A publication in Nature Protocols

A method for characterizing circular DNA molecules (virus and eukaryote) which play important roles (cancer, virosis)



Researchers from the Plant Genetics Laboratory (TERRA / Gembloux Agro-Bio Tech) of the University of Liège, in collaboration with researchers from the University of Alberta (Edmonton, Canada) and the University of Zurich (Switzerland), have developed a method for the precise sequencing of circular DNA molecules. This method, published in the scientific journal Nature Protocols, could contribute to a better characterization of important viral pathogens such as human papillomaviruses, animal circoviruses and plant geminiviruses. It also paves the way to the in-depth study of circular DNA molecules produced by eukaryotic cells.

C

ircular DNA is ubiquitous in nature in the form of plasmids, circular DNA viruses and extrachromosomal circular DNA (eccDNA) in eukaryotes. The sequencing of these molecules is essential for profiling the distribution of viruses, discovering new viruses, and understanding the role of eccDNA in eukaryotic cells. Circular DNA molecule enrichment sequencing (CIDER-Seq) is a technique that allows the precise enrichment and sequencing of circular DNA without the use of polymerase chain reaction amplification, cloning and computer sequence assembly. A new method, developed by Prof. Hervé Vanderschuren and his colleagues at the Plant Genetics Laboratory, using this technique allows the profiling of small circular DNA molecules such as viruses, as well as the characterization of so-called extrachromosomal circular DNA molecules (eccDNA) whose important functions in eukaryotic cells have been hypothesized and demonstrated, including the regulation of oncogenes in cancer cells.

CIDER Seq protocol 

Overview of the CIDER-Seq protocol.
 

The CIDER-Seq method - based on the combination of automated size selection, non-denaturing circular random amplification (RCA), linearization and repair of the RCA product, and real-time single molecule sequencing (SMRT) - achieves the goal of "one complete genome per single sequencing fragment", which is to produce high-quality complete circular DNA genomic sequences. The method could enable researchers to produce accurate complete genome data for a number of important viruses, including human papillomaviruses, animal circoviruses and plant geminiviruses. The CIDER-Seq method exceeds currently available methods in the field and reduces the cost per virus genome by at least 10 times.

The researchers were also able to demonstrate that the method allows the profiling of eccDNA in eukaryotic cells. The unambiguous identification of circular DNA molecules will help deciphering the roles of eccDNA molecules during various developmental processes as well as during exposure to various stress conditions. The Plant Genetics Laboratory is currently using this technology in FNRS-sponsored projects aimed at investigating the roles of eccDNA in plants.

Reference

Mehta, D., Cornet, L., Hirsch-Hoffmann, M., Shan-e-Ali Zaidi, S., Vanderschuren, H. (2020) Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq. Nature Protocols

Contact

Plant Genetics Laboratory | TERRA Taching and Research Center | Gembloux Agro-Bio Tech

Pr Hervé Vanderschuren

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