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Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples

Nature Methods volume 7, pages 913–915 (2010)Cite this article

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Abstract

Targeted genomic enrichment followed by next-generation DNA sequencing has dramatically increased efficiency of mutation-discovery efforts. We describe a protocol for genomic enrichment of pooled barcoded samples in a single assay that increases experimental flexibility and efficiency. We screened 770 genes (1.4 megabases) in thirty N-ethyl-N-nitrosourea (ENU)-mutagenized rats and identified known variants at >96% sensitivity as well as new mutations at a false positive rate < 1 in 8 megabases.

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Figure 1: Mutation discovery strategy.

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Acknowledgements

This work was supported by funds from the Cancer Genomics Centre, the award “Exploiting natural and induced genetic variation in the laboratory rat” to E.C. from the European Heads of Research Councils and European Science Foundation European Young Investigator Award scheme and the EU FP7 integrated project Euratrans.

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Author notes

  1. Isaäc J Nijman, Michal Mokry and Ruben van Boxtel: These authors contributed equally to this work.

Authors and Affiliations

  1. Hubrecht Institute, Developmental Biology and Stem Cell Research, Royal Netherlands Academy of Arts and Sciences and the University Medical Center Utrecht, Utrecht, The Netherlands

    Isaäc J Nijman, Michal Mokry, Ruben van Boxtel, Pim Toonen, Ewart de Bruijn & Edwin Cuppen

  2. Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands

    Edwin Cuppen

Authors
  1. Isaäc J Nijman
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  2. Michal Mokry
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  3. Ruben van Boxtel
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  4. Pim Toonen
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  5. Ewart de Bruijn
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  6. Edwin Cuppen
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Contributions

R.v.B., I.J.N., M.M. and E.C. designed the experiments. R.v.B. and P.T. performed rat ENU mutagenesis. M.M. performed library preparation and genomic enrichments. E.d.B. performed SOLiD sequencing. I.J.N. analyzed the data. R.v.B. performed capillary sequencing reconfirmation experiments. I.J.N. and E.C. wrote the manuscript.

Corresponding author

Correspondence to Edwin Cuppen.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2 and Supplementary Tables 2–5 (PDF 561 kb)

Supplementary Table 1

List of rat genes targeted in the next-generation reverse genetic. (XLS 2071 kb)

Supplementary Table 6

Complete list of polymorphic positions identified in the next-generation reverse genetics screen. (XLS 112 kb)

Supplementary Software

Custom SNP filtering PERL script. (ZIP 3 kb)

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Nijman, I., Mokry, M., van Boxtel, R. et al. Mutation discovery by targeted genomic enrichment of multiplexed barcoded samples. Nat Methods 7, 913–915 (2010). https://doi.org/10.1038/nmeth.1516

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