Illumina测序的原理和应用-ppt课件.pptx

1、Illumina 测序的原理和应用 1PPT课件Illumina测序流程 Data Analysis 数据分析 Sequencing 测序 Cluster Generation 簇生成 Library Preparation 文库制备测序流程2PPT课件3Adapter and Library 接头和文库接头：与flow cell结合区Read1和Read2测序引物结合区Index 用来区分不同样本3PPT课件4Illumina测序流程 Data Analysis 数据分析 Sequencing 测序 Cluster Generation 簇生成 Library Preparation 文库制

2、备测序流程4PPT课件5什么是 flow cell？Flow cell 即流动槽，如载玻片大小，有8个通道即8条lane。5PPT课件61. Single DNA libraries are hybridized to primer lawn and extended by polymerase6PPT课件72. Double-stranded molecule is denatured and original template is washed away7PPT课件83. New synthesized strand is covalently attached to flow cell

3、 surface to form a bridge and extended by polymerase4. Double-strand bridge is formed8PPT课件95. Double-strand bridge is denatured and formtwo copies of covalently bound single-stranded templates6. Bridge amplification cycle repeat9PPT课件107.Multiple bridges are formed8.dsDNA bridges are denatured and

4、reverse strands are cleaved10PPT课件119. Cleaved reverse strands are washed away and leaving a cluster with forward strands only在Illumina的测序过程中，无论是单单端测序端测序还是双端测序双端测序，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（甲酰胺基嘧啶糖苷酶（FpgFpg）”对“8-8-氧鸟嘌吟糖苷（氧鸟嘌

5、吟糖苷（8-oxo-G8-oxo-G）”的选择性切断作用。11PPT课件12Illumina测序流程 Data Analysis 数据分析 Sequencing 测序 Cluster Generation 簇生成 Library Preparation 文库制备测序流程12PPT课件13Hiseq2500Two flow cell13PPT课件1410. Sequencing primer is hybridized to adapter sequence测序引物3端5端14PPT课件1511. Only one nucleotide is added every cycle as the 3

6、 end is blocked15PPT课件1612. Cleave the fluor and free the 3 end then repeat above steps for next cycle16PPT课件1713. Sequenced strand is stripped off and the index primer is added for to seq index3端5端Index primer17PPT课件1814. Repeat bridge amplification to form reverse strand after index sequencing15. Sequencing the reverse strands 18PPT课件19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DGESmall RNAlncRNA19PPT课件20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-seq20PPT课件21 Thank you!21PPT课件