are packed in nucleosome/chromatosome particles (29).…”

“…We recently demonstrated that cirDNA are mainly compacted within mono-nucleosomes, which apparently constitutes their most stable form, while di- or oligo-nucleosomes or larger pieces of cirDNA constitute a very minor fraction of its population (28,29). In addition, we demonstrated that blunted and jagged double-stranded DNA (dsDNA) of size up to 220 bp and down to 70 bp are packed in nucleosome/chromatosome particles (29). Q-PCR assisted data on cirDNA distribution corresponded to data obtained from low-pass whole genome sequencing (WGS) performed using single-stranded DNA (ssDNA) library preparation (SSP) rather than dsDNA library preparation (DSP), allowing the harmonization of data obtained from both techniques (28).…”

“…To this end, we first explored cir-mtDNA’s structural features, using the analytical strategy of combined Q-PCR and low-pass WGS (LP-WGS) analysis, from both DSP and SSP, which provides a determination of the fractional distribution over a wide size range (28,29). This pointed to unexplained structural features, and led us to correlate these data with observations made using physical examination.…”