新一代测序技术的原理

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,2014-5-26,#,Next-generation sequencing technology overview,Agenda,测序技术简史,第一代测序技术,第二代测序技术,第三代测序技术,1,测序技术简史,1950,1960,1970,1980,1990,2000,2010,Development of Sanger Sequencing,(1977),Invention of Automated Fluorescent,Sequencer,(1985),Invention of,Capillary,Sequencer,(1996),Invention of,Applied Biosystems,Solid System,(2007),Invention of,Illumina Genome Analyzer,System,(2006),Invention of,454 GS 20,Sequencer,(2005),chemical degradation method by Maxam-Gilbert method,(1977),Chemical degradation method by Whitfield,(1954),Invention of Heliscope single molecular sequencer,Invention of Single molecule real time(SMRT) DNA sequencing,Invention of,Nanopore single molecular sequencing (,Oxford Nanopore corporation),Invention of personal genome machine,Invention of optical mappingsystem,1977,年，英国人,Fred Sanger,发现，如果在,DNA,复制过程中掺入,ddNTP,，就会产生一系列末端终止的,DNA,链，并能通过电泳按长度分辨。,不同末端终止,DNA,链的长度是由掺入到新合成链上随机位置的,ddNTP,决定的。,由此诞生了以,Sanger,命名的测序原理即双脱氧链终止法测序原理。,5,磷酸,3,羟基,3,5,-,磷酸二酯键,核苷酸形成,3,5,-,磷酸二酯键示意图,核酸序列形成的基础,“,双脱氧末端终止,”,的含义,Template,Primer,dNTP,Polymerase,Terminator,C,G,T,A,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。,3730xl Sequence Map,2005,2006,2007,Birthday,Principle,Pyrosequencing,Sequencing-by-Synthesis,Sequencing-by-Ligation,Roche 454,Genome Analyzer/,Hiseq 2000,ABI SOLiD,Next-generation sequencing/Deep sequencing technology main platforms,454,测序原理,测序反应以磁珠上大量扩增的,ssDNA,为模板，在每一轮测序反应中，依照,T,、,A,、,C,、,G,的顺序依次循环进入，每次只进入一个碱基。,如果这种,dNTP,能与待测序列配对，则会在合成后释放焦磷酸基团。释放的焦磷酸基团会与反应体系中的,ATP,硫酸化酶反应形成,ATP,。生成的,ATP,和荧光素酶共同氧化反应体系中的荧光素分子并发出荧光。,通过检测荧光信号释放的有无和强度，确定被测分子的序列。光信号由,CCD,摄像机检测并反应为峰。每个峰的高度（光信号）与反应中掺入的核苷酸数目成正比。,反应结束后，,ATP,和未掺入的,dNTP,由双磷酸酶降解，淬灭光信号，并再生反应体系。,454,测序流程,待测,DNA,文库的构建,将基因组,DNA/cDNA,片段打断至,400-800bp,，将用于后继的扩增测序的接头,A,和一段含有生物素的接头,B,连接到,DNA,片段上，会产生含有接头,AA,、,AB,、,BB,、,BA,四种,DNA,片段，然后与可结合生物素的磁珠结合，其中片段,AA,被洗脱掉，片段,ABBABB,结合到磁珠上，通过变性处理回收含有接头,A,和接头,B,的单链,DNA,。,EmulsionPCR,将这些,ssDNA,分别单独与水油包被的直径大约,28 m,的磁珠在一起孵育、退火，由于磁珠表面含有与接头互补的寡聚核苷酸序列，因此,ssDNA,会特异地连接到磁珠上。,同时孵育体系中含有,PCR,反应试剂，因此可以保证每一个与磁珠结合的小片段都会在各自的孵育体系内独立扩增,扩增产物仍可以结合到磁珠上。,反应完成后，破坏孵育体系并富集带有,DNA,的磁珠。经过扩增反应，每一个小片段都将被扩增大约,100,万倍，从而达到下一步测序反应所需的模板量。,测序,将携带,DNA,的磁珠与其他反应物混合物，放入,PTP,板中进行测序。,PTP,板上含有很多直径约为,44 m,的小孔，每个小孔仅能容纳一个磁珠，通过这种方法固定每个磁珠的位置以监测接下的测序反应。,454,的特点,较高的测序通量，每个循环能产生总量为,400-600 Mb,的序列。,提高测序读长，长度达到,400 bp,，使得后继的序列拼接工作更加高效、准确。,主要缺点是无法准确测量同聚物的长度。例如当待测序列中出现,Poly(A),的情况下，测序反应中会一次加上多个,T,，而加入,T,的数目只能从荧光信号的强度来推测，有可能造成结果不准确。因此,454,技术主要的错误不是来自核苷酸的替换，而是来自插入或缺失。,2005,2006,2007,Birthday,Principle,Pyrosequencing,Sequencing-by-Synthesis,Sequencing-by-Ligation,Roche 454,Genome Analyzer/,Hiseq 2000,ABI SOLiD,Next-generation sequencing/Deep sequencing technology main platforms,n=degenerate bases,z=universal bases,SOLiD,的特点,通量大,成本低,序列短,Ligase,的方式虽然能一定程度避免,phasing/pre-phasing,，但增加的复杂度也降低了效率,灵活性差，对小数据量测序不适用,Two-base coding,2005,2006,2007,Birthday,Principle,Pyrosequencing,Sequencing-by-Synthesis,Sequencing-by-Ligation,Roche 454,Genome Analyzer/,Hiseq 2000,ABI SOLiD,Next-generation sequencing/Deep sequencing technology main platforms,2 Hiseq 2000,（,Solexa,）测序原理,Solexa,是一种基于边合成边测序技术,(Sequencing-By-Synthesis，,SBS,),的新型测序方法。