Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells

Oncogenic extrachromosomal DNAs (ecDNA) are common in cancers, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe scEC&T-seq, a method for parallel isolation and sequencing of extrachromosomal circular DNAs and full-length mRNA from single human cells. By applying scEC&T-seq to cancer cells, we not only describe intercellular differences in ecDNA content, but also investigate their structural heterogeneity and transcriptional impact. We reveal that whereas oncogene-containing ecDNA elements are clonally present in cancer cells and drive intercellular oncogene expression differences, other small circular DNAs also captured by scEC&T-seq are mostly private to individual cancer cells, indicating differences in selection and propagation. Moreover, scEC&T-seq uncovers intercellular differences in ecDNA structure, which allowed the inference of ecDNA structural dynamics and point to circular recombination as a potential mechanism of ecDNA evolution. We envision that our method may enable the analysis of yet unknown prerequisites for the maintenance of both small and large circular DNA in cancers, but also in the context of other diseases and normal cellular development.EGA study EGAS00001007026