Illumina测序原理完整版.pptVIP

Illumina测序原理 目录概览制备文库 (Library Preparation)在测序芯片上生成DNA簇 (Cluster Generation)边合成边测序 (Sequencing by Synthesis)双侧测序，配对测序 (Paired-End, Mate Pair Multiplexing)数据分析 (Data Analysis) Library Preparation制备文库Sequencing测序 Data Analysis数据分析43Cluster Generation生成DNA簇2测序工作流程概览1 DNA(0.1-1.0 ug) Library preparationCluster generation5’5’3’GTCAGTCAGTCACAGTCATCACCTAGCGTAGT123789456Image acquisition Sequencing 测序过程概览 Sequencing OverviewBase calling T G C T A C G A T … 制备文库Library Preparation Library Prep Overview (gDNA)DNA Template1-5 μg，50 μl purified DNAOD260/280 = 1.8-2.0As intact as possibleLibraryYield 500-1000 ngOD260/280 =~1.8SR: 150-250 bpPE: 300-650 bp MW = size in bp x 650 DNA片段化 (Fragmentation)我们用的片段化方法CovarisBioruptor别的可选的片段化方法NebulizerSonicationHydroShear?EnzymaticOthers打碎Covaris修补末端T4, Klenow, T4 PNK加A 碱基AAAAAAKlenow exo- PPAA5’5’5’3’5’T3’TAAP3’5’T5’3’TT3’5’TA3’5’2% Agarose Gel 电泳切胶选取DNA ，对于单侧测序选取150-250 bp ，对于双侧测序选取600bp磁珠或Qiagen等回收试剂盒回收DNA片段Adaptors Ligation Excision PCR 扩增5’T3’A5’AT3’富集5’T3’A5’AT3’退火5’T3’A5’AT3’3’A5’T3’TA5’延伸5’3’5’T3’TAA10 - 12 Cycles Library QC双侧测序: 500 bp + 100 bp单侧测序: 250 bp + 100 bp Library Preparation SummaryRepair EndsAdd an A to the 3’ EndsFragment DNASize Select on GelLigate AdaptersPCRFragments 800 bpBlunt End Fragments with 5’ –Phosphorylated ends3’ - dA OverhangAdapter-Modified Ends300 - 600 bp FragmentsAmplified DNA with AdaptersGenomic DNA LibraryPurified Genomic DNAQC Library FAST LIBRARY PREP IN LESS THAN 2 HOURSClosed tube DNA fragmentationTransposon-mediated library preparationUltra-low input requirements (50 ng)ENABLES A RANGE OF CE AND NGS APPLICATIONSEpicentre Nextera TechnologySingle Tube, Rapid Library Prep 生成DNA簇Cluster Generation 测序芯片 (Flow cell) 生成DNA簇 (Cluster Generation)每个簇有~1000-6000 个DNA分子 OHOH Grafted flowcell种有引物的芯片表面diol P7 P5 Template HybridizationdioldiolTemplate Hybridization模板杂交dioldiol Initial extension启始延伸dioldiol Denaturation解链 1st cycle annealing第一次退火dioldioln=25/28total 1st cycle