Detecting Full-Length EccDNA with FLED and long-reads sequencing

AbstractReconstructing the full-length sequence of extrachromosomal circular DNA (eccDNA) from short sequencing reads has proved challenging given the similarity of eccDNAs and their corresponding linear DNAs. Previous sequencing methods were unable to achieve high-throughput detection of full-length eccDNAs. Here we describe a new strategy that combined rolling circle amplification (RCA) and nanopore long-reads sequencing technology to generate full-length eccDNAs. We further developed a novel algorithm, called Full-Length eccDNA Detection (FLED), to reconstruct the sequence of eccDNAs. We used FLED to analyze seven human epithelial and cancer cell line samples and identified over 5,000 full-length eccDNAs per sample. The structures of identified eccDNAs were validated by both PCR and Sanger sequencing. Compared to other published nanopore-based eccDNA detectors, FLED exhibited higher sensitivity. In cancer cell lines, the genes overlapped with eccDNA regions were enriched in cancer-related pathways andcis-regulatory elements can be predicted in the up-stream or downstream of intact genes on eccDNA molecules, and the expressions of these cancer-related genes were dysregulated in tumor cell lines, indicating the regulatory potency of eccDNAs in biological processes. Our method takes advantage of nanopore long reads and enables unbiased reconstruction of full-length eccDNA sequences. FLED is imple-mented using Python3 which is freely available on GitHub (https://github.com/FuyuLi/FLED).