Twr_Capture
Rapid Identification of Heterozygous Mutations in <I>Drosophila melanogaster</I> using Genomic Capture Sequencing
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 the second generation sequencing technology.  We first tested this method on heterozygous flies carrying either a previously characterized dac5 or sensE2 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 substitution in the gene spase18-21.  This targeted-genome-sequencing method greatly reduces time and effort required for mutation cloning by up to 80% compared to current approach and lower the cost to less than $1000 for each mutant.  Introduction of this and other sequencing based methods for mutation cloning will further unleash the power of forward genetics studies and have significant impacts in the field of model organisms including Drosophila.