Abstract
Extrachromosomal circular DNA (eccDNA) is a type of circular DNA that exists independently of chromosomes and has garnered significant attention in various fields, particularly in the context of smaller eccDNAs, which have considerable roles in gene regulation through various mechanisms. Current methods such as Circle-Seq and 3SEP can enrich small eccDNAs during sample preparation, but most bioinformatics pipelines remain challenging, exhibiting low accuracy and efficiency. This protocol describes the detailed workflow of a newly developed bioinformatics analysis pipeline, named EccDNA Caller based on Consecutive Full Pass (ECCFP), to accurately identify eccDNA from long-read Nanopore sequencing data. Compared to other pipelines, ECCFP significantly improves detection sensitivity, accuracy, and runtime efficiency. The process includes raw data quality control, trimming of adapters and barcodes, alignment to a reference genome, and identification of eccDNA, with detailed results encompassing accurate positioning of eccDNA, consensus sequences, and variants of individual eccDNA. Key features • This protocol provides a beginner-friendly, step-by-step workflow that enables researchers without bioinformatics experience to successfully execute the entire eccDNA identification process. • It offers an efficient computational pipeline for eccDNA detection from Nanopore sequencing data, integrating quality control, trimming, alignment, and eccDNA identification. • ECCFP exhibits sensitivity, accuracy, high efficiency, and low false-positive rates compared to existing long-read-based tools.