焦磷酸测序专题知识讲座专家讲座.pptx

焦磷酸测序法原理及应用焦磷酸测序法原理及应用（Pyrosequencing遗传分析技术）遗传分析技术）焦磷酸测序专题知识讲座第1页pyro=pyrophosphatepyro=pyrophosphate焦磷酸焦磷酸一个一个全新全新基于酶级联反应新基于酶级联反应新型型遗传分析技术遗传分析技术;一套完整试验方案；一套完整试验方案；很多领域广泛应用很多领域广泛应用 PyrosequencingPyrosequencing遗传分析技术遗传分析技术PyrosequencingPyrosequencing这项专利技术完全由这项专利技术完全由BiotageBiotage企业拥有企业拥有焦磷酸测序专题知识讲座第2页PPiATPPrinciple of Principle of P PyrosequencingyrosequencingAPSLuciferin硫酸化酶硫酸化酶双磷酸酶双磷酸酶荧光素酶荧光素酶oxyluciferin焦磷酸测序专题知识讲座第3页Detection of the lightDetection of the lightlighttime焦磷酸测序专题知识讲座第4页 C G TT CC A CC T N 每次向反应体系中加入一个每次向反应体系中加入一个dNTPdNTPN 对应位置峰代表该种对应位置峰代表该种dNTPdNTP掺入情况掺入情况N 峰高与掺入核苷酸数量成正比峰高与掺入核苷酸数量成正比N 多出多出dNTPdNTP在加入下一个在加入下一个dNTPdNTP前就被降解前就被降解焦磷酸测序专题知识讲座第5页A complete solution for accurate genetic determinationPyrosequencingPyrosequencing 系统平台系统平台Reagent Kits Sample Prep WorkstationSoftwareInstrumentation焦磷酸测序专题知识讲座第6页PyroMarkTM IDComplete solution for Clinical MicrobiologyPyrosequencingPyrosequencing 系统平台系统平台Assay Design SW Sample Prep PyrosequencingFeaturing Pyro Gold chemistryIdentiFireTM SW焦磷酸测序专题知识讲座第7页P Pyrosequencing yrosequencing 技术流程技术流程PCR试样预处理，取得单链模板P PP Pyrosequencingyrosequencingyrosequencingyrosequencing序列分析序列分析序列分析序列分析15 min/96 samplesSQA:35 min/96 samplesSNP:10min/96 samples1-2h/96 samples结果分析结果分析试验设计试验设计焦磷酸测序专题知识讲座第8页 Peak height is proportional to the number of incorporated nucleotidesAllele 1:ACTGAllele 2:GCTG杂合子标本杂合子标本:焦磷酸测序专题知识讲座第9页Genotypes are clearly distinguished纯合子纯合子杂合子杂合子ACTGCCTGCTGCCTA/GCTGCCT焦磷酸测序专题知识讲座第10页Insertions/deletionsSingle nucleotide InDels(e.g.C)Multiple nucleotide InDels(e.g.CGACGGT)The software supports analysis of insertions/deletions(InDels)焦磷酸测序专题知识讲座第11页CYP2D6-A2637del(Allele 3)Sequence(reverse):TCC T GTGT/TT/-/-焦磷酸测序专题知识讲座第12页Large deletion3ATGTGGCATTCCAA5.ACGTACGCTTACACCGTAAGGTTCTAAAGGTGCACCATGACTGGGGTTACAGTCATC.焦磷酸测序专题知识讲座第13页Microsatellites:TGAATGnTTTGGGCAAAT8/128/84 7 84 7 8 (9)(10)(11)(12)焦磷酸测序专题知识讲座第14页Tri-and tetra-allelic SNPsC/CT/TG/GC/TC/GT/GGenotyping of tri-allelic SNP(C/T/G)焦磷酸测序专题知识讲座第15页Multiple polymorphisms 3TAAGCCGAATG5AACATTCGGCTTACAT/GGAATGAA/C/G/TCGGTACGAACGATTTTAGA.焦磷酸测序专题知识讲座第16页MultiplexingSNPs located on different fragments.or on the same焦磷酸测序专题知识讲座第17页Principle of multiplexingDesign of sequencing primers and dispensDesign of sequencing primers and dispensatation ion orderorder3,5refref00,511,522,53Relative light unitsTAGCGCACGATGTA/GCCTGCCACCA/GTTGGG Combined TGGACCACCA/GTGGTTGAGA/GCTGCCTG1 12 2TTGAGA/GCTGCCTG00,511,52TAGCGCACGATGTTGGACCACCA/GTGG00,511,52TAGCTGCACGATG焦磷酸测序专题知识讲座第18页User creates a SNP/mutation sequence databaseSoftware calculates theoretical genotyping resultsEach DNA fragment is assigned a color焦磷酸测序专题知识讲座第19页Quantitative peak heightsRelative peak height=peak1 height peak1 height+peak2 