illumina基因测序仪中文测序原理

©2010Illumina,Inc.Allrightsreserved.Illumina,illuminaDx,Solexa,MakingSenseOutofLife,Oligator,Sentrix,GoldenGate,GoldenGateIndexing,DASL,BeadArray,ArrayofArrays,Infinium,BeadXpress,VeraCode,IntelliHyb,iSelect,CSPro,GenomeStudio,GeneticEnergy,HiSeq,andHiScanareregisteredtrademarksortrademarksofIllumina,Inc.Allotherbrandsandnamescontainedhereinarethepropertyoftheirrespectiveowners.SequencingTechnology张军磊13655172180南京才泰生物科技有限公司2COMPANYCONFIDENTIAL–INTERNALUSEONLY测序流程片段化的DNA末端修饰/添加一个A两端加接头选择加上接头的DNA扩增达到合适上样量文库制备(~6hrs)1自动簇生成(~5hrs)杂交到flowcell面延伸oligo接头完成桥式扩在模板序列链上加测序引物21-8个样品测序(~36hrsfor36循环)3对模板序列链执行测序1-8样品3COMPANYCONFIDENTIAL–INTERNALUSEONLY在末端加上接头末端修饰加上一个“A”到3’端使DNA片段化PCR扩增片段大小选择文库质控片段<800bp末端补平，在5’加上一个磷酸基团3’–小突出加上接头的DNA片段300–600bp片段扩增带接头的DNA基因组DNA文库制备完成纯化基因组DNADNA片段通过填充和核酸外切酶作用使末端补平磷酸化作用添加一个A碱基连上接头文库制备流程4COMPANYCONFIDENTIAL–INTERNALUSEONLY测序流程Libraryprep(~6hrs)1AutomatedClusterGeneration(~5hrs)2杂交到flowcell表面延伸杂交的oligo短序列进行桥式扩增1-8个样品Sequencing(~46hrsfor36cycles)3对forwardstrand执行测序1-8个样品DNA片段末端修饰/添加一个ADNA片段两端加接头选择加上接头的DNA片度6COMPANYCONFIDENTIAL–INTERNALUSEONLY流动槽（Flowcell）纯净的簇是在一个独立的环境中生成的（需要一个洁净的室）测序步骤也在flowcell的簇上进行简易流程中的关键点8个通道Flowcell表面结合一层oligo接头。7COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成:DNA片段杂交&延伸>50M的单分子DNA片段杂交到flowcell表面的oligo引物上。单分子DNA片段杂交在flowcell表面后，oligo引物就会在聚合酶作用下延伸。接头序列3’延伸Oligo引物8COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成:变性双链DNA双链分子变性分开。模板链被洗掉。新合成链以共价键的形式紧紧连接在flowcell表面。洗掉新合成链原始链9COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成:在flowcell上形成共价结合的单链DNA片段单链被以共价键的形式结合随机到flowcell上的模式。10COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成:桥式扩增单链弯曲杂交在相邻的oligo引物上形成一个桥式结构。杂交引物在聚合酶作用下延伸。11COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成:桥式扩增形成双链的桥式结构.12COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成:桥式扩增双链的桥式结构变性打开Result:形成2个copy的以共价键结合在flowcell的单链模板。13COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成:桥式扩增单链弯曲杂交在相邻的oligo引物上形成一个桥式结构。杂交oligo引物在聚合酶作用下延伸。14COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成:桥式扩增桥式扩增一直循环重复，直至形成5000-10000copies15COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成双链DNA桥变性分开。Reversestrands被剪切然后洗掉。16COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成…仅仅留下由forwardstrands组成的簇。17COMPANYCONFIDENTIAL–INTERNALUSEONLY簇生成游离的3ˋ端被封闭，防止不必要的DNA延伸。18COMPANYCONFIDENTIAL–INTERNALUSEONLY测序引物被杂交到接头序列上测序测序引物19COMPANYCONFIDENTIAL–INTERNALUSEONLYSingleReadSequencingprocessLibraryprep(~6hrs)1AutomatedClusterGeneration(~5hrs)21-8samplesSequencing(~46hrsfor36cycles)3对forwardstrand进行测序1-8samplesDNA片段末端修饰/添加一个ADNA片段两端加接头选择加上接头的DNA片度杂交到flowcell表面延伸oligo接头完成桥式扩在模板序列链上加测序引物20COMPANYCONFIDENTIAL–INTERNALUSEONLY测序forwardstrandAdd4Fl-NTP’s+PolymeraseIncorporatedFl-NTPisimagedTerminatorandfluorescentdyearecleavedfromtheFl-NTPX36杂交的测序引物21COMPANYCONFIDENTIAL–INTERNALUSEONLY读取碱基序列123789456TTTTTTTGT…TGCTACGAT…Theidentityofeachbaseofaclusterisreadofffromsequentialimages22COMPANYCONFIDENTIAL–INTERNALUSEONLYDNA(0.1-1.0ug)测序流程回顾SamplepreparationClustergrowth5’5’3’GTCAGTCAGTCACAGTCATCACCTAGCGTAGT循环1循环2循环3循环7循环8循环9循环4循环5循环6ImageacquisitionBasecallingTGCTACGAT…Sequencing’第一个碱基被合成循环1:加入测序试剂检测荧光信号切掉阻遏基团和荧光基团循环2-n:加入测序试剂，重复第一个循环©2009Illumina,Inc.Allrightsreserved.Illumina,illuminaDx,Solexa,MakingSenseOutofLife,Oligator,Sentrix,GoldenGate,GoldenGateIndexing,DASL,BeadArray,ArrayofArrays,Infinium,BeadXpress,VeraCode,IntelliHyb,iSelect,CSPro,andGenomeStudioareregisteredtrademarksortrademarksofIllumina,Inc.Allotherbrandsandnamescontainedhereinarethepropertyoftheirrespectiveowners.COMPANYCONFIDENTIAL–INTERNALUSEONLY小结©2009Illumina,Inc.Allrightsreserved.Illumina,illuminaDx,Solexa,MakingSenseOutofLife,Oligator,Sentrix,GoldenGate,GoldenGateIndexing,DASL,BeadArray,ArrayofArrays,Infinium,BeadXpress,VeraCode,IntelliHyb,iSelect,CSPro,andGenomeStudioareregisteredtrademarksortrademarksofIllumina,Inc.Allotherbrandsandnamescontainedhereinarethepropertyoftheirrespectiveowners.COMPANYCONFIDENTIAL–INTERNALUSEONLYThankyou!