ATAC-seq identifies thousands of extrachromosomal circular DNA in cancer and cell lines

Abstract

Extrachromosomal circular DNAs (eccDNAs) are somatically mosaic and contribute to intercellular heterogeneity in normal and tumor cells. Because short eccDNAs are poorly chromatinized, we hypothesized that they are sequenced by tagmentation in ATAC-seq experiments without any enrichment of circular DNA. Indeed, ATAC-seq identified thousands of eccDNAs in cell lines that were validated by inverse PCR and by metaphase FISH. ATAC-seq in gliomas and glioblastomas identify hundreds of eccDNAs, including one containing the well-known EGFR gene amplicon from chr7. More than 18,000 eccDNAs, many carrying known cancer driver genes, are identified in a pan-cancer analysis of ATAC-seq libraries from 23 tumor types. Somatically mosaic eccDNAs are identified by ATAC-seq even before amplification is recognized by genome-wide copy number variation measurements. Thus, ATAC-seq is a sensitive method to detect eccDNA present in a tumor at the pre-amplification stage and can be used to predict resistance to therapy.

Fig. 1. A schematic to show that a circle could be part of an ATAC-seq library.

(A) If circular DNA has open chromatin structure near or around the ligation point, then the library preparation method will cut and attach an adaptor into a DNA fragment from eccDNA. (B) One end of paired-end read mapping on the body of a circular DNA with read from the other end mapping on the ligation junction. (C) Detailed steps from mapping to identification of the new Circle_finder pipeline.

Fig. 2. eccDNA in C4-2 and OVCAR8 cell lines.

(A) Length distribution of identified eccDNA in C4-2 and OVCAR8 cell lines. (B) Karyotype plot showing chromosomal distribution of C4-2 and OVCAR8 cell lines.

Fig. 3. Experimental validation of randomly selected eccDNA identified by ATAC-seq in C4-2B and OVCAR8 cells.

(A) Schematic for isolation and detection of eccDNA. See Materials and Methods for details. (B) PCR detection of eccDNA. DNA bands marked with blue boxes were gel-purified and sequenced. (C) Description of eccDNAs validated in (B) on the basis of analysis of ATAC-seq data from OVCAR8 and C4-2B. (D) Junctional tags obtained after sequencing of PCR products in (B). Shaded (blue) and unshaded sequences depict 15 bases on either side of junctions. Numbers indicate chromosomal location on respective chromosomes. Note the match between numbers for each circle in (C) and (D). Some of the junction sequence identified by Sanger sequencing differ by few bases because of multiple species of eccDNA present in the given cell lines. Oval circles represent insertion, and boxed sequences represent mismatches. *Sequence obtained from the bottom strand.

Fig. 4. eccDNA in cell lines and LGG or GBM tumors.

(A) Detection of eccDNA in OVCAR8 cell line by FISH: Metaphase spread of chromosome (blue) from OVCAR8 cells were stained with the probe (green) against the eccDNA locus chr2:238136071-238170279 (top row) or chr10:103457331-103528085 (bottom row). The spreads on the left do not have an extrachromosomal signal, while the spreads on the right have extrachromosomal signals that are better seen in the magnified insets on the extreme right. White arrows mark the eccDNA signals. (B) For the negative control cell lines, C4-2, the spread does not have an extrachromosomal DNA signal. (C) The eccDNA signals in OVCAR8 (n = 28) and C4-2 (n = 24) (negative control) were quantified for locus chr10:103457331-103528085 and shown in the graph. P values were calculated using Student’s t test; **P < 0.01. (D) eccDNA/duplication loci identified in whole-genome sequencing (WGS) libraries show genomic amplification (median, 1.5-fold), suggesting at least one allele is duplicated in all the cells. eccDNA loci identified in ATAC-seq libraries do not show genomic amplification (median close to zero), suggesting that the eccDNA are apparent before a CNV can be detected at the locus. The value of copy number amplification (CNA) in the y axis is in log₂. (E) Length distribution of eccDNA identified in LGG and GBM TCGA ATAC-seq data. (F) Karyotype plot showing chromosomal distribution of eccDNA identified in LGG and GBM from TCGA ATAC-seq data.

Fig. 5. Properties of microDNA identified in this paper (eccDNA <1 kb) by ATAC-seq.

(A) Length distribution of eccDNA shows peaks at 180 and 380 bases. (B) GC content of eccDNA locus and regions immediately upstream and downstream from the eccDNA is higher than genomic average, as calculated from 1000 random stretches of the genome of equivalent length as the eccDNA (Random-1000). (C) The sites in the genome that give rise to small eccDNA are enriched relative to random expectation in genic sites, sequences 2 kb upstream from genes and in CpG islands. FC, Fold Change. (D) Direct repeats of 2 to 15 bp flanking the genomic locus of the eccDNA at ligation point are present for ~20% of the loci. (E) Gene classes enriched in the set of genes found on the circular DNAs in two or more cancers. The color scale indicates enrichment in pathway (blue color indicates pathway was enriched). If the genes found on the eccDNA/duplication loci in a cancer type are significantly enriched in the indicated pathways, then the color in the cell is blue. If the set of genes is not enriched in that cancer, then the cell is black.