Cell-free DNA comprises an in vivo, genome-wide nucleosome footprint that informs its tissue(s)-of-origin

Nucleosomes are the basic unit of packaging of eukaryotic chromatin, and nucleosome positioning can differ substantially between cell types. Here, we sequence 14.5 billion plasma-borne cell-free DNA (cfDNA) fragments (700-fold coverage) to generate genome-wide maps of in vivo nucleosome occupancy. We identify 13 million local maxima of nucleosome protection, spanning 2.53 gigabases (Gb) of the human genome, whose positions and spacings correlate with nuclear architecture, gene structure and gene expression. We further show that short cfDNA fragments - poorly recovered by standard protocols - directly footprint the in vivo occupancy of DNA-bound transcription factors such as CTCF. The sequence composition of cfDNA has previously been used to noninvasively monitor cancer, pregnancy and organ transplantation, but a key limitation of this paradigm is its dependence on genotypic differences to distinguish between contributing tissues. We show that nucleosome spacing in gene bodies and cis-regulatory elements, inferred from cfDNA in healthy individuals, correlates most strongly with transcriptional and epigenetic features of lymphoid and myeloid cells, consistent with hematopoietic cell death as the normal source of cfDNA. We build on this observation to show how in vivo nucleosome footprints can be used to infer the cell types that contribute to circulating cfDNA in pathological states such as cancer. Because it does not rely on genotypic differences, this strategy may enable the noninvasive cfDNA-based monitoring of a much broader set of clinical conditions than is currently possible.