Illumina-测序的原理和应用.pptVIP

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

AdapterandLibrary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

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

什么是flowcell？Flowcell即流动槽，如载玻片大小，有8个通道即8条lane。

1.SingleDNAlibrariesarehybridizedtoprimerlawnandextendedbypolymerase

2.Double-strandedmoleculeisdenaturedandoriginaltemplateiswashedaway

3.Newsynthesizedstrandiscovalentlyattachedtoflowcellsurfacetoformabridgeandextendedbypolymerase4.Double-strandbridgeisformed

5.Double-strandbridgeisdenaturedandformtwocopiesofcovalentlyboundsingle-strandedtemplates6.Bridgeamplificationcyclerepeat

MultiplebridgesareformeddsDNAbridgesaredenaturedandreversestrandsarecleaved

9.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。

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

Hiseq2500Twoflowcell

10.Sequencingprimerishybridizedtoadaptersequence测序引物3‘端5‘端

11.Onlyonenucleotideisaddedeverycycleasthe3’endisblocked

12.Cleavethefluorandfreethe3’endthenrepeatabovestepsfornextcycle

13.Sequencedstrandisstrippedoffandtheindexprimerisaddedfortoseqindex3‘端5‘端Indexprimer

14.Repeatbridgeamplificationtoformreversestrandafterindexsequencing15.Sequencingthereversestrands

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DGESmallRNAlncRNA

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-seq

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