pyrosequencing焦磷酸测序

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PPiATPPrincipleofPyrosequencingAPS＋Luciferin＋硫酸化酶双磷酸酶荧光素酶oxyluciferinDetectionofthelightlighttimeCGTTCCACCT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AcompletesolutionforaccurategeneticdeterminationPyrosequencing系统平台ReagentKitsSamplePrepWorkstationSoftwareInstrumentationPyroMarkTMIDCompletesolutionforClinicalMicrobiologyPyrosequencing系统平台AssayDesignSWSamplePrepPyrosequencingFeaturingPyroGoldchemistryIdentiFireTMSWPyrosequencing技术的流程PCR试样预处理，获得单链模板Pyrosequencing序列分析15min/96samplesSQA:35min/96samplesSNP:10min/96samples1-2h/96samples结果分析实验设计PeakheightisproportionaltothenumberofincorporatednucleotidesAllele1:ACTGAllele2:GCTG杂合子标本:Genotypesareclearlydistinguished杂合子ACTGCCTGCTGCCTA/GCTGCCTInsertions/deletionsSinglenucleotideInDels(e.g.[C])MultiplenucleotideInDels(e.g.[CGACGGT])Thesoftwaresupportsanalysisofinsertions/deletions(InDels)CYP2D6-A2637del(Allele3)Sequence(reverse):TCC[T]GTGT/TT/--/-Largedeletion3’ATGTGGCATTCCAA5’...ACGTACGCTTACACCGTAAGGTTCTAAAGGTG[CACCATGACTGGGGTTA]CAGTCATC...Microsatellites:TG[AATG]nTTTGGGCAAAT8/128/8478478(9)(10)(11)(12)Tri-andtetra-allelicSNPsC/CT/TG/GC/TC/GT/GGenotypingoftri-allelicSNP(C/T/G)Multiplepolymorphisms3’TAAGCCGAATG5’…AACATTCGGCTTACAT/GGAATGAA/C/G/TCGGTACGAACGATTTTAGA...MultiplexingSNPslocatedondifferentfragments…...…..oronthesamePrincipleofmultiplexingDesignofsequencingprimersanddispensationorder3,5refref00,511,522,53RelativelightunitsTAGCGCACGATGT[A/G]CCTGCCACC[A/G]TTGGGCombinedTGGACCACC[A/G]TGGTTGAG[A/G]CTGCCTG12TTGAG[A/G]CTGCCTG00,511,52TAGCGCACGATGTTGGACCACC[A/G]TGG00,511,52TAGCTGCACGATGUsercreatesaSNP/mutationsequencedatabaseSoftwarecalculatestheoreticalgenotypingresultsEachDNAfragmentisassignedacolorQuantitativepeakheightsRelativepeakheight==peak1heightpeak1height+peak2heightAllelefrequency==relativepeakheightx100(%)RelativepeakheightAllelefrequency(%)0,0000,1000,2000,3000,4000,5000,6000,7000,8000,9001,000010203040506070809010015%30%75%AdvantageandBenefitofPyeosequencingFastAccuracyFlexibilityMultiplexingSimpleSequenceEfficientShortOptimizationTimeDedicatedSoftwareDedicatedReagentKitScalable1.AssayDesignExampleofageneraltarget:16SgeneAssayDesignSWSpecies-specificregionin16SgenePCR-forwardPCR-reverseSequencingprimer2.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PCRproductimmobilizedonSepharosebeadsPSQplatewithsequencingprimerEtOHNaOHWashingbufferWater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.PyrosequencingPyroMarkIDInstrument5.PyrogramAnalysissequenceresultwithqualitygrading96samplesanalyzedsequenceresult(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？lighttime1分子的dNTP掺入，释放出1分子的PPi，生成1分子的ATP，产生单位强度的光信号TGGCCGGGTCACGAGGCCCTA...TripleDoubleSingleWhyPyrosequencingisquantitative？1.00.00.50.50.01.0Expected1.090.050.550.560.021.05Average0.02n.d.0.030.03n.d.0.03CVC/CC/GG/Gn=190Pyrosequencing：QuantitativeAccuracyTestPyrosequencing：AccurateQuantifyingRelativepeakheight==peak1heightpeak1height+peak2heightAllelefrequency==relativepeakheightx100(%)RelativepeakheightAllelefrequency(%)0,0000,1000,2000,3000,4000,5000,6000,7000,8000,9001,000010203040506070809010015%30%75%ESGCTAGTAGAGC/CESGCTAGTAGAGT/TESGCTAGTAGAGC/TWhatare”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alignmentQC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-SNPandmutationanalysis•AQ-IncludedinSNPsoftware-Easystatisticsfunction•SQA-Basecalling-SequencealignmentAllthreewithqualityassessmentRunningAnalyzingPlanningDocumentingPyrosequencing的应用Sequenceanalysis•Sequenceidentification•Microbialtyping•ClonedDNAre-sequencing•mtDNA/Forensics•Transgenetics•Expressionprofiling•OligoID•Microsatellites•Knownpositions•Unknownpositionswithinhotspots•Pointmutations•Insertions/deletions•Multiplemutations•MultiplexingMutationdetectionQuantification•Allelefrequencyassessment•SNPfrequency•Tri/tetraallelicSNPfrequency•Indelfrequency•CpG-methylation•Genecopynumber•Lossofheterozygosity•Polyploidgenomes•ExpressionanalysesGeneticvariation•SNPsandDIPs•Di-,tri-andtetraallelic•Multiple•Multiplexing•Haplotype•Out-of-phase•Allele-specificPCR已发表的文章使用Pyrosequencing技术发表的研究报告已近千篇0246810122000200120022003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.anthra