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2b-RAD: a simple and flexible method for genome-wide genotyping

Nature Methods volume 9, pages 808–810 (2012)Cite this article

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Abstract

We describe 2b-RAD, a streamlined restriction site–associated DNA (RAD) genotyping method based on sequencing the uniform fragments produced by type IIB restriction endonucleases. Well-studied accessions of Arabidopsis thaliana were genotyped to validate the method's accuracy and to demonstrate fine-tuning of marker density as needed. The simplicity of the 2b-RAD protocol makes it particularly suitable for high-throughput genotyping as required for linkage mapping and profiling genetic variation in natural populations.

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Figure 1: Preparation and sequencing of 2b-RAD tags.
Figure 2: Accuracy, sequencing requirements and adjustment of marker densities in 2b-RAD genotyping.

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Sequence Read Archive

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Sequence Read Archive

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Acknowledgements

This work was supported by the National Science Foundation grants DEB-1054766 to M.V.M. and DEB-1022196 to J.K.M., and by grants from the National Natural Science Foundation of China (31130054) and National High Technology Research and Development Program of China (2012AA10A405) to S.W. We are grateful to T.E. Juenger and D.L. Des Marais (University of Texas at Austin) for growing Arabidopsis samples.

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Author notes

  1. Shi Wang and Eli Meyer: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Biological Sciences, University of Texas at Austin, Austin, Texas, USA

    Shi Wang, Eli Meyer & Mikhail V Matz

  2. College of Marine Life Sciences, Ocean University of China, Qingdao, Shandong, China.,

    Shi Wang

  3. Department of Zoology, Oregon State University, Corvallis, Oregon, USA

    Eli Meyer

  4. Department of Bioagricultural Sciences, Colorado State University, Fort Collins, Colorado, USA

    John K McKay

Authors
  1. Shi Wang
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  2. Eli Meyer
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  3. John K McKay
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  4. Mikhail V Matz
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Contributions

S.W. and M.V.M. conceived and designed the study. S.W. developed the original protocol for 2b-RAD library preparation, and E.M. developed modifications for RTR. S.W. and E.M. prepared 2b-RAD libraries for SOLiD and Illumina sequencing. E.M. created bioinformatics scripts and conducted sequence analysis and genotype validation. J.K.M. provided Arabidopsis samples and contributed resequencing data for bioinformatic validation. S.W., E.M., J.K.M. and M.V.M. wrote the paper. M.V.M. supervised the whole study.

Corresponding authors

Correspondence to Shi Wang or Eli Meyer.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 , Supplementary Tables 1–11 and Supplementary Protocol (PDF 365 kb)

Supplementary Software

Perl scripts for 2b-RAD analysis. (ZIP 12 kb)

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Wang, S., Meyer, E., McKay, J. et al. 2b-RAD: a simple and flexible method for genome-wide genotyping. Nat Methods 9, 808–810 (2012). https://doi.org/10.1038/nmeth.2023

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