DNA sequencing based biophysical characterization of cell-free DNA in the blood circulation sheds light on its origin

A large number of small fragments of cell-free DNA circulate in human blood. These molecules are the product of cell death across the body, and offer a unique window into health and disease. Here, we have used next-generation DNA sequencers to probe the biophysical properties of cell-free DNA (cfDNA) in blood plasma, urine, and peritoneal fluid. We analyzed more than 250 samples and show that the biophysical properties of cell-free DNA, including fragment length, genome alignment, and dinucleotide frequency, depend strongly on the biochemical properties of the extracellular environment. We further show that cell-free DNA in urine, plasma and peritoneal fluid comprises “footprints” of proteins bound to the genome. The displacement of chromatin organization protein complexes, such as nucleosomes, near transcription start sites and strong protection signals of transcription binding factors, can be used to identify the tissues that contribute cfDNA toward the mixture in different bodily fluids. Last, we discuss the properties of non-human cfDNA, i.e. bacterial and viral cfDNA, in these different fluids and demonstrate that unbiased sequencing enables comprehensive monitoring of infection.