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Global analysis of repetitive DNA from unassembled sequence reads using RepeatExplorer2

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Abstract

RepeatExplorer2 is a novel version of a computational pipeline that uses graph-based clustering of next-generation sequencing reads for characterization of repetitive DNA in eukaryotes. The clustering algorithm facilitates repeat identification in any genome by using relatively small quantities of short sequence reads, and additional tools within the pipeline perform automatic annotation and quantification of the identified repeats. The pipeline is integrated into the Galaxy platform, which provides a user-friendly web interface for script execution and documentation of the results. Compared to the original version of the pipeline, RepeatExplorer2 provides automated annotation of transposable elements, identification of tandem repeats and enhanced visualization of analysis results. Here, we present an overview of the RepeatExplorer2 workflow and provide procedures for its application to (i) de novo repeat identification in a single species, (ii) comparative repeat analysis in a set of species, (iii) development of satellite DNA probes for cytogenetic experiments and (iv) identification of centromeric repeats based on ChIP-seq data. Each procedure takes approximately 2 d to complete. RepeatExplorer2 is available at https://repeatexplorer-elixir.cerit-sc.cz.

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Fig. 1: Schematic representation of RepeatExplorer (a) and TAREAN (b) pipelines.
Fig. 2: Decision tree for automatic annotation.
Fig. 3: Principle of comparative analysis.
Fig. 4: Structure of HTML reports.
Fig. 5: Graphical summary of clustering results for V. villosa.
Fig. 6: Comparative analysis summary.
Fig. 7: Design of an oligonucleotide probe and primers for PCR.
Fig. 8: Example visualization of ChIP-seq Mapper output.

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Data availability

Example datasets that include WGS reads and ChIP-Seq reads (Table 1) were published in refs. 18 and 19 and are freely available at the ENA database (https://www.ebi.ac.uk/ena).

Code availability

The source code for all pipelines is available for public use at https://bitbucket.org/petrnovak/repex_tarean/ and https://bitbucket.org/repeatexplorer/re_utilities/ under a GNU General Public License.

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Acknowledgements

This work was supported by the ERDF/ESF project ELIXIR-CZ - Capacity building (No. CZ.02.1.01/0.0/0.0/16_013/0001777) and the ELIXIR-CZ research infrastructure project (MEYS No: LM2015047) including access to computing and storage facilities.

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Authors and Affiliations

  1. Biology Centre, Czech Academy of Sciences, Institute of Plant Molecular Biology, České Budějovice, Czech Republic

    Petr Novák, Pavel Neumann & Jiří Macas

Authors
  1. Petr Novák
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  2. Pavel Neumann
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  3. Jiří Macas
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Contributions

P. Novák, P. Neumann and J.M. conceptualized, designed or developed analysis workflows, tools or procedures and wrote the manuscript.

Corresponding author

Correspondence to Jiří Macas.

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The authors declare no competing interests.

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

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Related links

Key references using this protocol

Macas, J. et al. PLoS ONE 10, e0143424 (2015): https://doi.org/10.1371/journal.pone.0143424

Ávila Robledillo, L. et al. Sci. Rep. 8, 5838 (2018): https://doi.org/10.1038/s41598-018-24196-3

Ávila Robledillo, L. et al. Mol. Biol. Evol. 37, 2341–2356 (2020): https://doi.org/10.1093/molbev/msaa090

Key data used in this protocol

Macas, J. et al. PLoS ONE 10, e0143424 (2015): https://doi.org/10.1371/journal.pone.0143424

Supplementary information

Supplementary Data 1

Complete list of classification categories used for annotation in Viridiplantae

Supplementary Data 2

Repeat quantification performed on example dataset

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Novák, P., Neumann, P. & Macas, J. Global analysis of repetitive DNA from unassembled sequence reads using RepeatExplorer2. Nat Protoc 15, 3745–3776 (2020). https://doi.org/10.1038/s41596-020-0400-y

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