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.
Highlights
Using Q-PCR to examine fragment size, we initially demonstrated that (a) cell-free DNA (cfDNA) is highly fragmented [16, 17]; (b) cfDNA quantification is more efficient at lower amplicon sizes; and (c) cfDNA fragments can be as small as 45 bp [9, 18]
Sizing by whole-genome sequencing (WGS) allows the precise measurement of cfDNA fragments below approximately 1000 bp
Conventional DSP sequencing (DSP-S)–derived size distribution relies on double-strand breaks in the DNA molecule, whereas size profiling by SSP-S can reveal the level of nicks on both strands and can artificially measure singlestranded cfDNA fragments
Summary
Amgen; honoraria from Amgen, Sanofi, Bristol Myers Squibb, Sandoz, and AAA; and travel, accommodations, and expenses paid by Amgen. ART is a shareholder of DiaDx SAS
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