Cell-free DNA fragmentation is a stepwise process and conserved across mammals

Circulating cell-free DNA (cfDNA) is a pool of short DNA fragments mainly released from apoptotic hematopoietic cells. Nevertheless, the precise physiological process governing the DNA fragmentation and molecular profile of cfDNA remains obscure. To dissect the DNA fragmentation process, we use a human leukemia cell line HL60 undergoing apoptosis to analyze the size distribution of DNA fragments by shallow whole-genome sequencing (sWGS). Meanwhile, we also scrutinize the size profile of plasma cfDNA in 901 healthy human subjects and 38 dogs by sWGS.Distinct size distribution profiles in HL60 cells and supernatant suggest cfDNA fragmentation is a stepwise process which happens successively inside and outside cells. Moreover, C-end preference of cfDNA fragments has been formed inside apoptotic HL60 cells prior to be released into the extracellular milieu. In addition, the cfDNA profiles are characteristic and conserved across species.Our study provides new insight into fragmentomics of circulating cfDNA processing.