Biological Fragmentation of Circulating Cell-free DNA Alters Genetic Representation

High-throughput sequencing of circulating cell-free DNA (cfDNA) as liquid biopsy has revolutionized tumor genome profiling by providing a more accurate, longitudinal, real-time and non-invasive mean for precision and personalized medicine. Current knowledge on cfDNA characteristics revealed that it exists mainly as double-stranded molecules, resulting from biological fragmentation into both short (<1 kb) and long segments (>10 kb) [1,2]. Short fractions are mostly derived from apoptosis via the activation of cellular endonucleases leading to the cleavage of chromatin DNA into inter-nucleosomal fragments [3], whereas necrosis generates relatively long fragments of DNA. It is now believed that circulating cfDNA pool of cancer patients is originated from a combination of apoptosis, necrosis, and active release [4]. Within the nucleosomal core, cfDNA is protected from blood nucleases by histones, whereas the linker is vulnerable to digestion. As a result, regions showing high or low frequency of fragmentation correspond to the sequences between or occupied by nucleosomes, respectively, with the majority of short-fragment length corresponds to single nucleosome size of 160-170 bp. These observations strongly support the notion that the patterns of cfDNA fragmentation are guided by chromatin structure particularly the interplay between nucleosome positioning, epigenetic regulation, and gene expression machinery [5,6].