Characteristics, origin, and potential for cancer diagnostics of ultrashort plasma cell-free DNA
- Irena Hudecova1,2,9,
- Christopher G. Smith1,2,9,
- Robert Hänsel-Hertsch1,3,4,5,9,
- Chandra S. Chilamakuri1,
- James A. Morris1,2,
- Aadhitthya Vijayaraghavan1,2,
- Katrin Heider1,2,
- Dineika Chandrananda1,2,11,
- Wendy N. Cooper1,2,
- Davina Gale1,2,
- Javier Garcia-Corbacho6,
- Simon Pacey2,7,
- Richard D. Baird2,7,
- Nitzan Rosenfeld1,2,10 and
- Florent Mouliere1,2,8,10
- 1Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge CB2 0RE, United Kingdom;
- 2Cancer Research UK Cambridge Centre, Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Cambridge CB2 0RE, United Kingdom;
- 3Center for Molecular Medicine Cologne CMMC, University of Cologne, 50931 Cologne, Germany;
- 4Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne and University Hospital Cologne, 50931 Cologne, Germany;
- 5Institute of Human Genetics, University Hospital Cologne, 50931 Cologne, Germany;
- 6Clinical Trials Unit, Clinic Institute of Hematological and Oncological Diseases, Hospital Clinic, 170 08036 Barcelona, Spain;
- 7Department of Oncology, University of Cambridge, Cambridge CB2 0QQ, United Kingdom;
- 8Department of Pathology, Amsterdam UMC, Vrije Universiteit Amsterdam, Cancer Centre Amsterdam, 1081 HV Amsterdam, The Netherlands
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↵9 These authors contributed equally to this work.
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 centered 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.
Footnotes
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↵10 These authors share senior authorship.
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.275691.121.
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Freely available online through the Genome Research Open Access option.
- Received May 3, 2021.
- Accepted December 16, 2021.
This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
