1、Illumina 測序的原理和應用 1教學育人Illumina測序流程 Data Analysis 數(shù)據(jù)分析 Sequencing 測序 Cluster Generation 簇生成 Library Preparation 文庫制備測序流程2教學育人Adapter and Library 接頭和文庫接頭：與flow cell結合區(qū)Read1和Read2測序引物結合區(qū)Index 用來區(qū)分不同樣本3教學育人Illumina測序流程 Data Analysis 數(shù)據(jù)分析 Sequencing 測序 Cluster Generation 簇生成 Library Preparation 文庫制備測序流程

2、4教學育人什么是 flow cell？Flow cell 即流動槽，如載玻片大小，有8個通道即8條lane。5教學育人1. Single DNA libraries are hybridized to primer lawn and extended by polymerase6教學育人2. Double-stranded molecule is denatured and original template is washed away7教學育人3. New synthesized strand is covalently attached to flow cell surface to f

3、orm a bridge and extended by polymerase4. Double-strand bridge is formed8教學育人5. Double-strand bridge is denatured and formtwo copies of covalently bound single-stranded templates6. Bridge amplification cycle repeat9教學育人Multiple bridges are formeddsDNA bridges are denatured and reverse strands are cl

4、eaved10教學育人9. Cleaved reverse strands are washed away and leaving a cluster with forward strands only在Illumina的測序過程中，無論是單端測序還是雙端測序，都會用到特異選擇性鏈切斷的過程。其中單端測序的要切斷1次，雙端測序中的兩條鏈要先后各切斷1次（共2次）。單端測序：高碘酸希夫反應 。雙端測序： Illumina巧妙地利用了“甲酰胺基嘧啶糖苷酶（Fpg）”對“8-氧鳥嘌吟糖苷（8-oxo-G）”的選擇性切斷作用。11教學育人Illumina測序流程 Data Analysis 數(shù)據(jù)分析

5、Sequencing 測序 Cluster Generation 簇生成 Library Preparation 文庫制備測序流程12教學育人Hiseq2500Two flow cell13教學育人10. Sequencing primer is hybridized to adapter sequence測序引物3端5端14教學育人11. Only one nucleotide is added every cycle as the 3 end is blocked15教學育人12. Cleave the fluor and free the 3 end then repeat above

6、steps for next cycle16教學育人13. Sequenced strand is stripped off and the index primer is added for to seq index3端5端Index primer17教學育人14. Repeat bridge amplification to form reverse strand after index sequencing15. Sequencing the reverse strands 18教學育人高通量測序的應用DNA水平RNA水平基因組denovo、重測序外顯子捕獲測序DNA甲基化測序Chip-Seq16s、宏基因組轉錄組測序數(shù)字表達譜DGESmall RNAlncRNA19教學