新一代测序技术的原理

1、会计学1新一代测序技术的原理新一代测序技术的原理1950196019701980199020002010Development of Sanger Sequencing(1977)Invention of Automated FluorescentSequencer(1985)Invention of CapillarySequencer(1996)Invention of Applied BiosystemsSolid System(2007)Invention of Illumina Genome Analyzer System(2006)Invention of 454 GS 20 Se

2、quencer(2005)chemical degradation method by Maxam-Gilbert method(1977)Chemical degradation method by Whitfield (1954)Invention of Heliscope single molecular sequencerInvention of Single molecule real time(SMRT) DNA sequencingInvention of Nanopore single molecular sequencing (Oxford Nanopore corporat

3、ion)Invention of personal genome machineInvention of optical mappingsystemONHHOHHOHHCH2OPOHOOH（H）ONHHOHHOHHCH2OPOHOOH（H）ONHHOHHOHHCH2OPOHOOH（H）ONHHOHHOHHCH2OPOHOOH（H）（H）ONHHOHHOHHCH2OPOOHOONHHOHHHCH2OPOHOOH（H）5 磷酸磷酸3 羟基羟基3 ,5 -磷酸二磷酸二酯键酯键核苷酸形成3,5-磷酸二酯键示意图核酸序列形成的基础TemplatePrimerdNTPPolymeraseTerminato

4、rCGTA终止物标记方法因为颜色不同，4种终止物可以在一起进行反应。200520062007BirthdayPrinciplePyrosequencingSequencing-by-SynthesisSequencing-by-LigationRoche 454Genome Analyzer/Hiseq 2000ABI SOLiDNext-generation sequencing/Deep sequencing technology main platforms200520062007BirthdayPrinciplePyrosequencingSequencing-by-Synthesis

5、Sequencing-by-LigationRoche 454Genome Analyzer/Hiseq 2000ABI SOLiDNext-generation sequencing/Deep sequencing technology main platformsn=degenerate bases z=universal bases200520062007BirthdayPrinciplePyrosequencingSequencing-by-SynthesisSequencing-by-LigationRoche 454Genome Analyzer/Hiseq 2000ABI SOL

6、iDNext-generation sequencing/Deep sequencing technology main platformsCTAGCTA5-3-5GT合成第一个碱基合成第一个碱基Cycle 1:按顺序加入反应试剂按顺序加入反应试剂清除未反应的碱基和试剂清除未反应的碱基和试剂激发碱基荧光并收集荧光信号激发碱基荧光并收集荧光信号 去除阻断基团和荧光基团去除阻断基团和荧光基团Cycle 2-n:重复前面的步骤重复前面的步骤G TCTA GT CTGCTAGA 制备芯片制备芯片模板杂交模板杂交桥式桥式PCR扩增扩增测序引物准备测序测序引物准备测序2 测序测序测序测序生成生成 base

7、 calls3 数据分析数据分析拍照图片拍照图片IntensitiesReadsAlignments4DNA打断打断末端修复末端修复加加A加接头加接头纯化纯化 文库制备文库制备1Fragment DNA535+355+53351.End repair T4 polymerase , DNA Pol 1 (Klenow fragment)35po4po453PhosphorylationT4 polynucleaotide kinase, ATP3.Ligation of adaptors3Apo45A35po42.Add 3 Adenosine with Klenow (3exo- ) and

8、 dATP+DNA insert5533SBS oligoP7 反向互补序列反向互补序列3T5po435SBS oligoP7反向互补序列反向互补序列PCR enrichment5533555533331st round PCRP7P5SBS oligoP7 反向互补序列反向互补序列P73PCR enrichment (cont.)5533535353532nd round PCRInsertP7P5SBS oligoP7 反向互补序列反向互补序列P5Cluster templateSBS oligoP7Cluster 生成步骤:固定扩增线性化 阻断引物杂交Cluster 生成化学步骤cBOT

9、进样孔出样孔OHFlow Cell接头diol P7 P5 dioldiol模板杂交dioldiol 延长dioldiol 变性dioldiol1st cycle 变性1st cycle 退火dioldiol1st cycle 延长dioldioldioldiol2nd cycle 变性2nd cycle 退火dioldioldioldioldioldiol2nd cycle 延长n=35总数线性化线性化OH用ddNTP ( )阻断阻断Cluster 扩增扩增完成完成OHdioldiolOHPrimer加测序引物加测序引物OHNaIO4YX123789456T T T T T T T G T

10、T G C T A C G A T Single ReadPaired EndTemplate Hybridization模板杂交UUOHOH Grafted flowcell芯片上的接头U P7 P5 Initial extension 第一链的延长UU Denaturation 变性UU1st cycle denaturation 变性n=25totalUU1st cycle annealing退火UU1st cycle extension延长UU2nd cycle denaturation再变性UU2nd cycle annealing退火UUU2nd cycle extension延长

11、UUUCluster Amplification扩增后的簇UUP5 Linearization(USER)线性化Block with ddNTPs阻断Primer加测序引物SBS进行测序Sequencing First Read第一端测序Denaturation and De-Phosphorylation(PNK)变性与去磷酸化OH OHResynthesis of P5 Strand合成OHP7 Linearization (fpg)线性化OHBlock with ddNTPs阻断Primer加测序引物SequencingSecond Read第二端测序The advantage and disadva