Illumina测序的原理和应用--ppt课件

Illumina测序的原理和应用1PPT课件Illumina测序流程DataAnalysis数据分析Sequencing测序ClusterGeneration簇生成LibraryPreparation文库制备测序流程2PPT课件3AdapterandLibrary接头和文库接头：与flowcell结合区Read1和Read2测序引物结合区Index用来区分不同样本3PPT课件4Illumina测序流程DataAnalysis数据分析Sequencing测序ClusterGeneration簇生成LibraryPreparation文库制备测序流程4PPT课件5什么是flowcell？Flowcell即流动槽，如载玻片大小，有8个通道即8条lane。5PPT课件61.SingleDNAlibrariesarehybridizedtoprimerlawnandextendedbypolymerase6PPT课件72.Double-strandedmoleculeisdenaturedandoriginaltemplateiswashedaway7PPT课件83.Newsynthesizedstrandiscovalentlyattachedtoflowcellsurfacetoformabridgeandextendedbypolymerase4.Double-strandbridgeisformed8PPT课件95.Double-strandbridgeisdenaturedandformtwocopiesofcovalentlyboundsingle-strandedtemplates6.Bridgeamplificationcyclerepeat9PPT课件107.Multiplebridgesareformed8.dsDNAbridgesaredenaturedandreversestrandsarecleaved10PPT课件119.Cleavedreversestrandsarewashedawayandleavingaclusterwithforwardstrandsonly在Illumina的测序过程中，无论是单端测序还是双端测序，都会用到特异选择性链切断的过程。其中单端测序的要切断1次，双端测序中的两条链要先后各切断1次（共2次）。单端测序：高碘酸希夫反应。双端测序：Illumina巧妙地利用了“甲酰胺基嘧啶糖苷酶（Fpg）”对“8-氧鸟嘌吟糖苷（8-oxo-G）”的选择性切断作用。11PPT课件12Illumina测序流程DataAnalysis数据分析Sequencing测序ClusterGeneration簇生成LibraryPreparation文库制备测序流程12PPT课件13Hiseq2500Twoflowcell13PPT课件1410.Sequencingprimerishybridizedtoadaptersequence测序引物3‘端5‘端14PPT课件1511.Onlyonenucleotideisaddedeverycycleasthe3’endisblocked15PPT课件1612.Cleavethefluorandfreethe3’endthenrepeatabovestepsfornextcycle16PPT课件1713.Sequencedstrandisstrippedoffandtheindexprimerisaddedfortoseqindex3‘端5‘端Indexprimer17PPT课件1814.Repeatbridgeamplificationtoformreversestrandafterindexsequencing15.Sequencingthereversestrands18PPT课件19高通量测序的应用DNA水平RNA水平基因组denovo、重测序外显子捕获测序DNA甲基化测序Chip-Seq16s、宏基因组转录组测序数字表达谱DGESmallRNAlncRNA19PPT课件20高通量测序的应用RNADNAProteinsRNALongncRNA转录组DGE原核真核有参无参比较转录组BSA-seq甲基化Chip-seq20PPT课件21Thankyou!21PPT课件