Fig. 4: Retention elements enable selection of oncogene-containing ecDNAs in cancer.

a, Mean frequency (>10 independent replicates) of cells with ≥1 ecDNA in simulations. Shaded area, s.e.m. b, Analysis of retention element co-amplification with oncogenes on ecDNA in patient tumours. c, ecDNA amplicons that contain retention elements and/or oncogenes. d, Top, schematic of an ecDNA segment without retention elements co-amplified with a retention element. Bottom, frequency of co-amplification with retention elements in BFB, ecDNA or linear amplicons for genomic segments without retention elements. e, Top to bottom, oncogene sizes on ecDNA, frequency of genomic segments that contain retention elements sorted by size, and total ecDNA amplicon sizes. f, Schematic of experiment to analyse the distribution of retention element numbers among ecDNAs. g, Correlation (Pearson’s R with 95% confidence intervals) between local density of retention elements (Methods) and amplicon size. The plot shows the linear fit using ordinary least squares with 95% confidence intervals. h, Circular microDNAs in five human cell lines that overlap with retention elements or matched-sized random genomic intervals detected using circle-seq. i, Increased WGS coverage of EGFR ecDNA in GBM39 cells and retention element positions. j, 5mC CpG methylation of retention elements (n = 9 segments) compared with matched-sized sequence intervals (n = 1,235 segments) in GBM39 ecDNA. k, 5mC methylation (Me⁺ or Me^–) and density of CpG sites surrounding a retention element on GBM39 ecDNA. l, Site-specific methylation of retention elements by CRISPRoff. m, Frequency of GBM39 cells that contain untethered ecDNA foci 5 days after transfection. n = 60 (nontargeting) and n = 50 (targeting) visual fields. Box plot parameters are as described in Fig. 2. n, Plasmid retention after methylation in COLO320DM cells, as assessed by qPCR (three biological replicates). o, Retention elements and oncogenes on ecDNA (left) confer retention and selection, respectively, two processes that shape the evolution of cancer cell lineages (right). P  values were calculated using one-sided tests of equal proportions (d), two-sided Fisher’s z-tests (g), two-sided Fisher’s exact tests (h), two-sided Wilcoxon rank-sum tests (j), two-sided Mann–Whitney–Wilcoxon tests (m) or one-sided t-tests (n).