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1、briefcommunications2b-rad:asimpleandHerewedescribeastreamlinedandflexibleapproachforRADgenotyping,called2b-RADforitsuseoftypeIIBrestric-flexiblemethodfortionenzymes.Theseenzymes(forexample,BsaXIandAlfI)cleavegenomicDNAupstreamanddownstreamofthetargetgenome-
2、widegenotypingsite,producingtagsofuniformlengththatareideallysuitedforsequencingonexistingnext-generationplatforms(Fig.1a).Tovalidatethenewmethod,wepreparedBsaXIlibrariesfromaShiWang1,2,5,EliMeyer1,3,5,JohnKMcKay4&MikhailsingleArabidopsisthalianaFplantprodu
3、cedbycrossingtworese-VMatz11quencedaccessions,Tsu-1andKas-1.LibrariesweresequencedonbothSOLiDandIlluminaplatforms;eachplatformgeneratedWedescribe2b-rad,astreamlinedrestrictionsite–associatedmorethan11millionhigh-qualityreads(SupplementaryTable1)dna(rad)geno
4、typingmethodbasedonsequencingandproducedevencoverageacrossthenucleargenomeexceptfortheuniformfragmentsproducedbytypeiibrestrictionextendedgapsincentromereregions(Fig.1b).Todemonstrateendonucleases.Well-studiedaccessionsofArabidopsisthesuitabilityof2b-RADfor
5、humansandotherspecieswithlargethalianaweregenotypedtovalidatethemethod’saccuracygenomes,wealsosequencedandanalyzedcommerciallyobtainedandtodemonstratefine-tuningofmarkerdensityasneeded.humanDNA(SupplementaryFig.1).thesimplicityofthe2b-radprotocolmakesitpart
6、icularlyInA.thaliana,2b-RADreproduciblydetectedandgenotypedsuitableforhigh-throughputgenotypingasrequiredforlinkagethemajorityofuniqueBsaXIsitesacrosslibrarypreparationsmappingandprofilinggeneticvariationinnaturalpopulations.andsequencingplatforms(Supplemen
7、taryTables2and3).Genotypeswereassignedtositeswith≥20×coverageusingeitherSingle-nucleotidepolymorphisms(SNPs)havebecometheasimplethresholdapproachadjustedtomaximizetheagreementgeneticmarkersofchoiceinmanybiomedical,ecologicalandbetweenSOLiDandIlluminadataset
8、s(seeOnlineMethods)orevolutionarystudies1,2.Untilrecently,profilingalargenumberofamaximum-likelihood(ML)approach9.BothoutputsagreedSNPswasfeasibleonlyformodelorganismswithwell-developedwithbioinformati
