pyrosequencing焦磷酸测序.ppt

,焦磷酸测序法的原理及应用（Pyrosequencing遗传分析技术）,pyro=pyrophosphate焦磷酸一种全新的基于酶级联反应的新型遗传分析技术;一套完整的实验方案；诸多领域的广泛应用,Pyrosequencing遗传分析技术,Pyrosequencing这项专利技术完全由Biotage公司拥有,PPi,ATP,PrincipleofPyrosequencing,APS,Luciferin,硫酸化酶,双磷酸酶,荧光素酶,oxyluciferin,Detectionofthelight,CGTTCCACCT,每次向反应体系中加入一种dNTP相应位置的峰代表该种dNTP的掺入情况峰高与掺入的核苷酸数量成正比多余的dNTP在加入下一种dNTP前就被降解,Acompletesolutionforaccurategeneticdetermination,Pyrosequencing系统平台,ReagentKits,SamplePrepWorkstation,Software,Instrumentation,PyroMarkTMIDCompletesolutionforClinicalMicrobiology,Pyrosequencing系统平台,Pyrosequencing技术的流程,PCR,试样预处理，获得单链模板,Pyrosequencing序列分析,15min/96samples,SQA:35min/96samples,SNP:10min/96samples,1-2h/96samples,结果分析,实验设计,Peakheightisproportionaltothenumberofincorporatednucleotides,Allele1:ACTGAllele2:GCTG,杂合子标本:,Genotypesareclearlydistinguished,Insertions/deletions,SinglenucleotideInDels(e.g.C)MultiplenucleotideInDels(e.g.CGACGGT),Thesoftwaresupportsanalysisofinsertions/deletions(InDels),CYP2D6-A2637del(Allele3),Largedeletion,Microsatellites:TGAATGnTTTGGGCAAAT,Tri-andtetra-allelicSNPs,Genotypingoftri-allelicSNP(C/T/G),Multiplepolymorphisms,Multiplexing,Principleofmultiplexing,Designofsequencingprimersanddispensationorder,UsercreatesaSNP/mutationsequencedatabaseSoftwarecalculatestheoreticalgenotypingresultsEachDNAfragmentisassignedacolor,Quantitativepeakheights,Allelefrequency=relativepeakheightx100(%),AdvantageandBenefitofPyeosequencing,Fast,Accuracy,Flexibility,Multiplexing,Simple,Sequence,Efficient,ShortOptimizationTime,DedicatedSoftware,DedicatedReagentKit,Scalable,1.AssayDesign,Exampleofageneraltarget:16Sgene,AssayDesignSW,Species-specificregionin16Sgene,PCR-forward,PCR-reverse,Sequencingprimer,2.PCR扩增,一对扩增引物中的一条带有生物素标记，另一条不带标记。,生物素标记,3.SingleStrandTemplatePreparation,PCR产物中加入streptavidine包被的sepharosebeads振荡温育5-10min变性，生物素标记的单链结合于beads并被真空吸附于VacuumPrepTool上洗涤，中和单链模板加入反应体系，与测序引物退火,真实的序列：遗传检测的黄金标准快速的实时输出：1碱基/分钟96个样本平行处理能力新一代的PyroGold试剂：表现大为提升,4.Pyrosequencing,PyroMarkIDInstrument,5.PyrogramAnalysis,sequenceresultwithqualitygrading,96samplesanalyzed,sequenceresult(canbeexportedinFASTAformat),Pyrosequencing技术的特点,得到定量序列结果的技术极高的准确性Built-InQC重现性极佳通用型技术平台功能多样，应用领域极广可以用于任何遗传多态性的分析甲基化研究的平台高通量、低成本的快速临床微生物诊断实验设计灵活自动化程度高，操作简单,WhyPyrosequencingisquantitative？,WhyPyrosequencingisquantitative？,1.0,0.0,0.5,0.5,0.0,1.0,Expected,1.09,0.05,0.55,0.56,0.02,1.05,Average,0.02,n.d.,0.03,0.03,n.d.,0.03,CV,C/C,C/G,G/G,n=190,Pyrosequencing：QuantitativeAccuracyTest,Pyrosequencing：AccurateQuantifying,Allelefrequency=relativepeakheightx100(%),Whatare”InbuiltControls”?,SincePyrosequencingshowspolymorphismswithsequencecontext,youcanalwaystrustthevalidityofthedata.,BenefitsofBuilt-inQualityControl,QC基于周边的序列信息鉴定的黄金标准从primer后的第一个碱基开始便于数据alignmentQC验证了实验的成功与否具体到每一轮的每一个样品万一实验失败，可以帮助分析原因，解决问题,Pyrosequencing焦磷酸测序系统的卓越表现：99.998%accurate(basedonanalysisof100,000wellswithknowngenotype),IntegratedSoftwarePackage,整合且不断扩展的软件包,SNP-SNPandmutationanalysisAQ-IncludedinSNPsoftware-EasystatisticsfunctionSQA-Basecalling-SequencealignmentAllthreewithqualityassessment,Running,Analyzing,Planning,Documenting,Pyrosequencing的应用,Sequenceanalysis,SequenceidentificationMicrobialtypingClonedDNAre-sequencingmtDNA/ForensicsTransgeneticsExpressionprofilingOligoIDMicrosatellites,KnownpositionsUnknownpositionswithinhotspotsPointmutationsInsertions/deletionsMultiplemutationsMultiplexing,Mutationdetection,Quantification,AllelefrequencyassessmentSNPfrequencyTri/tetraallelicSNPfrequencyIndelfrequencyCpG-methylationGenecopynumberLossofheterozygosityPolyploidgenomesExpressionanalyses,Geneticvariation,SNPsandDIPsDi-,tri-andtetraallelicMultipleMultiplexingHaplotypeOut-of-phaseAllele-specificPCR,已发表的文章,使用Pyrosequencing技术发表的研究报告已近千篇,发表在Nature、Science上的文献,快速鉴定炭疽热细菌Bacillusanthracis,炭疽是一种人畜共患疾病。引起炭疽病的炭疽杆菌以孢子形式存在于土壤中，在特定条件下，可以存活数十年。炭疽热的潜伏期为1到6天，彻底发病则在多达60天后。抗生素能够抑制炭疽热感染，但条件是必须在接触炭疽热细菌后的48小时以内使用。,炭疽杆菌Bacillusanthracis(B.anthracis)是引起炭疽热严重感染的病原细菌。从接触病原体到症状最初出现的感染发展过程一般为1到6天。如果确认受到B.anthracis攻击,利用抗生素或解毒剂可以防治其感染,因此快速鉴定病原微生物种类和其致病力状态具有很重要的意义。在起初24-48小时内摄入抗生素可以防治其传染。,B.anthracis的致病菌株有两个质粒,pXO1含炭疽热毒素产生的基因(lef,cy