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Mapping ribonucleotides embedded in genomic DNA to single-nucleotide resolution using Ribose-Map

Nature Protocols volume 16, pages 3625–3638 (2021)Cite this article

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Abstract

The most common nonstandard nucleotides found in genomic DNA are ribonucleotides. Although ribonucleotides are frequently incorporated into DNA by replicative DNA polymerases, very little is known about the distribution and signatures of ribonucleotides incorporated into DNA. Recent advances in high-throughput ribonucleotide sequencing can capture the exact locations of ribonucleotides in genomic DNA. Ribose-Map is a user-friendly, standardized bioinformatics toolkit for the comprehensive analysis of ribonucleotide sequencing experiments. It allows researchers to map the locations of ribonucleotides in DNA to single-nucleotide resolution and identify biological signatures of ribonucleotide incorporation. In addition, it can be applied to data generated using any currently available high-throughput ribonucleotide sequencing technique, thus standardizing the analysis of ribonucleotide sequencing experiments and allowing direct comparisons of results. This protocol describes in detail how to use Ribose-Map to analyze ribonucleotide sequencing data, including preparing the reads for analysis, locating the genomic coordinates of ribonucleotides, exploring the genome-wide distribution of ribonucleotides, determining the nucleotide sequence context of ribonucleotides and identifying hotspots of ribonucleotide incorporation. Ribose-Map does not require background knowledge of ribonucleotide sequencing analysis and assumes only basic command-line skills. The protocol requires less than 3 h of computing time for most datasets and ~30 min of hands-on time. Ribose-Map is available at https://github.com/agombolay/ribose-map.

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Fig. 1: Input/output of Ribose-Map’s modules.
Fig. 2: rNMP position in sequencing reads for each rNMP sequencing technique.
Fig. 3: rNMP composition in nuclear and mitochondrial yeast DNA.
Fig. 4: DNA sequence context of rNMPs in mitochondrial yeast DNA.
Fig. 5: Distribution of rNMPs in yeast mitochondrial DNA.
Fig. 6: Consensus sequences of hotspots in yeast mitochondrial DNA.

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Data availability

The ribose-seq dataset used for this study is available in NCBI’s Sequence Read Archive via accession number SRR113649337. The emRiboSeq dataset used for this study is available in NCBI’s Gene Expression Omnibus via accession number SRR17349677.

Code availability

Ribose-Map is available for download at GitHub (https://doi.org/10.5281/zenodo.4574403).

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Acknowledgements

We are grateful to P. Xu, T. Yang, K. Mukherjee and D. Kundnani for suggestions on this manuscript as well as M. Borodovsky, I.K. Jordan, S. Yi, F. Vannberg and all members of the Storici laboratory for their advice during the course of this study. This research was supported by the National Institutes of Health (R01ES026243-01 to F.S.); the Parker H. Petit Institute for Bioengineering and Bioscience at Georgia Institute of Technology (12456H2 to F.S.), and the Howard Hughes Medical Institute Faculty Scholar grant (55108574 to F.S.).

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Authors and Affiliations

  1. School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA

    Alli L. Gombolay & Francesca Storici

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  1. Alli L. Gombolay
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  2. Francesca Storici
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Contributions

A.L.G. is the lead developer of Ribose-Map and wrote the manuscript with input from F.S. A.L.G. and F.S. designed and developed the protocol and generated the example experiments.

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Correspondence to Alli L. Gombolay or Francesca Storici.

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The authors declare no competing interests.

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

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Key references using this protocol

Gombolay, A. L. et al. Nucleic Acids Res. 47, e5 (2019): https://doi.org/10.1093/nar/gky874

Balachander, S. et al. Nat. Commun. 11, 2447 (2020): https://doi.org/10.1038/s41467-020-16152-5

Key data used in this protocol

Balachander, S. et al. Nat. Commun. 11, 2447 (2020): https://doi.org/10.1038/s41467-020-16152-5

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Gombolay, A.L., Storici, F. Mapping ribonucleotides embedded in genomic DNA to single-nucleotide resolution using Ribose-Map. Nat Protoc 16, 3625–3638 (2021). https://doi.org/10.1038/s41596-021-00553-x

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