ATAC-Seq-based Identification of Extrachromosomal Circular DNA in Mammalian Cells and Its Validation Using Inverse...

Area of Science:

• Genomics
• Cancer Biology
• Molecular Biology

Background:

• Extrachromosomal circular DNA (eccDNA) is increasingly recognized for its biological significance, ranging from yeast to human systems.
• In cancer, eccDNA can confer a selective advantage by amplifying oncogenes, contributing to genomic instability.
• Previous methods for eccDNA identification, such as rolling circle amplification, are biased towards smaller circular DNA molecules.

Purpose of the Study:

• To develop and validate a novel, rolling circle-independent method for detecting extrachromosomal circular DNA (eccDNA) in human cancer cells.
• To identify novel eccDNAs that may be missed by traditional amplification techniques.
• To establish a robust workflow for eccDNA discovery and validation.

Main Methods:

• Utilized ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) to profile open chromatin regions associated with eccDNA.
• Employed the Circle_finder bioinformatics algorithm to predict potential eccDNA sequences from sequencing data.
• Validated predicted eccDNAs using inverse PCR and metaphase FISH (Fluorescence in situ Hybridization).

Main Results:

• Successfully implemented a sensitive and robust workflow for detecting eccDNA independent of rolling circle amplification.
• Identified novel eccDNAs in human cancer cells using the developed ATAC-seq and Circle_finder approach.
• Confirmed the presence and location of novel eccDNAs through independent validation methods.

Conclusions:

• The developed ATAC-seq and Circle_finder based method provides a powerful, unbiased approach for eccDNA discovery in cancer.
• This rolling circle-independent strategy overcomes limitations of previous methods, enabling the identification of a broader range of eccDNAs.
• The validated workflow facilitates deeper understanding of eccDNA's role in cancer progression and genomic instability.