Illumina-测序的原理和应用

Illumina测序的原理和应用

1精选2021版课件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2精选2021版课件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3精选2021版课件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4精选2021版课件什么是flowcell？Flowcell即流动槽，如载玻片大小，有8个通道即8条lane。5精选2021版课件1.SingleDNAlibrariesarehybridizedtoprimerlawnandextendedbypolymerase6精选2021版课件2.Double-strandedmoleculeisdenaturedandoriginaltemplateiswashedaway7精选2021版课件3.Newsynthesizedstrandiscovalentlyattachedtoflowcellsurfacetoformabridgeandextendedbypolymerase4.Double-strandbridgeisformed8精选2021版课件5.Double-strandbridgeisdenaturedandformtwocopiesofcovalentlyboundsingle-strandedtemplates6.Bridgeamplificationcyclerepeat9精选2021版课件MultiplebridgesareformeddsDNAbridgesaredenaturedandreversestrandsarecleaved10精选2021版课件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。11精选2021版课件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12精选2021版课件Hiseq2500Twoflowcell13精选2021版课件10.

Sequencingprimerishybridizedtoadaptersequence测序引物3‘端5‘端14精选2021版课件11.Onlyonenucleotideisaddedeverycycleasthe3’endisblocked15精选2021版课件12.Cleavethefluorandfreethe3’endthenrepeatabovestepsfornextcycle16精选2021版课件13.Sequencedstrandisstrippedoffandtheindexprimerisaddedfortoseqindex3‘端5‘端Indexprimer17精选2021版课件14.Repeatbridgeamplificationtoformreversestrandafterindexsequencing15.Sequencingthereversestrands

18精选2021版课件高通量测序的应用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DGESmallRNAlncRNA19精选2021版课件高通量测序的应用RNADN