Table 1.

Advantages and disadvantages of eccDNA purification and enrichment methods.

name	principles	advantages	disadvantages	applications
Column-based purification	Employing genomic or plasmid DNA extraction protocols to isolate eccDNA via column chromatography.	1. Effective for isolating both small and large eccDNA fragments. 2. Offers reproducibility across different samples.	1. Multiple steps involved can be time-consuming. 2. Risk of contamination requires careful handling.	Suitable for large-scale eccDNA studies from cell and tissue samples, where high precision and efficiency are required. Ideal for both short and long-read sequencing applications and studies involving both high and low eccDNA abundance.
3SEP procedure	Three steps are: 1) modified lysis to preserve circle integrity, 2) linearization of mtDNA before digesting linear DNA, and 3) further separation from linear DNA remnants using beads.	1. Enhances eccDNA purity and reproducibility. 2. Significantly improves the efficiency of isolating pure eccDNA.	1. Additional step increases the complexity of the procedure. 2. May require optimization for different sample types.	Particularly useful for high-fidelity applications where the purity of eccDNA is crucial, such as in diagnostic and clinical research settings. Effective for both short and long eccDNA molecules.
CRISPR-CATCH	Combines in-vitro CRISPR-Cas9 cleavage with PFGE for selectively enriching megabase-sized eccDNA.	1. Allows for the selective enrichment of megabase-sized eccDNA. 2. Effective in enriching large eccDNA, making it suitable for studies on large eccDNA molecules.	1. Requires specific equipment and expertise, which might not be available in all laboratories. 2. The method is resource-intensive and may not be feasible for all research setups.	Ideal for detailed studies on large eccDNA, particularly in cancer research where megabase-sized eccDNA is common. Suitable for long-read sequencing applications to study eccDNA dynamics and functions.