title: Rapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing.
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.
authors.affiliation: Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Abanti
authors.initials: A
authors.lastname: Chattopadhyay
authors.forename: Zhe
authors.initials: Z
authors.lastname: Li
authors.forename: Bryce
authors.initials: B
authors.lastname: Daines
authors.forename: Yumei
authors.initials: Y
authors.lastname: Li
authors.forename: Chunxu
authors.initials: C
authors.lastname: Gao
authors.forename: Richard
authors.initials: R
authors.lastname: Gibbs
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D003362:Cost-Benefit Analysis; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D004331:Drosophila melanogaster / Q000235:genetics*; D006580:Genetic Carrier Screening / Q000379:methods*; D049750:Genome, Insect; D006579:Heterozygote; D008954:Models, Biological; D008969:Molecular Sequence Data; D017354:Point Mutation; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors
references.citation: Nat Methods. 2007 Nov;4(11):931-6
references.pmid: 17934468
references.citation: Anal Bioanal Chem. 2009 Jan;393(1):171-5
references.pmid: 18958448
references.citation: Genetics. 2008 Sep;180(1):661-7
references.pmid: 18780737
references.citation: Genome Res. 2008 Oct;18(10):1638-42
references.pmid: 18775913
references.citation: Nat Biotechnol. 2008 Oct;26(10):1135-45
references.pmid: 18846087
references.citation: PLoS Genet. 2008 Aug 01;4(8):e1000139
references.pmid: 18670626
references.citation: Nat Biotechnol. 2009 Apr;27(4):353-60
references.pmid: 19330000
references.citation: Ann Hum Genet. 2009 Sep;73(Pt 5):502-13
references.pmid: 19573206
references.citation: Fly (Austin). 2009 Jul-Sep;3(3):192-203
references.pmid: 19690466
references.citation: Genetics. 1980 Jun;95(2):367-81
references.pmid: 17249041
references.citation: Genome Res. 2002 Apr;12(4):656-64
references.pmid: 11932250
references.citation: Nature. 2008 Nov 6;456(7218):66-72
references.pmid: 18987736
references.citation: Genetics. 1983 Nov;105(3):581-600
references.pmid: 17246168
references.citation: Bioinformatics. 2009 Aug 15;25(16):2078-9
references.pmid: 19505943
references.citation: Genetics. 2009 May;182(1):25-32
references.pmid: 19307605
references.citation: Genome Res. 2005 May;15(5):718-23
references.pmid: 15867432
references.citation: Nature. 2008 Apr 17;452(7189):872-6
references.pmid: 18421352
references.citation: Nat Methods. 2007 Nov;4(11):903-5
references.pmid: 17934467
references.citation: Nature. 2009 Sep 10;461(7261):272-6
references.pmid: 19684571
references.citation: Trends Genet. 2008 Mar;24(3):133-41
references.pmid: 18262675
references.citation: Annu Rev Genomics Hum Genet. 2008;9:387-402
references.pmid: 18576944
references.citation: N Biotechnol. 2009 Apr;25(4):195-203
references.pmid: 19429539
references.citation: Proc Natl Acad Sci U S A. 2007 May 29;104(22):9387-92
references.pmid: 17517648
references.citation: Genome Res. 2009 Sep;19(9):1606-15
references.pmid: 19525355
references.citation: Nat Biotechnol. 2009 Feb;27(2):182-9
references.pmid: 19182786
references.citation: Nature. 2008 Nov 6;456(7218):53-9
references.pmid: 18987734
references.citation: Nat Methods. 2008 Apr;5(4):323-9
references.pmid: 18327265
references.citation: Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):10860-5
references.pmid: 12960394
references.citation: Nat Methods. 2008 Oct;5(10):865-7
references.pmid: 18677319