Jan Müller1,2,3,4, Christina Hartwig1,5, Mirko Sonntag1,6, Lisa Bitzer1, Christopher Adelmann1, Yevhen Vainshtein1, Karolina Glanz1, Sebastian O. Decker7, Thorsten Brenner8, Georg F. Weber9,10, Arndt von Haeseler11,12 and Kai Sohn1 1Innovation Field In-vitro Diagnostics, Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, 70569 Stuttgart, Germany; 2Max Perutz Labs, Vienna Biocenter Campus, 1030 Vienna, Austria; 3University of Vienna, Max Perutz Labs, Department of Structural and Computational Biology, Center of Integrative Bioinformatics Vienna, 1030 Vienna, Austria; 4Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, 1030 Vienna, Austria; 5Institute for Interfacial Engineering and Plasma Technology, University of Stuttgart, 70569 Stuttgart, Germany; 6Interfaculty Graduate School of Infection Biology and Microbiology, Eberhard Karls University Tübingen, 72074 Tübingen, Germany; 7Heidelberg University, Medical Faculty Heidelberg, Department of Anesthesiology, 69120 Heidelberg, Germany; 8Department of Anesthesiology and Intensive Care Medicine, University Hospital Essen, University Duisburg-Essen, 45147 Essen, Germany; 9Department of Surgery, Friedrich-Alexander University Erlangen-Nürnberg and Universitätsklinikum Erlangen, 91054 Erlangen, Germany; 10Comprehensive Cancer Center Erlangen-EMN, Friedrich-Alexander University Erlangen-Nürnberg and Universitätsklinikum Erlangen, 91054 Erlangen, Germany; 11Center of Integrative Bioinformatics Vienna, Max Perutz Labs, University of Vienna and Medical University of Vienna, Vienna BioCenter, 1030 Vienna, Austria; 12University of Vienna, Faculty of Computer Science Bioinformatics and Computational Biology, 1090 Vienna, Austria Corresponding author: kai.sohnigb.fraunhofer.de Abstract

Here, we present a method for enrichment of double-stranded cfDNA with an average length of ∼40 bp from cfDNA for high-throughput DNA sequencing. This class of cfDNA is enriched at gene promoters and binding sites of transcription factors or structural DNA-binding proteins, so that a genome-wide DNA footprint is directly captured from liquid biopsies. In short double-stranded cfDNA from healthy individuals, we find significant enrichment of 203 transcription factor motifs. Additionally, short double-stranded cfDNA signals at specific genomic regions correlate negatively with DNA methylation, positively with H3K4me3 histone modifications and gene transcription. The diagnostic potential of short double-stranded cell-free DNA (cfDNA) in blood plasma has not yet been recognized. When comparing short double-stranded cfDNA from patient samples of pancreatic ductal adenocarcinoma with colorectal carcinoma or septic with postoperative controls, we identify 136 and 241 differentially enriched loci, respectively. Using these differentially enriched loci, the disease types can be clearly distinguished by principal component analysis, demonstrating the diagnostic potential of short double-stranded cfDNA signals as a new class of biomarkers for liquid biopsies.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.279326.124.

Freely available online through the Genome Research Open Access option.

Received March 15, 2024. Accepted August 12, 2024.