Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics
Cynthia Sanchez, Benoit Roch, Thibault Mazard, Philippe Blache, Zahra Al Amir Dache, Brice Pastor, Ekaterina Pisareva, Rita Tanos, Alain R. Thierry
Cynthia Sanchez, Benoit Roch, Thibault Mazard, Philippe Blache, Zahra Al Amir Dache, Brice Pastor, Ekaterina Pisareva, Rita Tanos, Alain R. Thierry
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Resource and Technical Advance Genetics Oncology

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.

Authors

Cynthia Sanchez, Benoit Roch, Thibault Mazard, Philippe Blache, Zahra Al Amir Dache, Brice Pastor, Ekaterina Pisareva, Rita Tanos, Alain R. Thierry

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Figure 2

CfDNA size distribution as determined by Q-PCR.

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CfDNA size distribution as determined by Q-PCR. Fractional size distribu...
Fractional size distribution was performed using nested Q-PCR primer systems to detect amplicons of 67, 145, and 320 bp in the 7 healthy individuals (Supplemental Methods, Appendix 2). Note, fractional size distribution as presented here was obtained from cfDNA concentrations quantified by targeting the KRAS DNA region and is only indicative, as described in the Methods section. The cfDNA size distribution was summarized by presenting the levels (data represent mean ± SEM) in the highly fragmented cfDNA fraction (HF, 67–145 bp), the levels in the mono-nucleosome–derived fragmented cfDNA (MF, 145–320 bp), and a lower proportion (3%–20%) in the weakly fragmented cfDNA (WF, >320 bp). (A). The DNA Integrity Index (DII) was calculated based on the Q-PCR–based determination of the ratio of the number of fragments over 320 bp to those over 67 bp within a KRAS intron 3/exon 2 region in a panel of 109 healthy individuals (B). The sample median DII was 0.119. Bar, median; box, 25% to 75%; brackets, 5% to 95%; see Statistics section in Methods.