Illumina-测序的原理和应用.ppt

Illumina 测序的原理和应用 Illumina测序流程测序流程 Data Analysis 数据分析数据分析 Sequencing 测序测序 Cluster Generation 簇生成簇生成 Library Preparation 文库制备文库制备测测序序流流程程Adapter and Library 接头和文库接头：与接头：与flow cell结合区结合区Read1和和Read2测序引物结合区测序引物结合区Index 用来区分不同样本用来区分不同样本Illumina测序流程测序流程 Data Analysis 数据分析数据分析 Sequencing 测序测序 Cluster Generation 簇生成簇生成 Library Preparation 文库制备文库制备测测序序流流程程什么是什么是 flow cell？Flow cell 即流动槽，如载玻片大小，有即流动槽，如载玻片大小，有8个通道个通道即即8条条lane。1.Single DNA libraries are hybridized to primer lawn and extended by polymerase2.Double-stranded molecule is denatured and original template is washed away3.New synthesized strand is covalently attached to flow cell surface to form a bridge and extended by polymerase4.Double-strand bridge is formed5.Double-strand bridge is denatured and formtwo copies of covalently bound single-stranded templates6.Bridge amplification cycle repeat7.Multiple bridges are formed8.dsDNA bridges are denatured and reverse strands are cleaved9.Cleaved reverse strands are washed away and leaving a cluster with forward strands only在在Illumina的测序过程中，无论是单端测的测序过程中，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切断的过程。其中单端测序的要切断1 1次，次，双端测序中的两条链要先后各切断双端测序中的两条链要先后各切断1 1次次（共（共2 2次）。次）。单端测序单端测序：高碘酸希夫反应：高碘酸希夫反应。双端测序：双端测序：Illumina巧妙地利用了巧妙地利用了“甲酰甲酰胺基嘧啶糖苷酶（胺基嘧啶糖苷酶（FpgFpg）”对对“8-8-氧鸟嘌氧鸟嘌吟糖苷（吟糖苷（8-oxo-G8-oxo-G）”的选择性切断作用。的选择性切断作用。Illumina测序流程测序流程 Data Analysis 数据分析数据分析 Sequencing 测序测序 Cluster Generation 簇生成簇生成 Library Preparation 文库制备文库制备测测序序流流程程Hiseq2500Two flow cell10.Sequencing primer is hybridized to adapter sequence测测序序引引物物3端端5端端11.Only one nucleotide is added every cycle as the 3 end is blocked12.Cleave the fluor and free the 3 end then repeat above steps for next cycle13.Sequenced strand is stripped off and the index primer is added for to seq index3端端5端端Index primer14.Repeat bridge amplification to form reverse strand after index sequencing15.Sequencing the reverse strands 高通量测序的应用高通量测序的应用DNADNA水平水平RNARNA水平水平基因组基因组denovodenovo、重测序、重测序外显子捕获测序外显子捕获测序DNADNA甲基化测序甲基化测序Chip-SeqChip-Seq16s16s、宏基因组、宏基因组转录组测序转录组测序数字表达谱数字表达谱DGEDGESmall RNASmall RNAlncRNAlncRNA高通量测序的应用高通量测序的应用RNADNAProteinsRNALong ncRNA转录组DGE原核真核有参无参比较转录组BSA-seq甲基化Chip-seq Thank you!