Purification, full-length sequencing and genomic origin mapping of eccDNA

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Extrachromosomal circular DNA (eccDNA) has been known for decades, but its formation and roles remain largely unknown.
• Current methods for isolating pure eccDNA are inefficient due to low abundance and size heterogeneity.
• Existing purification relies on exonuclease digestion, which struggles with complete removal of linear DNA contaminants.

Purpose of the Study:

• To develop a highly efficient and reproducible method for purifying extrachromosomal circular DNA (eccDNA).
• To establish a robust technique for sequencing full-length eccDNA molecules.
• To create a bioinformatic tool for analyzing eccDNA sequences and their genomic origins.

Main Methods:

• A novel three-step eccDNA purification (3SEP) procedure was developed, incorporating an additional step to specifically recover circular DNA.
• Rolling-circle amplification was combined with Nanopore sequencing to enable full-length eccDNA sequencing.
• A bioinformatic tool, Flec, was created to identify consensus sequences from amplified eccDNA and map them to the genome.

Main Results:

• The 3SEP method yields eccDNA preparations with significantly higher purity and reproducibility compared to existing techniques.
• The combined rolling-circle amplification and Nanopore sequencing approach allows for comprehensive analysis of full-length eccDNA.
• The Flec caller accurately determines eccDNA consensus sequences and their genomic origins.

Conclusions:

• The 3SEP protocol and associated sequencing/bioinformatic tools provide a powerful platform for eccDNA research.
• This methodology overcomes previous limitations in eccDNA isolation and characterization.
• The developed protocol has potential applications in eccDNA-based diagnostics and clinical settings.