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A systematic genome-wide analysis of zebrafish protein-coding gene function

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Abstract

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches1,2,3 and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes3,4, this number falls considerably short of the more than 22,000 mouse protein-coding genes5. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning6, insertional mutagenesis7,8,9, antisense morpholino oligonucleotides10, targeted re-sequencing11,12,13, and zinc finger and TAL endonucleases14,15,16,17 have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes18. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence18,19, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.

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Figure 1: Exome sequencing.
Figure 2: Mutation detection.
Figure 3: Phenotypic analysis of alleles.
Figure 4: Confirmation of causality through complementation crosses.

Accession codes

Accessions

European Nucleotide Archive

Data deposits

Detailed information on alleles and their availability can be found online (http://www.sanger.ac.uk/Projects/D_rerio/zmp/). All sequencing data are deposited in the European Molecular Biology Laboratory (EMBL) European Nucleotide Archive under accession ERP000426.

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Acknowledgements

We thank P. Ellis, E. Markham, H. van Roekel, P. Toonen, J. van de Belt, J. Mudde and S. Widaa for technical assistance, and B. Novak, Y. Yi and E. LeProust from Agilent Technologies. We thank everyone at The Zebrafish International Resource Center and the European Zebrafish Resource Center for stocking and distributing alleles. We thank members of the Wellcome Trust Sanger Institute RSF and DNA pipelines. We also thank G. Powell, J. Collins and F. L. Marlow for critical reading of the manuscript. This work was funded through a core grant to the Sanger Institute by the Wellcome Trust (grant number 098051), the US National Institutes of Health (5R01HG004819), the EU Sixth Framework Programme (ZF-MODELS, contract number LSHG-CT-2003-503496) and the EU Seventh Framework Programme (ZF-HEALTH). F.v.E. is supported by the UK Medical Research Council (grant number G0777791) and E.C. is supported by the SmartMix program (SSM06010) from the Dutch government.

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Contributions

R.N.W.K., E.M.B.-N. and S.A.H. initiated, organized and executed the work (equal contributions). R.N.W.K., E.M.B.-N., S.A.H. and D.S. designed the experiments. R.N.W.K., E.M.B.-N. and S.A.H. wrote the manuscript with assistance from I.S., R.J.W., C.M.D. and D.L.S. Mutagenesis was carried out by R.N.W.K., F.v.E. and E.d.B. The mutation analysis pipeline was developed by I.S. and I.J.N., and maintained by R.J.W. C.H. and F.F. implemented and improved genotyping procedures, F.F. and E.M.B.-N. developed the cryopreservation procedure, S.A.H., S.M., C.S., C.M.D. and N.W. carried out the phenotyping, J.Y. designed and tested the first Agilent SureSelect exome set, R.G. helped to maintain and distribute alleles. S.C. and A.H. provided assistance for cryopreservation and genotyping. E.C. and D.L.S. collaborated in the initiation, design and process development of the project. All authors read the manuscript and provided comments.

Corresponding authors

Correspondence to Edwin Cuppen or Derek L. Stemple.

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The authors declare no competing financial interests.

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Supplementary Information

This file contains Supplementary Tables 1-3 and guidance notes for the Zebrafish Mutation Project website. (PDF 346 kb)

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Kettleborough, R., Busch-Nentwich, E., Harvey, S. et al. A systematic genome-wide analysis of zebrafish protein-coding gene function. Nature 496, 494–497 (2013). https://doi.org/10.1038/nature11992

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