Liquid footprinting: A novel approach for DNA footprinting using short double-stranded cell-free DNA from plasma

The diagnostic potential of short double-stranded cell-free DNA (cfDNA) in blood plasma has not been recognized, yet. Here, we present a method for enrichment of double-stranded cfDNA with an average length of about 40 base pairs 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 found 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. When comparing short double-stranded cfDNA from patient samples of pancreatic ductal adenocarcinoma with colorectal carcinoma or septic with post-operative controls, we identified 731 and 1,107 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.