ECCsplorer: a pipeline to detect extrachromosomal circular DNA (eccDNA) from next-generation sequencing data

Tony Heitkam,Beatrice Weber,Ludwig Mann,Kathrin M. Seibt

doi:10.1186/s12859-021-04545-2

Abstract

BackgroundExtrachromosomal circular DNAs (eccDNAs) are ring-like DNA structures physically separated from the chromosomes with 100 bp to several megabasepairs in size. Apart from carrying tandemly repeated DNA, eccDNAs may also harbor extra copies of genes or recently activated transposable elements. As eccDNAs occur in all eukaryotes investigated so far and likely play roles in stress, cancer, and aging, they have been prime targets in recent research—with their investigation limited by the scarcity of computational tools.ResultsHere, we present the ECCsplorer, a bioinformatics pipeline to detect eccDNAs in any kind of organism or tissue using next-generation sequencing techniques. Following Illumina-sequencing of amplified circular DNA (circSeq), the ECCsplorer enables an easy and automated discovery of eccDNA candidates. The data analysis encompasses two major procedures: first, read mapping to the reference genome allows the detection of informative read distributions including high coverage, discordant mapping, and split reads. Second, reference-free comparison of read clusters from amplified eccDNA against control sample data reveals specifically enriched DNA circles. Both software parts can be run separately or jointly, depending on the individual aim or data availability. To illustrate the wide applicability of our approach, we analyzed semi-artificial and published circSeq data from the model organisms Homo sapiens and Arabidopsis thaliana, and generated circSeq reads from the non-model crop plant Beta vulgaris. We clearly identified eccDNA candidates from all datasets, with and without reference genomes. The ECCsplorer pipeline specifically detected mitochondrial mini-circles and retrotransposon activation, showcasing the ECCsplorer’s sensitivity and specificity.ConclusionThe ECCsplorer (available online at https://github.com/crimBubble/ECCsplorer) is a bioinformatics pipeline to detect eccDNAs in any kind of organism or tissue using next-generation sequencing data. The derived eccDNA targets are valuable for a wide range of downstream investigations—from analysis of cancer-related eccDNAs over organelle genomics to identification of active transposable elements.

Highlights

Extrachromosomal circular DNAs are ring-like DNA structures physically separated from the chromosomes with 100 bp to several megabasepairs in size
EccDNAs often consist of repetitive DNAs such as tandem repeats or transposable elements [3, 11,12,13]
For the control data the same amount of 200 bp PE reads was generated from the reference only

Summary

Introduction

Extrachromosomal circular DNAs (eccDNAs) are ring-like DNA structures physically separated from the chromosomes with 100 bp to several megabasepairs in size. In animals and human they have been reported as drivers for aging [5, 6] and cancer [7,8,9], whereas in plants they play a role in gradual development of glyphosate resistance [10]. Many of these physiological consequences can be traced to extra gene copies residing extrachromosomally within the DNA circles. EccDNAs often consist of repetitive DNAs such as tandem repeats (satellite DNAs) or (active) transposable elements [3, 11,12,13]. Due to their relationship with transposable elements and genes, eccDNAs may be involved in transcriptional regulation, e.g. mediated by small RNAs [14]

Methods

Results

Conclusion