Whole Genome Sequencing-Based Mapping and Candidate Identification of Mutations from Fixed Zebrafish Tissue
- Sanchez, N.E., Harty, B.L., O'Reilly-Pol, T., Ackerman, S.D., Herbert, A.L., Holmgren, M., Johnson, S.L., Gray, R.S., Monk, K.R.
- G3 (Bethesda) 7(10): 3415-3425 (Journal)
- Registered Authors
- Gray, Ryan, Johnson, Stephen L., Monk, Kelly, O'Reilly-Pol, Thomas
- fixed tissue sequencing, genetic screen, linkage, mapping, whole genome sequencing, zebrafish
- MeSH Terms
- Chromosome Mapping
- Polymorphism, Single Nucleotide
- Tissue Fixation
- Whole Genome Sequencing/methods*
- 28855284 Full text @ G3 (Bethesda)
Sanchez, N.E., Harty, B.L., O'Reilly-Pol, T., Ackerman, S.D., Herbert, A.L., Holmgren, M., Johnson, S.L., Gray, R.S., Monk, K.R. (2017) Whole Genome Sequencing-Based Mapping and Candidate Identification of Mutations from Fixed Zebrafish Tissue. G3 (Bethesda). 7(10):3415-3425.
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 in situ 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, gpr126st49 and egr2bfh227 , both of which cause myelin defects. As further proof of concept we mapped a novel mutation, stl64, identified in a zebrafish WISH screen for myelination defects. We linked stl64 to chromosome 1 and identified a candidate nonsense mutation in the F-box and WD repeat domain containing 7 (fbxw7) gene. Importantly, stl64 mutants phenocopy previously described fbxw7vu56 mutants, and knockdown of fbxw7 in wild-type animals produced similar defects, demonstrating that stl64 disrupts fbxw7 Together, these data show that our mapping protocol can map and identify causative lesions in mutant screens that require tissue fixation for phenotypic analysis.
Genes / Markers
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Engineered Foreign Genes
Errata and Notes