FREQ-Seq A Rapid, Cost-Effective, Sequencing-Based Method to Determine Allele Frequencies Directly from Mixed Populations 英文参考文献.docVIP

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FREQ-Seq A Rapid, Cost-Effective, Sequencing-Based Method to Determine Allele Frequencies Directly from Mixed Populations 英文参考文献.doc

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FREQ-Seq A Rapid, Cost-Effective, Sequencing-Based Method to Determine Allele Frequencies Directly from Mixed Populations 英文参考文献

FREQ-Seq:ARapid,Cost-Effective,Sequencing-Based MethodtoDetermineAlleleFrequenciesDirectlyfrom MixedPopulations LonM.Chubiz1.,Ming-ChunLee1.¤,NigelF.Delaney1,ChristopherJ.Marx1,2* 1DepartmentofOrganismicandEvolutionaryBiology,HarvardUniversity,Cambridge,Massachusetts,UnitedStatesofAmerica,2FacultyofArtsandSciencesCenterfor SystemsBiology,HarvardUniversity,Cambridge,Massachusetts,UnitedStatesofAmerica Abstract Understanding evolutionary dynamics within microbial populations requires the ability to accurately follow allele frequencies through time. Here we present a rapid, cost-effective method (FREQ-Seq) that leverages Illumina next- generationsequencingforlocalized,quantitativeallelefrequencydetection.AnalogoustoRNA-Seq,FREQ-Seqreliesupon countsfromthe.105readsgeneratedperlocuspertime-pointtodetermineallelefrequencies.Lociofinterestaredirectly amplified froma mixed population via two rounds of PCR using inexpensive, user-designed oligonucleotides anda bar- coded bridging primer system that can be regenerated in-house. The resulting bar-coded PCR products contain the adaptersneededforIlluminasequencing,eliminatingfurtherlibrarypreparation.WedemonstratetheutilityofFREQ-Seqby determiningtheorderanddynamicsofbeneficialallelesthataroseasamicrobialpopulation,foundedwithanengineered strain of Methylobacterium, evolved to grow on methanol. Quantifying allele frequencies with minimal bias down to 1% abundanceallowedeffectiveanalysisofSNPs,smallin-delsandinsertionsoftransposableelements.Ourdatareveallarge- scaleclonalinterferenceduringtheearlystagesofadaptationandillustratetheutilityofFREQ-Seqasacost-effectivetool fortrackingallelefrequenciesinpopulations. Citation:ChubizLM,LeeM-C,DelaneyNF,MarxCJ(2012)FREQ-Seq:ARapid,Cost-Effective,Sequencing-BasedMethodtoDetermineAlleleFrequenciesDirectly fromMixedPopulations.PLoSONE7(10):e47959.doi:10.1371/journal.pone.0047959 Editor:Ju¨rgBa¨hler, UniversityCollegeLondon,UnitedKingdom ReceivedJuly31,2012;AcceptedSeptember17,2012;Publis