Identification of eccDNA in Extracellular Vesicles Derived from Human Dermal Fibroblasts Through Nanopore Sequencing

Abstract

Extrachromosomal circular DNAs (eccDNAs) are heterogeneous circular DNA molecules derived from genomic DNA, and believed to be involved in intercellular communication and in natural biological processes. Extracellular vesicles (EVs) are membrane-bound particles released from all cells, and have been shown to contain various classes of nucleic acids. EVs can play a role in intercellular communication and may be used as biomarkers. This constitutes the first study to demonstrate that EVs derived from healthy human dermal fibroblasts carry eccDNA. eccDNA from EVs and their corresponding donor cells were isolated and sequenced on the Oxford Nanopore MinIon platform, followed by the identification of potential eccDNAs through four different bioinformatic pipelines, namely ecc_Finder, cyrcular-calling, CReSIL, and Flec. Our main findings demonstrate that EVs derived from human dermal fibroblasts carry eccDNA; there is variability in the number of eccDNAs identified in the same sample through different pipelines; and there is variability in the identified eccDNAs across biological replicates. Additionally, eccDNAs characterized in this research had (a) sequences as small as 306 base pairs and as large as 28,958 base pairs across all samples, (b) uneven chromosomal distribution, and (c) an average of 49.7% of the identified eccDNAs harboring gene fragments. Future implications for this novel research include using this framework method to elucidate factors and conditions that may influence the skin aging process and related biogenesis in human dermal cells.