Three CEN.PK Saccharomyces cerevisiae populations (10^10 cells/polulation), purified by the Circle-Seq method, reporting hundreds of eccDNA profiles in sizes larger than 1 kb.. Circle-Seq on Saccharomyces cerevisiae CEN.PK113-7D strain.

Chromosomal DNA deletions in eukaryotic genomes can lead to the formation of extrachromosomal circular DNA (eccDNA). Autonomous replication and missegregation of eccDNA at mitosis might cause genetic variation between somatic cells in multicellular organisms and could be important for evolution of unicellular eukaryotes. However, the role of eccDNAs at the genomic level, how they form and what their mutation frequencies are remains to be elucidated. Here we describe a very sensitive eccDNA purification method called Circle-Seq, encompassing column purification of eccDNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, high-throughput sequencing and mapping. We validate the Circle-Seq method on three CEN.PK populations (10^10 cells/population), reporting hundreds of eccDNA profiles in sizes larger than 1 kb. We also find that extensive exonuclease treatment, using more than 100 units, is typically required for sufficient linear chromosomal DNA degradation and that the rolling-circle amplification by φ29 polymerase enriches for circular DNA. We believe that the Circle-Seq method has broad applicability in genome-scale screening for eccDNA in eukaryotes as well as for detection of specific eccDNA types.