Molecular characterization of cell-free eccDNAs in human plasma

Abstract

Extrachromosomal circular DNAs (eccDNAs) have been reported in most eukaryotes. However, little is known about the cell-free eccDNA profiles in circulating system such as blood. To characterize plasma cell-free eccDNAs, we performed sequencing analysis in 26 libraries from three blood donors and negative controls. We identified thousands of unique plasma eccDNAs in the three subjects. We observed proportional eccDNA increase with initial DNA input. The detected eccDNAs were also associated with circular DNA enrichment efficiency. Increasing the sequencing depth in an additional sample identified many more eccDNAs with highly heterogenous molecular structure. Size distribution of eccDNAs varied significantly from 31 bp to 19,989 bp. We found significantly higher GC content in smaller eccDNAs (<500 bp) than the larger ones (>500 bp) (p < 0.01). We also found an enrichment of eccDNAs at exons and 3′UTR (enrichment folds from 1.36 to 3.1) as well as the DNase hypersensitive sites (1.58–2.42 fold), H3K4Me1 (1.23–1.42 fold) and H3K27Ac (1.33–1.62 fold) marks. Junction sequence analysis suggested fundamental role of nonhomologous end joining mechanism during eccDNA formation. Further characterization of the extracellular eccDNAs in peripheral blood will facilitate understanding of their molecular mechanisms and potential clinical utilities.

Cell-Free eccDNAs: A New Type of Nucleic Acid Component for Liquid Biopsy?

“…The enrichment is involved in exonuclease digestion (such as Plasmid-Safe-ATP-dependent DNase and/or exonuclease V) to remove linear DNA and multiple displacement amplification (MDA) to preferentially amplify circular DNAs (1, 3, 5, 6, 16, 27, 36–38). This method has been proved to be effective for eccDNA detection and enrichment, especially when the initial DNA amount is very limited, for example the cell-free DNA (cfDNA) in the circulating system (27, 37). Other reported methods for eccDNA enrichment include multiple rounds of extensive exonuclease V digestion and chloride ethidium bromide density gradient centrifugation.…”

“…Mapping of the split reads has to be inconsistent, i.e. first part of sequence read being mapped to 3’ end while second part of the split read being mapped to 5’ end of a defined genomic region (27). This entire procedure is illustrated in Figure 1.…”

“…For the recombination-dependent mechanism, DNA double strand breaks are repaired by homologous recombination (24–26), non-homologous end joining and microhomology-mediated end joining (3, 6, 16, 27). The circular DNA are formed by looping out or being excised from the genomes.…”

“…It is believed that the larger eccDNAs are basically located inside of the nucleus while the smaller ones spread in nucleus, cytoplasm, and extracellular sites. Recent findings have shown highly abundant smaller eccDNAs in the form of cfDNAs in human circulation system in both healthy and cancerous conditions (27, 37). Consistent with the origin features of eccDNAs in tissues and cell lines, the cell-free eccDNAs also showed some distribution preference in the genome, such as UTRs, exons, DNase-hypersensitivity sites and histone markers-enriched regions (27, 37).…”

“…Recent findings have shown highly abundant smaller eccDNAs in the form of cfDNAs in human circulation system in both healthy and cancerous conditions (27, 37). Consistent with the origin features of eccDNAs in tissues and cell lines, the cell-free eccDNAs also showed some distribution preference in the genome, such as UTRs, exons, DNase-hypersensitivity sites and histone markers-enriched regions (27, 37). By comparing the sizes of microDNAs in circulation, studies have found that cell-free microDNAs have different sizes under different conditions.…”