Advances in sequencing technologies have allowed for genome-scale identification and mapping of eccDNAs that range in size from 0.1 kilobase (kb) to 2 kb (named microDNA) from human cell lines and mice 20, 21. EccDNA has also been isolated using linear DNA-specific exonucleases 18, 19 or targeted probes for specific eccDNA types and imaging after two-dimensional gel electrophoresis 12 or fluorescence in situ hybridization 10. Identification of smaller circular DNA has been by achieved by density separation of DNA by cesium-chloride ultracentrifugation, followed by electron microscopy 17. In addition to oncogenes, 5 S ribosomal DNA (rDNA) and repetitive DNA have been found on eccDNA in human cell lines 12, 13, 14, showing that a fraction of the genome can exist as eccDNA, at least under certain conditions.Ĭircular DNA structures of megabase (Mb) sizes are also known as ring chromosomes and can be visualized microscopically by staining metaphase DNA 15, 16. EccDNA was later shown to be common in many types of cancers, forming from chromosomal genes and promoting oncogenesis 8, 9, 10, 11. Evidence of eccDNA in humans has existed for more than half a century since double minutes were discovered in tumor specimens from children 7. We hypothesized that deleted DNA as well as by-products of damaged DNA can circularize and co-exist in genomes as semi-stable extrachromosomal circular DNA (eccDNA see Supplementary Note 1 for review of abbreviations for circular DNA). Deleted acentric DNA may be removed from cells by targeted degradation or exclusion from replication. However, the fate of the deleted chromosomal DNA in human somatic cells is unknown. Genome-scale sequencing has revealed that insertions, deletions, and amplifications are common in humans 3, 4, 5 and even single cells from healthy somatic tissues contain large structural variations 6. This genome structure was previously thought to be highly stable, with minimal divergence between cells 1, 2.

The human genome contains 22 linear autosome pairs and a pair of sex-determining chromosomes. Thus, somatic genomes are rich in chromosome-derived eccDNAs that may influence phenotypes through altered gene copy numbers and transcription of full-length or truncated genes. Gene-rich chromosomes contribute to more eccDNAs per megabase and the most transcribed protein-coding gene in muscle, TTN (titin), provides the most eccDNAs per gene. Transcription from eccDNAs suggests that eccDNAs reside in nuclei and recurrence of certain eccDNAs in several individuals implies DNA circularization hotspots. Half of these structures carry genes or gene fragments and the majority are smaller than 25 kb. Here, we purify and sequence eccDNAs from muscle and blood samples from 16 healthy men, detecting ~100,000 unique eccDNA types from 16 million nuclei. However, it must be expected that kb eccDNAs exist in normal cells as a result of mutations. The human genome is generally organized into stable chromosomes, and only tumor cells are known to accumulate kilobase (kb)-sized extrachromosomal circular DNA elements (eccDNAs).

Serial cloner linear to circular