Decoding the genomic enigma: Approaches to studying extrachromosomal circular DNA

Abstract

Extrachromosomal circular DNA (eccDNA), a pervasive yet enigmatic component of the eukaryotic genome, exists autonomously from its chromosomal counterparts. Ubiquitous in eukaryotes, eccDNA plays a critical role in the orchestration of cellular processes and the etiology of diseases, particularly cancers. However, the full scope of its influence on health and disease remains elusive, presenting a rich vein of research yet to be mined. Unraveling the complexities of eccDNA necessitates a distillation of methodologies - from biogenesis to functional analysis - a landscape we overview in this study with precision and clarity. Here, we systematically outline cutting-edge methodologies from high-throughput sequencing and bioinformatics to experimental validations, showcasing the intricate world of eccDNAs. We combed through a treasure trove of auxiliary research resources and analytical tools. Moreover, we chart a course for future inquiry, illuminating the horizon with potential groundbreaking strategies for designing eccDNA research projects and pioneering new methodological frontiers.

Keywords: Analytical tool; Approach; Experimental method; Extrachromosomal circular DNA; High-throughput sequencing; Methodology; Single cell; ecDNA; eccDNA.

Fig. 1

The pivotal role of eccDNA in various physiological processes and its implications in human health and disease. (A) The importance of eccDNA in physiological processes. This section underscores the significance of eccDNA in key biological processes, such as genome rearrangements, DNA damage and repair, and cellular evolution and fitness. It also highlights the role of eccDNA in fundamental life processes including development and aging, as well as its involvement in the regulation of immune responses. These diverse functions underscore eccDNA's influence on the cellular and molecular architecture, emphasizing its importance in maintaining cellular homeostasis and responding to environmental challenges. (B) Implications of eccDNA in human health and diseases. In this part, the focus shifts to the impact of eccDNA on various aspects of human health, particularly in the context of oncology. It delineates how eccDNA influences oncogene expression and transcription, contributing to tumor heterogeneity and the development of drug resistance. This section also explores the emerging potential of eccDNA to serve as a liquid biopsy-derived biomarker. The visual elements in the image were created by BioRender.com.

Fig. 2

Systematic categorization of methods for investigating eccDNA. (A) Methods of eccDNA isolation and purification prior to NGS sequencing or visualization. These methods are crucial for preparing samples for subsequent high-throughput sequencing and ensuring the integrity and specificity of the eccDNA being studied. (B) High-throughput sequencing for eccDNA study. This part is further subdivided into two categories: bulk high-throughput techniques and single-cell-based high-throughput sequencing. (C) Experimental methodologies for identifying and exploring eccDNA. It includes various techniques used to confirm the presence of eccDNA in samples, as well as to investigate the biological function of eccDNA: This part focuses on the approaches including DAPI/FISH staining, microscopy, gel, southern blot, sanger sequencing and in-vito synthesis of eccDNA, employed to understand the biological roles and functional implications of eccDNA. The visual elements in the image were created by BioRender.com.

Fig. 3

The evolutionary journey of various eccDNA sequencing methods. The timeline format effectively maps out key milestones and breakthroughs in this field, illustrating the transformative impact these methods have had on our understanding of eccDNA. It begins with the inception of basic techniques (such as Circle-seq and WGS) for eccDNA detection and gradually transitions to more sophisticated methods, including bulk high-throughput approaches (such as scCircle-seq and scWGS) which have allowed for the study of eccDNA in populations of cells. Further along the timeline, the focus shifts to single-cell high-throughput technologies. This distinction between bulk and single-cell methodologies highlights the increasing precision and specificity with which researchers can now study eccDNA. The visual elements in the image were created by BioRender.com.

Fig. 4

Two candidate designs for eccDNA research. (A) Phenotype-driven approach. This design begins with the selection of a biologically or clinically relevant phenotype, such as cellular drug resistance, and involves a group design incorporating both resistant and non-resistant cells. It includes high-throughput sequencing (e.g., WGS or Circle-seq) to identify eccDNA and associated genes, followed by a selection process for potential target eccDNA based on bioinformatics analysis. Verification of eccDNA presence is achieved through FISH and advanced imaging. Functional studies using gain-of-function and loss-of-function techniques further explore the role of eccDNA in phenotypes like drug resistance, aiming to elucidate underlying mechanisms. (B) High-throughput sequencing and bioinformatics-driven approach. This method utilizes extensive sequencing data or database-driven selection (e.g., from eccDNAdb) to identify eccDNA and genes on eccDNA. It involves a comprehensive bioinformatics analysis, including gene expression abundance, prognosis analysis, and the integration of AI models for enhanced predictive analytics and expression profiling. The existence verification of eccDNA in experimental models is optional, emphasizing flexibility in research design. The visual elements in the image were created by BioRender.com.