Zebrafish Tilling by Illumina Sequencing

Target selected mutagenesis, or TILLING (for targeting induced local lesions in genomes), is a proven method that allows mutations to be identified in virtually any gene. We have previously shown that capillary-sequencing can be used to effectively identify mutations in a high throughput manner. A library of Zebrafish non-sense alleles is being created by the Zebrafish Mutation Resource, each with an associated morphological and molecular phenotype. Phenotypic data will be published via ZFIN (using an adapted version of the Phenote database). Information about the mutations identified by the project can be found on our website: http://www.sanger.ac.uk/Projects/D_rerio/mutres/. The advent of next generation sequencing technologies has significantly changed the amount of mutations that can be identified and has opened up the possibility of identifying a non-sense allele in every protein coding gene within a reasonable time frame and budget. We have designed a strategy that will allow us to amplify and sequence thousands of exonic fragments on a single lane of the Illumina sequencing platform. A primer design program has been written that selects 100bp exonic fragments that are most likely to give a non-sense allele by ENU mutagenesis. These fragments (up to 4000 exons per experiment) are amplified across DNA pools from 24 mutagenised Zebrafish, and each pool is run on one lane of the Illumina platform. Automated data analysis then searches the sequence for all potential non-sense alleles in the amplified fragments, and putative non-sense alleles are confirmed by capillary sequencing. Using this method the Zebrafish Mutation Resource aims to produce >1000 knockouts over the next 2 years, creating a rich resource for the Zebrafish research community.