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

Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas 77030, USA.
Genome Research (Impact Factor: 14.63). 07/2010; 20(7):981-8. DOI: 10.1101/gr.102921.109
Source: PubMed


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.

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Available from: Rui Chen, Oct 20, 2014
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    • "We have successfully demonstrated the use of next generation sequencing to identify an EMS-induced mutation responsible for cyanogenic glycoside breakdown in the complex genome of a nonmodel crop species. Previous use of NGS in targeted gene cloning has focused on model organisms [e.g., A. thaliana (Mokry et al. 2011; Uchida et al. 2011; Austin et al. 2011), Drosophila melanogaster (Blumenstiel et al. 2009; Wang et al. 2010), Caenorhabditis elegans (Sarin et al. 2008), and fission yeast (Irvine et al. 2009)]. Sorghum, while sequenced (Paterson et al. 2009), has not been used successfully to clone genes using NGS tools. "
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    ABSTRACT: Whole genome sequencing has allowed rapid progress in the application of forward genetics in model species. In this study, we demonstrated an application of Next-Generation Sequencing for forward genetics in a complex crop genome. We sequenced an ethyl methanesulfonate-induced mutant of Sorghum bicolor defective in hydrogen cyanide release and identified the causal mutation. A workflow identified the causal polymorphism relative to the reference BTx623 genome by integrating data from single nucleotide polymorphism identification, prior information about candidate gene(s) implicated in cyanogenesis, mutation spectra, and polymorphisms likely to affect phenotypic changes. A point mutation resulting in a premature stop codon in the coding sequence of dhurrinase2, a protein involved in the dhurrin catabolic pathway, was responsible for the acyanogenic phenotype. Cyanogenic glucosides are not cyanogenic compounds but their cyanohydrins derivatives do release cyanide. The mutant accumulated the glucoside, dhurrin, but failed to efficiently release cyanide upon tissue disruption. Thus, we tested the effects of cyanide release on insect herbivory in a genetic background in which accumulation of cyanogenic glucoside is unchanged. Insect preference choice experiments and herbivory measurements demonstrate a deterrent effect of cyanide release capacity, even in the presence of wild-type levels of cyanogenic glucoside accumulation. Our gene cloning method substantiates the value of 1.) a sequenced genome; 2.) a strongly penetrant and easily measurable phenotype; and 3.) a workflow to pinpoint a causal mutation in crop genomes and accelerate in the discovery of gene function in the post genomic era.
    Full-text · Article · Jul 2013 · Genetics
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    • "Exon capture sequencing has transformed genetic analysis in humans and has demonstrated utility in mice. However, to date, exon capture HTS has been scarcely reported in other genetic models [42,43] despite the significant need to facilitate cloning of mutants identified in forward genetic screens. Very recently, WGS with BSA has identified causative mutations in both zebrafish and mouse validating HTS and BSA as an effective method [4,5]. "
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    ABSTRACT: Background Exome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like Xenopus tropicalis, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of X. tropicalis mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step. Results Here we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping. Conclusion Exon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.
    Full-text · Article · Nov 2012 · BMC Genomics
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    • "With the reduction in costs of next generation sequencing (NGS), it is now possible to TILL several genes simultaneously using TILLING-by-sequencing [10,38,39]. Screening for mutations using NGS can be done along two lines; first by sequencing pooled amplicons [10,38] and second by sequence capture [39,40]. The former has high throughput for the number of individuals for some genes, whereas the latter is suitable for scanning many genes within a few individuals [10]. "
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    ABSTRACT: Background Triticum monococcum L., an A genome diploid einkorn wheat, was the first domesticated crop. As a diploid, it is attractive genetic model for the study of gene structure and function of wheat-specific traits. Diploid wheat is currently not amenable to reverse genetics approaches such as insertion mutagenesis and post-transcriptional gene silencing strategies. However, TILLING offers a powerful functional genetics approach for wheat gene analysis. Results We developed a TILLING population of 1,532 M2 families using EMS as a mutagen. A total of 67 mutants were obtained for the four genes studied. Waxy gene mutation frequencies are known to be 1/17.6 - 34.4 kb DNA in polyploid wheat TILLING populations. The T. monococcum diploid wheat TILLING population had a mutation frequency of 1/90 kb for the same gene. Lignin biosynthesis pathway genes- COMT1, HCT2, and 4CL1 had mutation frequencies of 1/86 kb, 1/92 kb and 1/100 kb, respectively. The overall mutation frequency of the diploid wheat TILLING population was 1/92 kb. Conclusion The mutation frequency of a diploid wheat TILLING population was found to be higher than that reported for other diploid grasses. The rate, however, is lower than tetraploid and hexaploid wheat TILLING populations because of the higher tolerance of polyploids to mutations. Unlike polyploid wheat, most mutants in diploid wheat have a phenotype amenable to forward and reverse genetic analysis and establish diploid wheat as an attractive model to study gene function in wheat. We estimate that a TILLING population of 5, 520 will be needed to get a non-sense mutation for every wheat gene of interest with 95% probability.
    Full-text · Article · Nov 2012 · BMC Plant Biology
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