通过利用单分子阵列实现在小型芯片,(Flow Cell),上进行,桥式,PCR,反应。由于新的,可逆阻断技术,可以实现每次只合成一个碱基，并标记荧光基团，再利用相应的激光激发荧光基团，捕获激发光，从而读取碱基信息。,可逆阻断技术,C,T,A,G,C,T,A,5-,3-,-5,G,T,合成第一个碱基,Cycle 1:,按顺序加入反应试剂,清除未反应的碱基和试剂,激发碱基荧光并收集荧光信号,去除阻断基团和荧光基团,Cycle 2-n:,重复前面的步骤,G,T,C,T,A,G,T,C,T,G,C,T,A,G,A,基于,SBS,的,Solexa,测序技术,3 Hiseq 2000,测序技术流程,制备芯片,模板杂交,桥式,PCR,扩增,测序引物准备测序,2,测序,测序,生成,base calls,3,数据分析,拍照图片,Intensities,Reads,Alignments,4,DNA,打断,末端修复,加,A,加接头,纯化,文库制备,1,3.1,文库制备,Solexa,有三种不同的测序种类：,Single-Read Sequencing,单向测序,Paired-End Sequencing,双向测序,Indexed Sequencing,混合样品测序,这三种测序类型的文库构建大体一致，主要区别在于接头。,Single-Read Sequencing,Fragment DNA,5,3,5,+,3,5,5,+,5,3,3,5,1.End repair,T4 polymerase , DNA Pol 1 (Klenow fragment),3,5,po,4,po,4,5,3,Phosphorylation,T4 polynucleaotide kinase, ATP,3.Ligation of adaptors,3A,po,4,5,A3,5,po,4,2.Add 3 Adenosine,with,Klenow (3exo,-,),and dATP,+,DNA insert,5,5,3,3,SBS oligo,P7,反向互补序列,3T,5,po,4,3,5,SBS oligo,P7,反向互补序列,PCR enrichment,5,5,3,3,5,5,5,5,3,3,3,3,1,st,round PCR,P7,P5,SBS oligo,P7,反向互补序列,P7,3,PCR enrichment (cont.),5,5,3,3,5,3,5,3,5,3,5,3,2,nd,round PCR,Insert,P7,P5,SBS oligo,P7,反向互补序列,P5,Cluster template,SBS oligo,P7,Paired-End Sequencing,双向测序,Indexed Sequencing,混合样品测序,3.2,芯片制备和测序,Cluster,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,Flow Cell,想像图,进样孔,出样孔,Single-Read Sequencing(SR,单向测序,),OH,Flow Cell,接头,diol,P7,P5,diol,diol,模板杂交,diol,diol,延长,diol,diol,变性,碱基片段杂交固定,diol,diol,1,st,cycle,变性,1,st,cycle,退火,diol,diol,1,st,cycle,延长,diol,diol,diol,diol,2,nd,cycle,变性,2,nd,cycle,退火,diol,diol,diol,diol,diol,diol,2,nd,cycle,延长,n=35,总数,碱基片段扩增成簇,线性化,OH,用,ddNTP (,),阻断,Cluster,扩增,完成,OH,diol,diol,OH,Cluster Station,结束,Primer,加测序引物,OH,NaIO,4,每一个基因簇就是信号收集照片上一个亮点,Y,X,通过荧光信号读取序列信息,1,2,3,7,8,9,4,5,6,T T T T T T T,G,T,T,G,C,T,A,C,G,A,T,通过荧光信号读取序列信息,3.3,测序,Paired-End Sequencing (PE,双向测序,),和,Indexed Sequencing,SR,与,PE,的,Flow Cell,接头对比,Single Read,Paired End,Template,Hybridization,模板杂交,U,U,OH,OH,Grafted flowcell,芯片上的接头,U,P7,P5,DNA,片段杂交和固定,Initial extension,第一链的延长,U,U,Denaturation,变性,U,U,1,st,cycle,denaturation,变性,n=,25,total,碱基片段扩增成簇,U,U,1,st,cycle,annealing,退火,U,U,1,st,cycle,extension,延长,U,U,2,nd,cycle,denaturation,再变性,U,U,2,nd,cycle,annealing,退火,U,U,U,2,nd,cycle,extension,延长,U,U,U,Cluster Station,结束,Cluster,Amplification,扩增后的簇,U,U,P5 Linearization,(,USER,),线性化,Block with,ddNTPs,阻断,Primer,加测序引物,Paired End,双向测序,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,OH,Resynthesis,of P5 Strand,合成,OH,P7 Linearization,(,fpg,),线性化,OH,Block with,ddNTPs,阻断,Primer,加测序引物,Sequencing,Second Read,第二端测序,The advantage and disadvantage of three platform,第三代测序技术,单分子测序技术,Heliscope,Pacific Biosciences,Life Technologies,Ion Torrent,Oxford Nanopore,基因组光图谱系统,谢谢！,