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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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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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.
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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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DOI: https://doi.org/10.1038/nmeth.1516
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