title: Fast homozygosity mapping and identification of a zebrafish ENU-induced mutation by whole-genome sequencing.
abstract: Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish.
authors.affiliation: Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Sart Tilman, Belgium. mvoz@ulg.ac.be
authors.forename: Marianne L
authors.initials: ML
authors.lastname: Voz
authors.forename: Wouter
authors.initials: W
authors.lastname: Coppieters
authors.forename: Isabelle
authors.initials: I
authors.lastname: Manfroid
authors.forename: Ariane
authors.initials: A
authors.lastname: Baudhuin
authors.forename: Virginie
authors.initials: V
authors.lastname: Von Berg
authors.forename: Carole
authors.initials: C
authors.lastname: Charlier
authors.forename: Dirk
authors.initials: D
authors.lastname: Meyer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Driever
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Martial
authors.forename: Bernard
authors.initials: B
authors.lastname: Peers
mesh_terms: D000465:Algorithms; D000477:Alkylating Agents / Q000633:toxicity*; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D018389:Codon, Nonsense; D004252:DNA Mutational Analysis / Q000379:methods*; D005038:Ethylnitrosourea / Q000633:toxicity*; D005260:Female; D006720:Homozygote; D008297:Male; D008969:Molecular Sequence Data; D046790:Pancreas, Exocrine; D020641:Polymorphism, Single Nucleotide / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: D000477:Alkylating Agents; D018389:Codon, Nonsense; C572194:Snapc4 protein, zebrafish; D014157:Transcription Factors; D029961:Zebrafish Proteins; D005038:Ethylnitrosourea