Illumina測(cè)序的原理和應(yīng)用

1、Illumina 測(cè)序的原理和應(yīng)用 Illumina測(cè)序流程 Data Analysis 數(shù)據(jù)分析 Sequencing 測(cè)序 Cluster Generation 簇生成 Library Preparation 文庫(kù)制備測(cè)序流程3Adapter and Library 接頭和文庫(kù)接頭：與flow cell結(jié)合區(qū)Read1和Read2測(cè)序引物結(jié)合區(qū)Index 用來(lái)區(qū)分不同樣本4Illumina測(cè)序流程 Data Analysis 數(shù)據(jù)分析 Sequencing 測(cè)序 Cluster Generation 簇生成 Library Preparation 文庫(kù)制備測(cè)序流程5什么是 flow cel

2、l？Flow cell 即流動(dòng)槽，如載玻片大小，有8個(gè)通道即8條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 repeat107.Multiple bridges are formed8.dsDNA bridges are denatured and reverse strands are cleaved119. Cleaved reverse strand

4、s are washed away and leaving a cluster with forward strands only在Illumina的測(cè)序過(guò)程中，無(wú)論是單單端測(cè)序端測(cè)序還是雙端測(cè)序雙端測(cè)序，都會(huì)用到特異選擇性鏈切斷的過(guò)程。其中單端測(cè)序的要切斷1次，雙端測(cè)序中的兩條鏈要先后各切斷1次（共2次）。單端測(cè)序：高碘酸希夫高碘酸希夫反應(yīng) 。雙端測(cè)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）”對(duì)“8-8-氧鳥嘌吟糖苷（氧鳥嘌吟糖苷（8-oxo-G8-oxo-G）”的選擇性切斷作用。12Illumina測(cè)序流程 Data Analysis 數(shù)據(jù)分

5、析 Sequencing 測(cè)序 Cluster Generation 簇生成 Library Preparation 文庫(kù)制備測(cè)序流程13Hiseq2500Two flow cell1410. Sequencing primer is hybridized to adapter sequence測(cè)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

6、 cycle1713. 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高通量測(cè)序的應(yīng)用DNA水平RNA水平基因組denovo、重測(cè)序外顯子捕獲測(cè)序DNA甲基化測(cè)序Chip-Seq16s、宏基因組轉(zhuǎn)錄組測(cè)序數(shù)字表達(dá)譜DGESmall R