An enhancement of extrachromosomal circular DNA enrichment and amplification to address the extremely low overlap between replicates
Issued Date
2026-04-01
Resource Type
ISSN
00219258
eISSN
1083351X
Scopus ID
2-s2.0-105034623274
Pubmed ID
41724384
Journal Title
Journal of Biological Chemistry
Volume
302
Issue
4
Rights Holder(s)
SCOPUS
Bibliographic Citation
Journal of Biological Chemistry Vol.302 No.4 (2026)
Suggested Citation
Burnham C.M., Kurilung A., Wanchai V., Regenberg B., Delgado-Calle J., Basnakian A.G., Nookaew I. An enhancement of extrachromosomal circular DNA enrichment and amplification to address the extremely low overlap between replicates. Journal of Biological Chemistry Vol.302 No.4 (2026). doi:10.1016/j.jbc.2026.111302 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/116077
Title
An enhancement of extrachromosomal circular DNA enrichment and amplification to address the extremely low overlap between replicates
Corresponding Author(s)
Other Contributor(s)
Abstract
Extrachromosomal circular DNA (eccDNA) of chromosomal origin is present in all eukaryotic organisms and tissues that have been tested. Populations of eccDNA exhibit immense diversity and a characteristically low degree of overlap between samples, suggesting low inheritance of eccDNA between cells or a deficiency in the methods by which eccDNA is detected. This study revisits the Circle-Seq approach for enrichment of eccDNA to address these limitations, hypothesizing that experimental procedures significantly contribute to the observed low eccDNA overlap. We optimized the protocol by reducing the time needed to complete the procedure. Linear DNA is digested by increasing Exonuclease V activity. We employed CRISPR-Cas9 for mitochondrial linearization, which proved superior to using restriction enzymes. A key finding is the critical role of random hexamer primer concentration and genomic DNA input in rolling circle amplification (RCA) for generating high-quality long concatemeric tandem copy amplicons from eccDNA, essential for confident de novo eccDNA construction from long-read sequencing data. Lower primer concentrations substantially increased the percentage of concatemer-derived eccDNA and improved the overlap of identified eccDNAs in technical replicates. Applying this revised approach to human myeloma and breast cancer cell lines, as well as xenograft models, demonstrated >50% overlap in detected eccDNA, a substantial improvement over the <1% overlap observed in previous studies. Additionally, the oncogenic signature of eccDNAs can be identified across all replicates. These findings provide guidelines for developing standardized procedures for eccDNA profiling, advancing our understanding of eccDNA biology, and its potential clinical applications.