ATAC-Seq-based Identification of Extrachromosomal Circular DNA in Mammalian Cells and Its Validation Using Inverse PCR and FISH

Recent studies from multiple labs including ours have demonstrated the importance of extrachromosomal circular DNA (eccDNA) from yeast to humans ( Shibata et al., 2012 ; Dillon et al., 2015 ; Moller et al., 2016 ; Kumar et al., 2017 ; Turner et al., 2017 ; Kim et al., 2020 ). More recently, it has been found that cancer cells obtain a selective advantage by amplifying oncogenes on eccDNA, which drives genomic instability ( Wu et al., 2019 ; Kim et al., 2020 ). Previously, we have purified circular DNA and enriched the population using rolling circle amplification followed by high-throughput sequencing for the identification of eccDNA based on the unique junctional sequence. However, eccDNA identification by rolling circle amplification is biased toward small circles. Here, we report a rolling circle-independent method to detect eccDNA in human cancer cells. We demonstrate a sensitive and robust step-by-step workflow for finding novel eccDNAs using ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) combined with a Circle_finder bioinformatics algorithm to predict the eccDNAs, followed by its validation using two independent methods, inverse PCR and metaphase FISH (Fluorescence in situ Hybridization).