A new single-cell sequencing method has been published in Nature Genetics. Single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq) can achieve parallel sequencing of circular DNAs and full-length mRNA from single cells. scEC&T-seq was used to characterise single cancer cells, facilitating the analysis of circular DNA in cancer and beyond.
Circular DNA and cancer
Three types of circular DNAs have been described in human cells. (1) small circular DNAs, also referred to as microDNAs or apoptotic DNAs, among other names; (2) T cell receptor excision circles; (3) large oncogenic copy number-amplified circular extrachromosomal DNAs (ecDNAs).
Oncogenic amplification of ecDNAs potentially drives copy number heterogeneity in cancer. Copy number heterogeneity enables tumours to adapt and evade therapies. As such, patients with ecDNA-harbouring cancers have been shown to have worse clinical outcomes.
The measurement of multiple parameters in the same cell helps to accurately understand the biological changes occurring in disease. With circular DNAs, it is critical to integrate DNA sequence with transcriptional output to assess their functional impact on cells.
Targeting circular DNA and the transcriptome
In the current study, scEC&T-seq was introduced as a method that enables parallel sequencing of all circular DNA types and full-length mRNA in individual cells. The utility of the method was demonstrated by profiling single cancer cells containing both structurally complex multi-fragmented ecDNAs and small circular DNAs.
The results showed that scEC&T-seq was able to detect circular DNA and mRNA in single cells. scEC&T-seq also distinguished the transcriptional consequences of ecDNA-driven intercellular oncogene copy number heterogeneity and has the potential to uncover the principles of ecDNA structural evolution.
Looking in more detail at cancer cells, small circular DNAs were identified as exclusive to single cells, whereas oncogenic ecDNAs were clonally present in single cells. The authors suggest that these results indicate that small circular DNAs do not offer a selective advantage to cancer cells, but highlight unknown prerequisites for their selection, propagation and maintenance.
A complementary method for future research
The number of single-cell DNA and RNA sequencing methods has been steadily growing. scEC&T-seq complements these, as it can distinguish extrachromosomal circular DNA. It is possible to pair the method with other high throughput single-cell technologies, which may increase the spectrum of somatic variation detected by scEC&T-seq.
The robust integration of circular DNA and mRNA sequencing in single cancer cells performed by scEC&T-seq indicates that the same approach can be applied to a diverse range of biological systems. This can be used to explore the diversity of circular DNA in individual cells and the authors anticipate that the technology will be a resource for future research in many fields beyond cancer biology.