The power of the zebrafish system stems from its utility as a developmental biology model combined with the ease of its genetic manipulation and experimentation. Our understanding of key genetic mechanisms of vertebrate development has been propelled by the phenotypic characterization, genetic mapping and positional cloning of induced and spontaneous mutations in zebrafish. However, the potential of this system has not been fully realized, as inefficient microsatellite-based mapping remains the primary method in the field. We propose to apply technological and computational advances of present day genomics to genetic mapping in the zebrafish system. Specifically, we propose to develop a method for rapid and accurate mapping of recessive zebrafish mutants using Next Generation Sequencing (NGS) of pooled samples. We also propose to investigate parameters of screen design and sample analysis to optimize the use of this protocol. Finally, we aim to develop methods for identification of the causal mutation among the variants discovered within the mapping interval. Application of NGS technology, complemented by specifically developed computational techniques, will provide an efficient, accurate and inexpensive method for genetic mapping in zebrafish. This approach will enable the simultaneous identification of informative genetic markers, mapping of the mutation position, and potential identification of the causal sequence change in a single experiment. The data obtained in these genomic analyses and the methods developed will be made available to the zebrafish community. Importantly, these approaches will also be widely applicable to genetic analysis of other model systems.

Public Health Relevance

Analysis of zebrafish mutants has enabled the identification of genes contributing to fundamental biological processes, including human diseases;however, the methods used for mutant mapping and gene discovery are inefficient. Here, we propose a fast and cost-effective method for genetic mapping and mutation identification using Next Generation Sequencing. This will facilitate the more rapid discovery of gene function and the translation of this knowledge to biomedical investigation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK095721-02
Application #
8549217
Study Section
Special Emphasis Panel (ZRG1-CB-Z (56))
Program Officer
Rasooly, Rebekah S
Project Start
2012-09-21
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
2
Fiscal Year
2013
Total Cost
$419,650
Indirect Cost
$135,683
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Ryan, Sean; Willer, Jason; Marjoram, Lindsay et al. (2013) Rapid identification of kidney cyst mutations by whole exome sequencing in zebrafish. Development 140:4445-51