Illumina测序的原理和应用

Illumina测序的原理和应用

当前1页，总共21页。Illumina测序流程DataAnalysis

数据分析Sequencing

测序ClusterGeneration

簇生成LibraryPreparation

文库制备测序流程当前2页，总共21页。AdapterandLibrary接头和文库接头：与flowcell结合区Read1和Read2测序引物结合区Index

用来区分不同样本当前3页，总共21页。Illumina测序流程DataAnalysis

数据分析Sequencing

测序ClusterGeneration

簇生成LibraryPreparation

文库制备测序流程当前4页，总共21页。什么是flowcell？Flowcell即流动槽，如载玻片大小，有8个通道即8条lane。当前5页，总共21页。1.SingleDNAlibrariesarehybridizedtoprimerlawnandextendedbypolymerase当前6页，总共21页。2.Double-strandedmoleculeisdenaturedandoriginaltemplateiswashedaway当前7页，总共21页。3.Newsynthesizedstrandiscovalentlyattachedtoflowcellsurfacetoformabridgeandextendedbypolymerase4.Double-strandbridgeisformed当前8页，总共21页。5.Double-strandbridgeisdenaturedandformtwocopiesofcovalentlyboundsingle-strandedtemplates6.Bridgeamplificationcyclerepeat当前9页，总共21页。MultiplebridgesareformeddsDNAbridgesaredenaturedandreversestrandsarecleaved当前10页，总共21页。9.Cleaved

reversestrandsarewashedawayandleavingaclusterwithforwardstrandsonly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。当前11页，总共21页。Illumina测序流程DataAnalysis

数据分析Sequencing

测序ClusterGeneration

簇生成LibraryPreparation

文库制备测序流程当前12页，总共21页。Hiseq2500Twoflowcell当前13页，总共21页。10.

Sequencingprimerishybridizedtoadaptersequence测序引物3‘端5‘端当前14页，总共21页。11.Onlyonenucleotideisaddedeverycycleasthe3’endisblocked当前15页，总共21页。12.Cleavethefluorandfreethe3’endthenrepeatabovestepsfornextcycle当前16页，总共21页。13.Sequencedstrandisstrippedoffandtheindexprimerisaddedfortoseqindex3‘端5‘端Indexprimer当前17页，总共21页。14.Repeatbridgeamplificationtoformreversestrandafterindexsequencing15.Sequencingthereversestrands

当前18页，总共21页。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当前19页，总共21页。高通量测序的应用RNADN