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Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq

An Author Correction to this article was published on 27 May 2022

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Abstract

Circular DNA is ubiquitous in nature in the form of plasmids, circular DNA viruses, and extrachromosomal circular DNA (eccDNA) in eukaryotes. Sequencing of such molecules is essential to profiling virus distributions, discovering new viruses and understanding the roles of eccDNAs in eukaryotic cells. Circular DNA enrichment sequencing (CIDER-Seq) is a technique to enrich and accurately sequence circular DNA without the need for polymerase chain reaction amplification, cloning, and computational sequence assembly. The approach is based on randomly primed circular DNA amplification, which is followed by several enzymatic DNA repair steps and then by long-read sequencing. CIDER-Seq includes a custom data analysis package (CIDER-Seq Data Analysis Software 2) that implements the DeConcat algorithm to deconcatenate the long sequencing products of random circular DNA amplification into the intact sequences of the input circular DNA. The CIDER-Seq data analysis package can generate full-length annotated virus genomes, as well as circular DNA sequences of novel viruses. Applications of CIDER-Seq also include profiling of eccDNA molecules such as transposable elements (TEs) from biological samples. The method takes ~2 weeks to complete, depending on the computational resources available. Owing to the present constraints of long-read single-molecule sequencing, the accuracy of circular virus and eccDNA sequences generated by the CIDER-Seq method scales with sequence length, and the greatest accuracy is obtained for molecules <10 kb long.

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Fig. 1: Profile of eccDNA sequences obtained via CIDER-Seq on three independent 6-week-old wild-type A. thaliana plants (WT1–3).
Fig. 2: Overview of the CIDER-Seq protocol.
Fig. 3: Example output plots for annotated virus genomes produced by the CIDER-Seq data analysis software for three sequencing reads from a circular DNA virus, East African cassava mosaic virus (EACMV).

Data availability

Raw and processed data from the original publication are freely available at https://doi.org/10.5281/zenodo.1009036. Data from Fig. 1 are available upon request. Fig. 3 has associated raw data that are provided in the software installation.

Code availability

The CIDER-Seq Data Analysis Software package is available under a GNU Affero Public License v. 3.0 at https://github.com/devang-mehta/ciderseq2. A static copy of the software version used in developing this protocol has been deposited at https://doi.org/10.5281/zenodo.3386568. A Singularity image of the software is also available at https://doi.org/10.5281/zenodo.3401571.

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Acknowledgements

We thank A. Patrignani (Functional Genomics Center Zurich, Switzerland) for assistance with SMRT sequencing, as well as for helpful advice and discussion. The protocol for geminivirus sequencing was initially developed during a project funded by the European Union’s Seventh Framework Programme for research, technological development, and demonstration (EUGA–2013–608422-IDP BRIDGES to H.V. & D.M.). The authors acknowledge financial support from the Belgian FNRS (grant M.i.S. F.4515.17 to H.V.; grant 1.B456.20 to S.S.Z. and H.V.), LEAPAgri grant 288 to H.V., and a Swiss National Science Foundation Early Postdoc Mobility grant (181602) to D.M.

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Conceptualization: D.M. and H.V.; methodology and formal analysis: D.M., M.H.-H., L.C., S.S.Z.; software: M.H.-H., L.C. and D.M.; visualization and writing: D.M.; resources and supervision: H.V. All authors agreed with the final version of the manuscript.

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Correspondence to Devang Mehta or Hervé Vanderschuren.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Simon Roux and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Mehta, D. et al. Nucleic Acids Res. 47, e9 (2019): https://doi.org/10.1093/nar/gky914

Integrated supplementary information

Supplementary Fig. 1 Example profiles of enriched circular DNA.

a. Cassava DNA samples after CIDER-Seq enrichment run on a Bioanalyzer 2100 High-sensitivity DNA chip. b. Arabidopsis DNA samples after CIDER-Seq enrichment run on a Tapestation Genomic DNA Screentape.

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Mehta, D., Cornet, L., Hirsch-Hoffmann, M. et al. Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq. Nat Protoc 15, 1673–1689 (2020). https://doi.org/10.1038/s41596-020-0301-0

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