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

Abstract

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 <i>in situ</i> 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, <i>gpr126^st49</i> and <i>egr2b^fh227</i> , both of which cause myelin defects. As further proof of concept we mapped a novel mutation, <i>stl64</i>, identified in a zebrafish WISH screen for myelination defects. We linked <i>stl64</i> to chromosome 1 and identified a candidate nonsense mutation in the <i>F-box and WD repeat domain containing 7</i> (<i>fbxw7</i>) gene. Importantly, <i>stl64</i> mutants phenocopy previously described <i>fbxw7^vu56</i> mutants, and knockdown of <i>fbxw7</i> in wild-type animals produced similar defects, demonstrating that <i>stl64</i> disrupts <i>fbxw7</i> Together, these data show that our mapping protocol can map and identify causative lesions in mutant screens that require tissue fixation for phenotypic analysis.