Rapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing.

TitleRapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing.
Publication TypeJournal Article
Year of Publication2010
AuthorsWang, H, Chattopadhyay, A, Li, Z, Daines, B, Li, Y, Gao, C, Gibbs, R, Zhang, K, Chen, R
JournalGenome Res
Volume20
Issue7
Pagination981-8
Date Published2010 Jul
ISSN1549-5469
KeywordsAnimals, Animals, Genetically Modified, Base Sequence, Cost-Benefit Analysis, DNA Mutational Analysis, Drosophila melanogaster, Genetic Carrier Screening, Genome, Insect, Heterozygote, Models, Biological, Molecular Sequence Data, Point Mutation, Sequence Homology, Nucleic Acid, Time Factors
Abstract

One of the key advantages of using Drosophila melanogaster as a genetic model organism is the ability to conduct saturation mutagenesis screens to identify genes and pathways underlying a given phenotype. Despite the large number of genetic tools developed to facilitate downstream cloning of mutations obtained from such screens, the current procedure remains labor intensive, time consuming, and costly. To address this issue, we designed an efficient strategy for rapid identification of heterozygous mutations in the fly genome by combining rough genetic mapping, targeted DNA capture, and second generation sequencing technology. We first tested this method on heterozygous flies carrying either a previously characterized dac(5) or sens(E2) mutation. Targeted amplification of genomic regions near these two loci was used to enrich DNA for sequencing, and both point mutations were successfully identified. When this method was applied to uncharacterized twr mutant flies, the underlying mutation was identified as a single-base mutation in the gene Spase18-21. This targeted-genome-sequencing method reduces time and effort required for mutation cloning by up to 80% compared with the current approach and lowers the cost to <$1000 for each mutant. Introduction of this and other sequencing-based methods for mutation cloning will enable broader usage of forward genetics screens and have significant impacts in the field of model organisms such as Drosophila.

DOI10.1101/gr.102921.109
Alternate JournalGenome Res.
PubMed ID20472684
PubMed Central IDPMC2892099
Grant ListF32 EY019430 / EY / NEI NIH HHS / United States
EY19430-01 / EY / NEI NIH HHS / United States
U54 HG003273 / HG / NHGRI NIH HHS / United States
R01 EY016853 / EY / NEI NIH HHS / United States
T32 EY07102-16 / EY / NEI NIH HHS / United States
R01EY016853 / EY / NEI NIH HHS / United States
T32 EY007102 / EY / NEI NIH HHS / United States