Whole Genome Sequencing-Based Mapping and Candidate Identification of Mutations from Fixed Zebrafish Tissue

• Published in: G3 : genes - genomes - genetics Vol. 7; no. 10; pp. 3415 - 3425
• Main Authors: Sanchez, Nicholas E, Harty, Breanne L, O'Reilly-Pol, Thomas, Ackerman, Sarah D, Herbert, Amy L, Holmgren, Melanie, Johnson, Stephen L, Gray, Ryan S, Monk, Kelly R
• Format: Journal Article
• Language: English
• Published: United States Genetics Society of America 01.10.2017; Oxford University Press
• Subjects: Animals; Chromosome Mapping; fixed tissue sequencing; genetic screen; Investigations; linkage; mapping; Mutation; Polymorphism, Single Nucleotide; Tissue Fixation; whole genome sequencing; Whole Genome Sequencing - methods; zebrafish; Zebrafish - genetics; whole genome sequencing; mapping; linkage; zebrafish; genetic screen; fixed tissue sequencing
• ISSN: 2160-1836; 2160-1836
• DOI: 10.1534/g3.117.300212

As forward genetic screens in zebrafish become more common, the number of mutants that cannot be identified by gross morphology or through transgenic approaches, such as many nervous system defects, has also increased. Screening for these difficult-to-visualize phenotypes demands techniques such as whole-mount hybridization (WISH) or antibody staining, which require tissue fixation. To date, fixed tissue has not been amenable for generating libraries for whole genome sequencing (WGS). Here, we describe a method for using genomic DNA from fixed tissue and a bioinformatics suite for WGS-based mapping of zebrafish mutants. We tested our protocol using two known zebrafish mutant alleles, and , both of which cause myelin defects. As further proof of concept we mapped a novel mutation, , identified in a zebrafish WISH screen for myelination defects. We linked to chromosome 1 and identified a candidate nonsense mutation in the ( ) gene. Importantly, mutants phenocopy previously described mutants, and knockdown of in wild-type animals produced similar defects, demonstrating that disrupts Together, these data show that our mapping protocol can map and identify causative lesions in mutant screens that require tissue fixation for phenotypic analysis.