Illumina 测序的原理和应用

1、1Illumina测序的原理和应用2Illumina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4Illumina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5什么是 flow cel

2、l？Flow cell 即流动槽，如载玻片大小，有8个通道即8条lane。61. Single DNA libraries are hybridized to primer lawn and extended by polymerase72. Double-stranded molecule is denatured and original template is washed away83. New synthesized strand is covalently attached to flow cell surface to form a bridge and extended by

3、polymerase4. Double-strand bridge is formed95. Double-strand bridge is denatured and formtwo copies of covalently bound single-stranded templates6. Bridge amplification cycle repeat10 Multiple bridges are formed dsDNA bridges are denatured and reverse strands are cleaved119. Cleaved reverse strands

4、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（氧鸟嘌吟糖苷（8-oxo-G8-oxo-G）”的选择性切断作用。12Illumina测序流程 Data Analysis 数据分析

5、Sequencing 测序 Cluster Generation 簇生成 Library Preparation 文库制备测序流程13Hiseq2500Two flow cell1410. Sequencing primer is hybridized to adapter sequence测序引物3端5端1511. Only one nucleotide is added every cycle as the 3 end is blocked1612. Cleave the fluor and free the 3 end then repeat above steps for next c

6、ycle1713. Sequenced strand is stripped off and the index primer is added for to seq index3端5端Index primer1814. Repeat bridge amplification to form reverse strand after index sequencing15. Sequencing the reverse strands 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 RN