Illumina 测序的原理和应用

Illumina测序的原理和应用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。61.SingleDNAlibrariesarehybridizedtoprimerlawnandextendedbypolymerase72.Double-strandedmoleculeisdenaturedandoriginaltemplateiswashedaway83.Newsynthesizedstrandiscovalentlyattachedtoflowcellsurfacetoformabridgeandextendedbypolymerase4.Double-strandbridgeisformed95.Double-strandbridgeisdenaturedandformtwocopiesofcovalentlyboundsingle-strandedtemplates6.Bridgeamplificationcyclerepeat107.Multiplebridgesareformed8.dsDNAbridgesaredenaturedandreversestrandsarecleaved119.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。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13Hiseq2500Twoflowcell1410.Sequencingprimerishybridizedtoadaptersequence测序引物3‘端5‘端1511.Onlyonenucleotideisaddedeverycycleasthe3’endisblocked1612.Cleavethefluorandfreethe3’endthenrepeatabovestepsfornextcycle1713.Sequencedstrandisstrippedoffandtheindexprimerisaddedfortoseqindex3‘端5‘端Indexprimer1814.Repeatbridgeamplificationtoformreversestrandafterindexsequencing15.Sequencingthereversestrands19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DGESmallRNAlncRNA20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-seq21Thankyou!