Extrachromosomal circular DNA (eccDNA) was discovered more than half a century ago. However, its biogenesis and function have just begun to be elucidated. One hurdle that has prevented our understanding of eccDNA is the difficulty in obtaining pure eccDNA from cells. The current eccDNA purification methods mainly rely on depleting linear DNAs by exonuclease digestion after obtaining crude circles by alkaline lysis. Owing to eccDNA’s low abundance and heterogeneous size, the current purification methods are not efficient in obtaining pure eccDNA. Here we describe a new three-step eccDNA purification (3SEP) procedure that adds a step to recover circular DNA, but not linear DNA that escape from the exonuclease digestion, whereby 3SEP results in eccDNA preparations with high purity and reproducibility. Additionally, we developed a full-length eccDNA sequencing technique by combining rolling-circle amplification with Nanopore sequencing. Accordingly, we developed a full-length eccDNA caller (Flec) to call the consensus sequence of multiple tandem copies of eccDNA contained within the debranched rolling-circle amplification product and map the consensus to its genomic origin. Collectively, our protocol will facilitate eccDNA identification and characterization, and has the potential for diagnostic and clinical applications. For a well-trained molecular biologist, it takes ~1–2 d to purify eccDNAs, another 5–6 d to carry out Nanopore library preparation and sequencing, and 1–5 d for an experienced bioinformatic scientist to analyze the data.
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The example dataset of Nanopore sequencing of RCA eccDNA can be downloaded at https://figshare.com/articles/dataset/Nanopore_reads_of_eccDNA/17046158 or https://doi.org/10.6084/m9.figshare.17046158.v1. This example dataset is a subset of the data in Gene Expression Omnibus with accession GSM5058061.
All scripts used for data analysis described in this paper are available at https://github.com/YiZhang-lab/eccDNA_RCA_nanopore or https://doi.org/10.5281/zenodo.7045025.
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We thank P. Yin for providing the AFM instrument. This work was supported by the Howard Hughes Medical Institute. Y.Z. is an Investigator at the Howard Hughes Medical Institute.
A patent application covering the eccDNA purification method and eccDNA’s application as an immunostimulant and diagnostic marker has been filed by the Boston Children’s Medical Center Corporation.
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Key reference using this protocol
Wang, Y. et al. Nature 599, 308–314 (2021): https://doi.org/10.1038/s41586-021-04009-w
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Wang, Y., Wang, M. & Zhang, Y. Purification, full-length sequencing and genomic origin mapping of eccDNA. Nat Protoc 18, 683–699 (2023). https://doi.org/10.1038/s41596-022-00783-7
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