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Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics
Cynthia Sanchez, … , Rita Tanos, Alain R. Thierry
Cynthia Sanchez, … , Rita Tanos, Alain R. Thierry
Published February 11, 2021
Citation Information: JCI Insight. 2021;6(7):e144561. https://doi.org/10.1172/jci.insight.144561.
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Technical Advance Genetics Oncology

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

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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 1

CfDNA size profile as determined from DSP-S and SSP-S.

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CfDNA size profile as determined from DSP-S and SSP-S.
CfDNA size profil...
CfDNA size profiles of 7 healthy individuals, obtained by sequencing either from double- or single-strand DNA library preparations (A and B, respectively). Mean size profiles of the 7 individuals, as determined by DSP-S (black lines) and SSP-S (red lines) (C); curves of the cumulative frequencies between SSP-S and DSP-S (D); the difference in cumulative frequencies, denoted as ΔS, between SSP-S minus DSP-S (E); and the curve of the difference of % values, denoted as ΔV, between SSP-S minus DSP-S (F). The increasing part of the ΔS curve indicates the fragment size range, in which SSP-S detected fragment number is proportionally higher than DSP-S detected fragments; while the decreasing part of the ΔS curve indicates the fragment size range in which SSP-S–detected fragment number is proportionally lower than for DSP-S–detected fragments (E). Positive ΔV values for cfDNA size indicate where more fragments were detected by SSP-S than by DSP-S (F). Negative ΔV values for cfDNA size indicate where less fragments were detected by SSP-S than by DSP-S. More fragments are detected by SSP-S up to 158 bp (nt) as compared with DSP-S, and more fragments are detected by DSP-S over 158 bp (nt) (F).

Copyright © 2021 American Society for Clinical Investigation
ISSN 2379-3708

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