title: Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing.
abstract: Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing approximately 70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.
authors.affiliation: Department of Physiology, Kansas University Medical Center, Kansas City, Kansas 66160, USA.
authors.forename: Justin P
authors.initials: JP
authors.lastname: Blumenstiel
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Noll
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Griffiths
authors.forename: Anoja G
authors.initials: AG
authors.lastname: Perera
authors.forename: Kendra N
authors.initials: KN
authors.lastname: Walton
authors.forename: William D
authors.initials: WD
authors.lastname: Gilliland
authors.forename: R Scott
authors.initials: RS
authors.lastname: Hawley
authors.forename: Karen
authors.initials: K
authors.lastname: Staehling-Hampton
mesh_terms: D000818:Animals; D002874:Chromosome Mapping; D004252:DNA Mutational Analysis; D004331:Drosophila melanogaster / Q000235:genetics*; D005020:Ethyl Methanesulfonate / Q000494:pharmacology*; D016678:Genome; D055106:Genome-Wide Association Study; D006720:Homozygote; D009153:Mutagens / Q000494:pharmacology*; D009154:Mutation / Q000187:drug effects*; D020641:Polymorphism, Single Nucleotide
chemical_list: D009153:Mutagens; D005020:Ethyl Methanesulfonate