Journal
BIO-PROTOCOL
Volume 11, Issue 9, Pages -Publisher
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.4003
Keywords
Circular DNA; eccDNA; ATAC-seq; Inverse PCR; FISH
Categories
Funding
- [R01 CA060499]
- [P30 CA044579]
- [T32 CA009109]
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Recent studies have highlighted the importance of eccDNA from yeast to humans, particularly in cancer cells where oncogenes are amplified on eccDNA causing genomic instability. A new method independent of rolling circle amplification was developed by the research team, utilizing ATAC-seq and a Circle_finder algorithm to predict eccDNA, followed by validation through 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).
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