Where do I get the fish carrying alleles from?
What is the history of the Zebrafish Mutation Project?
The Zebrafish Mutation Project was led by Derek Stemple at the Wellcome Sanger Institute and was funded by the Wellcome Trust, the NIH and ZF-HEALTH. Our collaborators included Freek van Eeden and Edwin Cuppen. The project ended in 2016, but the alleles are available from ZIRC and/or EZRC.
What does each allele status mean?
Mutation detected in F1 DNA
A heterozygous nonsense or splice mutation has been detected in DNA prepared from F1 individuals. We expect about 90% of such mutations to be confirmed by KASP genotyping.
F2 line generated
An F2 population has been generated by outcrossing F1 carriers.
Confirmed mutation in F2 line
The allele has been confirmed by KASP genotyping in the F2 population.
Available for shipment
Carriers have been identified and the allele is available for shipment from ZIRC and/or EZRC.
- Mutation detected in F1 DNA
How should I cite the Zebrafish Mutation Project?
The Zebrafish Mutation Project has been published:
A systematic genome-wide analysis of zebrafish protein-coding gene function.
Kettleborough RN, Busch-Nentwich EM, Harvey SA, Dooley CM, de Bruijn E, van Eeden F, Sealy I, White RJ, Herd C, Nijman IJ, Fényes F, Mehroke S, Scahill C, Gibbons R, Wali N, Carruthers S, Hall A, Yen J, Cuppen E and Stemple DL.
Nature 496, 494–497 (2013).
How do I genotype the fish I receive from ZIRC or EZRC?
We routinely genotype individuals by allele-specific amplification using KASP genotyping. Many alleles have an associated KASP assay that can be ordered directly from LGC using the ID listed with the allele. Alternatively, standard PCR primers can be designed around the allele and the product can be sequenced to identify the genotype of each individual.
How did you determine the phenotypes of alleles?
We performed morphological and behavioural phenotypic analysis of F3 embryos from F2 incrosses during the first five days of development. This was followed by genotype analysis of phenotypic and non-phenotypic embryos to determine associations between observed phenotypes and nonsense or essential splice site mutations present in the family.
We consider a genotype-phenotype correlation to be true when all of at least 12 phenotypic embryos are homozygous mutant and non-phenotypic siblings are either heterozygous or wild type for that allele. A 10% error margin allows for partial penetrance and pipetting mistakes. Phenotypic alleles were then outcrossed and the association was confirmed on F4 embryos from 12 independent clutches.
This linkage analysis does not constitute proof of causality. We cannot guarantee the assocation and a phenotype might be moved to a different gene in the future.
For more detailed information, please see our phenotyping publication. The process is also illustrated below.
What is a 'normal' phenotype?
A phenotype is described as 'normal' where no morphological or behavioural difference was observed using a dissecting microscope during the first five days of development.
Can you outline the mutation detection process?
DNA was prepared from F1 individuals which are heterozygous for mutations across the genome. There are an average of 10 nonsense and 5 splice mutations per F1 individual. A sequencing library was prepared and enriched for coding exons using Agilent SureSelect and sequenced on the Illumina platform. An F2 population was generated by outcrossing F1 carriers and each individual was genotyped for all nonsense and splice mutations. F2 carriers were frozen to capture mutations and were made available via ZIRC and/or EZRC. F2 carriers were also incrossed and phenotyped morphologically for the first five days of development. Approximately 5% to 10% of mutations cause a morphological phenotype during the first five days of development.
For more detailed information, please see our publication. The process is also illustrated below.