Whole genome sequencing-based mapping and candidate identification of mutations from fixed zebrafish tissue

Nicholas E. Sanchez, Breanne L. Harty, Thomas O'Reilly-Pol, Sarah D. Ackerman, Amy L. Herbert, Melanie Holmgren, Stephen L. Johnson, Ryan S. Gray, Kelly Monk

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)3415-3425
Number of pages11
JournalG3: Genes, Genomes, Genetics
Volume7
Issue number10
DOIs
StatePublished - Oct 1 2017

Fingerprint

Zebrafish
Genome
Tissue Fixation
Mutation
In Situ Hybridization
Wild Animals
Genomic Library
Nonsense Codon
Chromosomes, Human, Pair 1
Myelin Sheath
Computational Biology
Nervous System
Alleles
Staining and Labeling
Phenotype
Antibodies
DNA
Genes

Keywords

  • Fixed tissue sequencing
  • Genetic screen
  • Linkage
  • Mapping
  • Whole genome sequencing
  • Zebrafish

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Genetics(clinical)

Cite this

Sanchez, N. E., Harty, B. L., O'Reilly-Pol, T., Ackerman, S. D., Herbert, A. L., Holmgren, M., ... Monk, K. (2017). Whole genome sequencing-based mapping and candidate identification of mutations from fixed zebrafish tissue. G3: Genes, Genomes, Genetics, 7(10), 3415-3425. https://doi.org/10.1534/g3.117.300212

Whole genome sequencing-based mapping and candidate identification of mutations from fixed zebrafish tissue. / 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.

In: G3: Genes, Genomes, Genetics, Vol. 7, No. 10, 01.10.2017, p. 3415-3425.

Research output: Contribution to journalArticle

Sanchez, NE, Harty, BL, O'Reilly-Pol, T, Ackerman, SD, Herbert, AL, Holmgren, M, Johnson, SL, Gray, RS & Monk, K 2017, 'Whole genome sequencing-based mapping and candidate identification of mutations from fixed zebrafish tissue', G3: Genes, Genomes, Genetics, vol. 7, no. 10, pp. 3415-3425. https://doi.org/10.1534/g3.117.300212
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. / Whole genome sequencing-based mapping and candidate identification of mutations from fixed zebrafish tissue. In: G3: Genes, Genomes, Genetics. 2017 ; Vol. 7, No. 10. pp. 3415-3425.
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