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1、测序技术原理介绍,化学测序Sanger测序NGS（二代测序）Form the next to the third（三代测序）,测序方法分类,手工测序自动测序,Sanger双脱氧链终止法,Maxam-Gilbert化学降解法,3100/3100-a,3130/3130XL,3700,3730/3730XL,Solexa,454,焦磷酸法,SOLiD,Gilbert化学降解法1. 目的DNA制备2. 单侧末端标记待测DNA片段3. 碱基的特异性修饰及化学降解4. 聚丙烯酰胺凝胶电泳5. 放射自显影6. 阅读测序结果,Sanger测序法1. 单链DNA模板制备2. DNA模板与测序引物退火3. 掺入
2、法标记反应4. 延伸终止反应5. 变性聚丙烯酰胺凝胶电泳6. 放射自显影7. 阅读测序结果,Chain-terminator reaction,deoxynucleotides, dNTP,dideoxynucleotides, ddNTP,Frederick Sanger,Original Sanger sequencing,1980-1990Radio gelRead length: 102bp 103 bp / day,Automated Sanger sequencing,1990-Fluorescent capillaryRead length: 103bp106bp / day,A
3、BI 3730 DNA Analyzer,$376K/instrument650 bp/read0.06 Mb/run2h/run$10K/Mb,Hierarchical shotgun (map-based) method,Long fragment,BAC library,Physical map,Shotgun,Sequencing and assembly,Human Genome Project$3 billion, 11 years,Whole-genome shotgun method,Short fragment,Plasmid library,Mate-pair sequen
4、cing,3 years$300 million,Craig Venter,Sanger vs next-generation sequencing,Nat Biotech 26:1135(2008),Roche 454,Since 2004,Genome Sequencer / FLX,Template preparation- emulsion PCR,Trends in Genet 24:133(2008),Pyrosequencing,Single dNTP type flows per cycleInorganic pyrophosphate (PPi) drives visible
5、 light through a series of reactionsRemove unincorporated nucleotide,Trends in Genet 24:133(2008),Base calling,Homopolymer error,GV6330,Illumina Solexa,Since 2006,Genome Analyzer,Template preparation-bridge RCR,Adaptor ligation,Surface attachment,Bridge amplification,Denaturation,Trends in Genet 24:
6、133(2008),Cyclic reversible termination,All four labeled reversible terminators are added per cycleRemove unincorporated bases and detect signalRemove the terminating group and the fluorescent dye,Trends in Genet 24:133(2008),Terminating group,Fluorophore cleavage,Nat Rev Genet 11:31(2010),Base call
7、ing,Solexa测序原理,Main Illumina noise factors,(a) In the ideal situation, after several cycles the signal (green arrows) is strong, coherent and corresponds to the interrogated position.(b) Phasing noise introduces lagging (blue arrows) and leading (red arrow) nascent strands, which transmit a mixture
8、of signals.(c) Fading is attributed to loss of material that reduces the signal intensity.(d) Changes in the fluorophore cross-talk cause misinterpretation of the received signal (teal arrows).,Nat Methods 5: 679 (2008),ABI SOLiD,Since 2007,SOLiD Analyzer,Template preparation-emulsion PCR,Nature Rev
9、 Genet 11:31(2010),Annu Rev Geno Hum Genet 9:387(2008),SOLiD color space,fluorescent dye,N: Degenerated basesZ: Universal bases,Annu Rev Geno Hum Genet 9:387(2008),Sequencing by ligation,Prime and ligate,Image,Cleave off fluor,Sequencing by ligation,Extend sequence,Reset primer,Extend with new prime
10、r,Annu Rev Geno Hum Genet 9:387(2008),Base decoding,Instrument comparison,Journal of Genetics and Genomics 38:95(2011)Molecular Ecology Resources (2011),Pair-end sequencing,Mate-pair sequencing,Molecular Ecology Resources (2011),Science 327:1190 (2010),Nature Methods 8:41 (2011),Jonathan Rothberg,Pe
11、rsonal Genome Machine,$50K/instrument100bp/read1Gb/run2h/run$1.2/Mb,Ion Torrent,Detect H+ released as a voltage changefast Common microchip design standardslow-cost manufacturingSequencing volume is increasing,Semiconductor sequencing,Helicos BioSciences,1.将待测DNA随机打成200bp左右的小片段，在每个小片段末端加上ploy-dA，最后一
12、个A有荧光标记。2.与玻璃片上随机固定的多个poly-dT引物结合。3.激光刺激，标记荧光的A发光，确定位置。4.洗去荧光物质。,Helicos BioSciences,逐一加入荧光标记的末端终止子（这个终止子与Illumina终止子不一样，它所有的终止子都标有同一种单色染料），然后经过洗涤单色成像之后切开荧光染料和抑制基团，洗涤，加帽，允许下一个核酸进入。如此循环。,Paired-End Reads,Hybridize,25cycle,To end,Primer,25 cycle,melt,Pacific Biosciences,模板制备,将待测DNA随机打成250bp-6kb的片段，两端加
13、上发卡状的引物序列。,ZMW(zero-mode waveguide),短测序 长测序讨论？,主要技术特点,1.荧光标记到磷酸上，合成后自动脱落（传统的是标记到核苷酸的碱基上，大分子染料会干扰DNA合成酶的活性或者造成聚合反应提前终止）2.ZMW(zero-mode waveguide):十几纳米的小孔能让激光进入后迅速衰减，只有底部30纳米可见。排除背景影响。此外由于dNTP在荧光信号检测区停留的时间（毫秒级）与它进入和离开的时间（ 微秒级） 相比会很长，所以信号强度会很大。3.荧光脉冲的到达时间和持续时间反映了聚合酶动力学信息，而各种修士对聚合酶动力学的影响不一，从而将它们（N6-甲基腺嘌
14、呤、5-甲基胞嘧啶和5-羟甲基胞嘧啶等）区分开来。,整个过程所用到的软件,Life Technologies,基于荧光共振能的单分子测序合成技术。（fluorescence resonance energy transfer (FRET)-based single-molecule sequencing-by-synthesis technology ）技术概要：量子点与聚合酶结合，当有带有荧光标记的核苷酸结合时，在激光的照射下，量子点与核苷酸相互作用，发出荧光，同时被检测到。,Oxford Nanopore Technologie,O x f o r d N a n o p o r e Te
15、chnologies公司正在研究的纳米孔单分子技术是一种基于电信号测序的技术。他们设计了一种以-溶血素为材料制作的纳米孔，在孔内共价结合有分子接头环糊精。用核酸外切酶切割ssDNA时，被切下来的单个碱基会落入纳米孔，并和纳米孔内的环糊精相互作用，短暂地影响流过纳米孔的电流强度，这种电流强度的变化幅度就成为每种碱基的特征。碱基在纳米孔内的平均停留时间是毫秒级的，它的解离速率常数与电压有关，180 mV的电压就能够保证在电信号记录后将碱基从纳米孔中清除。,电信号,成像,Third-generation sequencing,Single molecular templateReal-time sequencing Low costLong reads,Nat Biotech 28:426 (2010),
