EccDNA identification by long-read sequencing. Step 0. The experimental workflow begins with purified genomic DNA; chromosomal DNA (blue), mitochondrial DNA (magenta), eccDNA (red), restriction enzyme (green), CRISPR-Cas9 (cyan), and exonuclease V (yellow). Step 1. A read (left panel) can be aligned on regions of 2 chromosomes (green and red) of the reference genome (blue) with breakpoint reads (dashed line) linking the aligned chromosomal regions and trimmed unmapped portions (gray). Self-read dot plots (right panels) showing read regions that align to the reference genome (blue ovals); reads without CTCs (left dot plot) and with CTCs (right dot plot). Step 2. Merged regions R and S with the reference sequences (green and red bars) and the breakpoint event (dashed lines) that links the two regions; arrows for non-breakpoint reads (light blue), breakpoint reads (blue), and reads with CTCs (magenta) point in the direction of the aligned orientation on the plus strand of the reference sequence (arrows above the bars) or the minus strand (arrows below the bars). Step 3. The information of the read alignments was converted to directed graphs. Step 4. The reads were assembled using our developed regions/linkages algorithm; the assembled sequences were polished, variants were identified, and eccDNA was annotated with genomic features such as exon, repeat, and CpG. Step 5. Circos visualization to present the eccDNA architecture.