Fast and accurate resolution of ecDNA sequence using Cycle-Extractor

Abstract Extrachromosomal DNA (ecDNA) plays a key role in cancer pathology. EcDNAs mediate high oncogene amplification and expression and worse patient outcomes. Accurately determining the structure of these circular molecules is essential for understanding their function, yet reconstructing ecDNA cycles from sequencing data remains challenging. We introduce Cycle-Extractor (CE) for reconstruction. CE accepts a breakpoint graph derived from either short or long read sequencing data as input and extracts a cycle with the maximum length-weighted-copy-number. CE utilizes a mixed-integer linear program (MILP) and a separate traversal procedure, enabling fast optimization and compatibility with free solvers. We evaluated CE against CoRAL (long-read-based quadratic optimization), Decoil (long-reads), and AmpliconArchitect (AA for short reads) on both simulated data and real cancer cell lines. On simulated ecDNA, CE achieves performance comparable to CoRAL across three accuracy metrics and consistently outperforms AA and Decoil. On cancer cell lines, CE produces longer and heavier cycles than AA, and achieves performance similar to CoRAL. Moreover, CE is, on average, 40× faster than CoRAL. These results demonstrate that CE accurately reconstructs ecDNA from both short- and long-read sequencing data, while long-read inputs allow CE to recover more complete and higher-confidence ecDNA structures. CE improved the prediction of many ecDNA structures. On a PC3 ecDNA containing MYC , CE uses ONT data to reconstruct a substantially larger and higher-copy sequence (4.2 Mbp) compared to the short-read-derived reconstruction (690 Kbp). CRISPR-CATCH experiments confirm the presence of a large ecDNA molecule, validating the long-read-based CE reconstruction.