heightAllelefrequency=relative peak height x 100(%)Relative peak heightAllele frequency(%)0,0000,1000,2000,3000,4000,5000,6000,7000,8000,9001,000010203040506070809010015%30%75%焦磷酸测序专题知识讲座第20页Advantage and Benefit of PyeosequencingFastAccuracyFlexibilityMultiplexingSimpleSequenceEfficientShort Optimization TimeDedicated SoftwareDedicated Reagent KitScalable焦磷酸测序专题知识讲座第21页1.Assay Design1.Assay DesignExample of a general target:16S geneAssay Design SWSpecies-specific region in 16 S genePCR-forwardPCR-reverseSequencing primer焦磷酸测序专题知识讲座第22页2.PCR2.PCR扩增扩增 一对扩增引物中一条带有生物素标识，另一条不带标识。生物素标识焦磷酸测序专题知识讲座第23页3.Single Strand Template Preparation3.Single Strand Template Preparation PCR产物中加入streptavidine包被sepharose beads 振荡温育 5-10 min 变性，生物素标识单链结合于beads并被真空吸附于Vacuum Prep Tool上 洗涤，中和 单链模板加入反应体系，与测序引物退火PCR product immobilized on Sepharose beadsPSQ plate with sequencing primerEtOHNaOHWashingbufferWater焦磷酸测序专题知识讲座第24页J真实序列：遗传真实序列：遗传检测黄金标准检测黄金标准J快速实时输出：快速实时输出：1 1 碱基碱基/分钟分钟J9696个样本平行处个样本平行处理能力理能力J新一代新一代Pyro Gold试剂：表现试剂：表现大为提升大为提升4.4.PyrosequencingPyrosequencingPyroMark ID Instrument焦磷酸测序专题知识讲座第25页5.5.PyroPyrogram Analysisgram Analysissequence result with quality grading96 samples analyzed sequence result(can be exported in FASTA format)焦磷酸测序专题知识讲座第26页PyrosequencingPyrosequencing技术特点技术特点J得到定量序列结果技术J极高准确性Built-In QCJ重现性极佳J通用型技术平台功效多样，应用领域极广能够用于任何遗传多态性分析甲基化研究平台高通量、低成本快速临床微生物诊疗J试验设计灵活J自动化程度高，操作简单焦磷酸测序专题知识讲座第27页Why Pyrosequencing is Why Pyrosequencing is quantitativequantitative？lighttime焦磷酸测序专题知识讲座第28页 1 1分子分子dNTPdNTP掺入，释放出掺入，释放出1 1分子分子PPiPPi，生成，生成1 1分子分子ATPATP，产生单位强度光信号，产生单位强度光信号T GG CC GGG T C A C G A GG CCC TA.Triple DoubleSingleWhy Pyrosequencing is quantitativeWhy Pyrosequencing is quantitative？焦磷酸测序专题知识讲座第29页1.00.00.50.50.01.0Expected1.09 0.050.550.560.021.05Average0.02 n.d.0.030.03n.d.0.03CVC/CC/GG/Gn=190PyrosequencingPyrosequencingPyrosequencingPyrosequencing：Quantitative Accuracy Quantitative Accuracy Quantitative Accuracy Quantitative Accuracy TestTestTestTest焦磷酸测序专题知识讲座第30页PyrosequencingPyrosequencing：Accurate Accurate QuantifyingQuantifyingRelative peak height=peak1 height peak1 height+peak2 heightAllelefrequency=relative peak height x 100(%)Relative peak heightAllele frequency(%)0,0000,1000,2000,3000,4000,5000,6000,7000,8000,9001,000010203040506070809010015%30%75%焦磷酸测序专题知识讲座第31页E S G C T A G T A G A GC/CE S G C T A G T A G A GT/TE S G C T A G T A G A GC/TWhat are”Inbuilt Controls”?What are”Inbuilt Controls”?Since Pyrosequencing shows polymorphisms with sequence context,you can always trust the validity of the data.