Extended Data Fig. 1: Characterization of isogenic cell line pairs.

(a-c) The structure of one dominant ecDNA detected in COLO320DM (a), GBM39ec (b) and PC3-DM (c). COLO320DM ecDNA reconstruction is adapted from Hung, et al. Nature 2021¹⁰ using hg38 coordinates; GBM39ec ecDNA reconstruction is adapted from Turner, et al. Nature 2017² using hg38 coordinates; PC3-DM ecDNA was reconstructed by short-read WGS aligned to hg38, capturing the MYC-containing genome cycle identified by AmpliconArchitect. Some complexities of the PC3 DM ecDNA may not be captured from short read sequencing alone. (d) Amplicon similarity analysis of 3 near-isogenic cell line pairs as computed from whole genome sequencing data. Major oncogene copy number was extracted for individual cell line: COLO320 pairs: MYC; GBM39 pairs: EGFR; PC3: MYC. (e) Genomic intervals in COLO320 and GBM39 cells used in this study for ecDNA amplicons. (f) Representative metaphase-FISH images of PC3-DM and PC3-HSR cells confirming MYC gene amplification on ecDNA and chromosome respectively. For each clone, about 6–15 metaphase spread images were collected from a one-off validation experiment to check amplicon status. Scale bar: 10 µm (g) Amplicon structure and absolute gene copy of MYC between PC3-DM and PC3-HSR based on WGS. (h) Comparison of STR profiles between PC-3 from ATCC, PC3-DM and PC3-HSR.