High-confidence structural predictions of extrachromosomal DNA with ecDNAInspector

Abstract

Extrachromosomal DNA (ecDNA) are circularized genomic elements that reside outside canonical chromosomes. ecDNA amplify oncogene copy number, enhance chromatin accessibility, and act as mobile enhancers through cis- and trans-regulatory interactions, collectively boosting oncogene expression. ecDNA has been implicated in tumor progression, intratumoral heterogeneity, and poor patient prognosis. Despite various lines of evidence that ecDNA promotes aggressive disease, the mechanisms and selective pressures leading to ecDNA formation and propagation remain poorly understood as are their structures. While several computational tools have been developed to infer ecDNA presence or absence from short read sequencing data, accurate identification of large or complex ecDNA structures remains challenging. Here we introduce ecDNAInspector, a novel computational framework to systematically assess the confidence of ecDNA predictions from existing inference tools. Leveraging abundant short-read whole genome sequencing (WGS) data from population-scale cohorts, we demonstrate that ecDNAInspector accurately identifies high-confidence ecDNA calls, improving interpretability and facilitating the association with clinical features. As an illustrative example, applied to a cohort of 250 breast cancers, ecDNAInspector identifies associations between ecDNA structure and molecular subgroups of disease. These findings are supported by orthogonal omic data and experimental characterization of ecDNA captured in representative cell lines. ecDNAInspector provides a scalable, data-driven approach to characterize ecDNA structure, enabling integrative studies of the clinical and biological impact of this non-mendelian mode of oncogene amplification and inheritance.