Fig. 1

CCDA-seq for profiling chromatin accessibility and nucleosome position in ecDNAs. A Intact chromatin was treated with m⁶A methyltransferase (EcoGII), which preferentially methylates DNA bases in the open chromatin region on ecDNAs and linear DNAs. High molecular weight DNA was then isolated and subjected to exonuclease digestion to remove partially linear DNAs. The remaining DNA molecules were subjected to nanopore library construction and nanopore sequencing. The data were aligned to the hg19 genome to identify ecDNAs based on head-to-tail pattern. The methylated bases were used to reconstruct nucleosomes in ecDNAs and other linear DNAs. In contrast, the ATAC-seq used the transposon to attack the open chromatin. The tagmentated short fragments were amplified and subjected to NGS. The short reads were aligned with genome to identify ecDNA bases. The mapped reads were calling as peaks representing the open chromatin region. B CCDA-seq bioinformatics pipeline is illustrated. The signal data were processed through guppy base calling to generate sequence. The sequences were aligned to the genome to identify linear DNAs and ecDNAs. We assembled the ecDNA sequence reference. Based on the ecDNA and linear DNA references, we used Megalodon to call the m⁶A sites based on ecDNA and linear DNA sequences. Then, we performed the accessibility analysis, gene element annotation, gene expression analysis, and co-accessibility assessment. C Large aggregate CCDA-seq signal enrichments match closely with DNase-seq accessibility peaks. (Chr20: 49220090–58167461)