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Anoja Perera([email protected])Molecular Biology Core FacilityStowers Institute for Medical ResearchDateKansas City, US1
Initial mapping to a chromosomeUsing Bal stocks Labor intensive Time Consuming(over 6 months) Often difficult Sometimes costlyDateFine mapping to a region ofa chromosome using Df stocksSelect candidate genesNextGenerationSequencing?Amplify and sequence genesMutation2
Ethylmethane Sulfonate (EMS)- Common chemical mutagen- Randomly induces single base pair mutations orsmall insertions or deletions- Usually generates G/C to A/T point mutationsDateWild TypeMutant3
1. Obtained genomic DNA: F3 homozygous mutantsand wild-type males.2. Performed the following steps:a) Made two Illumina genomic fragment libraries(1.5 day)b) Hybridized the fragments on to two flowcell –“cluster generation” (2 day)c) Sequenced the flowcells (1-2 weeks)3. Analyzed data to find mutations.DateMay 2008 using an Illumina GAI4
Primary Analysis using Illumina data analysis pipeline.ImagesSequencesAlignment using ELANDUniquely mapping sequencesDate5
1. Aligned each strain individually to reference genomeusing MAQ (LI et al., 2008)2. Generated consensus sequences for both mutant andwild-type.3. Compared the consensus sequences of both mutant andwild type strains to generate a list of polymorphisms.4. Annotated the polymorphisms to find possiblemutations causing phenotype.Date6
DateMay 2008: Using Illumina GAI7
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HAWKINS et al., 1996, 1997; VANBUSKIRK et al., 2000;OHLMEYER et al., 2003-Plays a role in regulation of cyclin E during oogenesis-Known to have an effect on dorsal appendage formation-Mutation results in the replacement of a glutamine with astop codonDate9
Validation:Complementation crosses with Df stocks resultedin the same “dorsal appendage phenotype”Why is this significant? What does this mean tothe Drosophila community?Date10
Date“Well, if I need to find a mutation right now, I would not usetraditional mapping techniques, I will find the mutations bysequencing the whole genome because it has been done and itworks!”- Dr. Scott Hawley11
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Next generation whole genome sequencingapproach can be used to easily map EMSinduced mutations in organisms. Cost-effective:- Currently (September, 2009), 2*36bp PElanes 20X coverage of the Drosophila or C.elegans genomes. Time efficient:- Opposed to the traditional ways ofmapping, mutation(s) can be discovered in abouta month!Date13
AcknowledgementsDate14
Stowers Institute for Medical Research Kansas City, US . Date 2 Initial mapping to a chromosome Using Bal stocks Fine mapping to a region of . Dr. Scott Hawley . Date 12 . Date 13 Next generation whole genome sequencing approach can be used to easily map EMS-induced mutations in organisms.
