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

Abstract

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond.

Decoil: Reconstructing extrachromosomal DNA structural heterogeneity from long-read sequencing data

“…The reconstructed ecDNA elements varied greatly in their complexity (Figure 4f) and ranged from very simple (Figure 4a) or multi-region (Figure 4b) to heavily rearranged complex structures (Figure 4c,d,e). Decoil reconstructs for Patient 4 two ecDNA elements with an individual estimated proportions of more than 700x, resolving the same breakpoints as previously published ([8], Fig. 7a).…”

“…The reconstructed ecDNA elements varied greatly in their complexity (Figure 4f) and ranged from very simple (Figure 4a) or multi-region (Figure 4b) to heavily rearranged multi-fragmented structures (Figure 4c,d). Decoil reconstructed two ecDNA elements with an individual estimated proportions of more than 700x in patient #4, resolving the same breakpoints as previously described in single cell ecDNA sequencing data from this tumor ( [7]). For samples with a very high structural-variant density at the genomic site of ecDNA origin, Decoil reconstructed multiple circular elements with di↵erent estimated relative proportions, which indicates ecDNA structural heterogeneity (Figure 4e).…”

“…Due to their random mitotic segregation, many ecDNA elements, which may structurally differ, co-occur in the same cancer cells [8]. Disentangling ecDNA with shared genomic regions has not yet been addressed by other methods, and it cannot be resolved by de-novo assemblers (e.g.…”

“…To assess Decoil's reconstruction performance, we generated an in-silico collection of ecDNA elements, spanning various sequence complexities for systematic evaluation. We introduced a ranking system and defined seven topologies of increasing computational complexity, based on the SV's contained on the ecDNA element: (1) Simple circularization -no structural variants on the ecDNA template, (2) Simple SV's -ecDNA contains either a series of inversions or deletions, (3) Mixed SV's -ecDNA has a combination of inversions and deletions, (4) Multi-region -ecDNA contains di↵erent genomic regions from the same chromosome (DEL, INV and TRA allowed), (5) Multi-chromosomal -ecDNA originates from multiple chromosomes (DEL, INV and TRA allowed), (6) Duplications -ecDNA contains duplications defined as a region larger than 50 bp repeated on the amplicon (DUP's + other simple rearrangements), (7) Foldbacks -ecDNA contains a foldback defined as a two consecutive fragments which overlap in the genomic space, with di↵erent orientations (INVDUP's + all other simple SV's). Every topology can contain a mixture of all other low-rank topologies.…”

“…repeats), which pose a challenge to mapping and de-novo assembly based methods. On the other hand, one tumor can contain different ecDNA elements [7, 8], which can either originate from different or shared genomic locations [9]. The latter scenario may be very challenging for ecDNA reconstruction, as different co-occurring ecDNA elements have overlapping genomic footprints, making it difficult to attribute the overlapping features to each of the different circular elements.…”