A Systematic Genome-Wide Analysis of Zebrafish Protein-Coding Gene Function

Over the past nine years we have been establishing and applying methods for the systematic identification of disruptive mutations in zebrafish. With the release of the Zv8 and Zv9 assemblies of the zebrafish genome and their detailed protein annotation, we were able to design reagents (Agilent SureSelect) to extract all of the annotated exons from zebrafish genomic DNA.  By sequencing the exon-enriched DNA from mutagenised F1 individuals we could then identify induced mutations by using a modified version of the 1000 Genomes project variant calling pipeline. For each F1 we predict protein-coding consequences of these induced mutations and for each nonsense and essential splice-site mutation we generate a single nucleotide polymorphism (SNP) genotyping assay to facilitate retrieval after crossing. To date, we have analysed the exome sequences of 1554 F1 individuals and identified 16149 nonsense and essential splice alleles in 9634 genes (37% of total protein-coding genes).

To yield the most value from this resource it is important to capture any phenotypic consequence of homozygous mutations. To this end we have established a high-throughput systematic phenotypic analysis of alleles to assess the developmental consequences of any given mutation. Using this approach, we have performed a phenotypic analysis of 936 nonsense and essential splice mutations. Of these 840 were deemed to have no phenotype at 5 dpf, 34 mutations caused a phenotype and 62 alleles were linked to a phenotype. The mutation discovery and multiallelic phenotyping pipeline we have developed systematically annotates zebrafish gene function.

ZMP website (http://www.sanger.ac.uk/Projects/D_rerio/zmp/).