焦磷酸测序专题知识讲座第32页Benefits of Benefits of Built-in Built-in Quality Quality ControlControlJQCQC基于周围序列信息基于周围序列信息判定黄金标准判定黄金标准从从primerprimer后第一个碱基开始后第一个碱基开始便于数据便于数据alignmentalignmentJQCQC 验证了试验成功是否验证了试验成功是否详细到每一轮每一个样品详细到每一轮每一个样品万一试验失败，能够帮助分析原因，处理万一试验失败，能够帮助分析原因，处理问题问题焦磷酸测序专题知识讲座第33页PyrosequencingPyrosequencing焦磷酸测序系统卓越表现：99.998%accurateaccurate(based on analysis of 100,000 wells based on analysis of 100,000 wells with known genotype)with known genotype)焦磷酸测序专题知识讲座第34页Integrated Software PackageIntegrated Software Package整合且不停扩展软件包整合且不停扩展软件包 SNP-SNP and mutation analysis AQ -Included in SNP software-Easy statistics function SQA-Base calling-Sequence alignmentAll three with quality assessmentRunningAnalyzingPlanningDocumenting焦磷酸测序专题知识讲座第35页PyrosequencingPyrosequencing应用应用Sequence analysis Sequence identification Microbial typing Cloned DNA re-sequencing mtDNA/Forensics Transgenetics Expression profiling Oligo ID Microsatellites Known positions Unknown positions within hot spots Point mutations Insertions/deletions Multiple mutations MultiplexingMutation detectionQuantification Allele frequency assessment SNP frequency Tri/tetra allelic SNP frequency Indel frequency CpG-methylation Gene copy number Loss of heterozygosity Polyploid genomes Expression analysesGenetic variation SNPs and DIPs Di-,tri-and tetra allelic Multiple Multiplexing Haplotype Out-of-phase Allele-specific PCR焦磷酸测序专题知识讲座第36页已发表文章已发表文章焦磷酸测序专题知识讲座第37页使用使用PyrosequencingPyrosequencing技术发表研究汇报已近千篇技术发表研究汇报已近千篇焦磷酸测序专题知识讲座第38页发表在Nature、Science上文件焦磷酸测序专题知识讲座第39页快速判定炭疽热细菌快速判定炭疽热细菌Bacillus anthracis 炭疽是一个人畜共患疾病。引发炭炭疽是一个人畜共患疾病。引发炭疽病炭疽杆菌以孢子形式存在于土壤中，疽病炭疽杆菌以孢子形式存在于土壤中，在特定条件下，能够存活数十年。在特定条件下，能够存活数十年。炭疽热潜伏期为炭疽热潜伏期为1到到6天，彻底发病天，彻底发病则在多达则在多达60天后。抗生素能够抑制炭疽天后。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热感染，但条件是必须在接触炭疽热细菌后菌后48小时以内使用。小时以内使用。焦磷酸测序专题知识讲座第40页 炭疽杆菌炭疽杆菌Bacillus anthracis(B.anthracis)是引发炭疽热严重感染病原细是引发炭疽热严重感染病原细菌。从接触病原体到症状最初出现感染菌。从接触病原体到症状最初出现感染发展过程普通为发展过程普通为1到到6天。假如确认受到天。假如确认受到B.anthracis攻击攻击,利用抗生素或解毒剂能够利用抗生素或解毒剂能够防治其感染防治其感染,所以快速判定病原微生物种所以快速判定病原微生物种类和其致病力状态含有很主要意义。在类和其致病力状态含有很主要意义。在起初起初24-48小时内摄入抗生素能够防治其小时内摄入抗生素能够防治其传染。传染。焦磷酸测序专题知识讲座第41页 B.anthracis致病菌株有两个质粒致病菌株有两个质粒,pXO1含炭疽热毒素产生基因含炭疽热毒素产生基因(lef,cya,pag).pXO2编码包装和孢子形成所必须产物编码包装和孢子形成所必须产物.被称为被称为Ba813一段一段DNA序列是序列是B.anthracis一个特异染色体标识一个特异染色体标识,是是B.anthracis区分于与其紧密相关其它土壤杆菌种标志区分于与其紧密相关其它土壤杆菌种标志.pXO1Anthrax toxinpXO2Encapsulation factor焦磷酸测序专题知识讲座第42页B.anthracis assay design2)determine presence of pXO1 and/or pXO21)identify bacteriumFragment Ba813ChromosomeATACCGTATC AGCAAGCTAT pXO1AAGATATTAT CAAGGGATATpXO2ACCACTCATT AAGTTCTTCGProf.Lars Engstrand et al.,Uppsala University焦磷酸测序专题知识讲座第43页B.anthracis:raw dataCalled sequence:ATACCGTATC AGCAAGCTAT TCCCalled sequence:AAGATATTAT CAAGGGATAT TTTAAGTAAA ATTAATCAAC CATCalled sequence:ACCACTCATT AAGTTCTTCG CACCGCTAAA CASpecies-specific chromosomal fragment(Ba813)lef gene:anthrax toxin production(pXO1)cap gene:capsule formation(pXO2)焦磷酸测序专题知识讲座第44页B.anthracis:Result焦磷酸测序专题知识讲座第45页谢谢！焦磷酸测序专题知识讲座第46页