高通量测序的应用与进展.pdf

生工生物工程(上海)有限公司Sangon Biotech(Shanghai)Co.,Ltd.蒯田庖局同邈屐高通量测序服务部报告纲要 高通量测序简介 高通量测序平台的介绍 高通量测序的应用范围及案例分析 相关生物信息学分析软件介绍pANGON高通量测序简介高通量测序:一次性对，DNA分子进行并行测序，又称为下一代测序技术,其使得可对一个物种的转录组和基因组进 行深入、细致、全貌的分析，所以又被称为 深度测序。High-throughput Sequencing Next Generation Sequencing Deep S3高通量测序流程bDNA fragmentationDNA tra9taitionIn vitro adaptor ligation文库扩增In vivo clonvtg and ampkftcatjonCycle sequencingGACTAGAWCGAGCGTGA.-5(tempatei无需建立文库，两端加测序接头/PCR扩增低通量T-y-CTGATPcymerase dNTPa Labe lea ddNTPaQ ipamen-CTGATC-.CTGAICT CTGATCTA-.CTGATC7AT.CTGATC7ATG CTGATCTATGC.CTGATCATGCT 2 CTGATCWGCTC/CTGATCWGCTUGElectrophorseBtfi(1 read/capii!aryGenetabon of polony arrayCyclic array sequencing(10 reada.(Brray)一 CyOe，Cycle 2 Cyoe 3Wtei ts base 17 What is case 2?WMS 拓 Hase 3?并行测序 高通量A Sang er测序B高通量测序报告纲要 高通量测序简介 高通量测序平台的介绍 高通量测序的应用范围及案例分析 相关生物信息学分析软件介绍高通量测序技术的起源与发展 1992年Lynx Therapeutics MPSS 2003年Polony Sequencing（哈佛）2005年454 Pyrosequencing 2006年 Solexa Sequencing-by-Synthesis 2007年 ABI SOLiD 2008年 Helicos tSMS Sequencing 2010 年Ion torrent Semiconductor Sequensing 2011 年Pacific Biosciences SMRT S6高通量测序技术的传承关系图现有主要高通量测序仪开发商测序仪品牌技术原理开发商Roche 454焦磷酸测序RocheIllumina Solexa边合成边测序IlluminaABI SOLiD基于磁珠的大规 模并行连接测序ABIHelicos单分子荧光测序HelicosIon Torrent半导体测序ABISMRT单分子实时测序Pacific Bio 454 Pyrosequencing基于磁珠的焦磷酸测序:A磁珠制备设备q3cM imMt;uuiumB 454测序仪C 454测序原理Arp luatermjcihl any lucifitnn 454测序流程Load beads onto PicoTrter plateLoad enzyme beads 454测序流程与Base Calling454的特点与主要应用 读长较长，400600bp 通量较彳氐，IRunIM序歹U,400-600Mb 相对成本较高 主要应用：de novo测序 Illumina Solexa简介桥式PCR边合成边测序可逆终止物Debloc k;fluor removalLibrary Preparat ion Clust er Generat ion-2 h 15 mt n hands-on(Next era)J-5 h(10 min hands-on)6 h 3 h hands-on(TruSeq)Sequenc ing by Synt hesis 1.5 t o 11 daysCASAVA2 days(30 min hands-on)ijuON Illumina Solexa 测序流程MOLECULES2.ATTACH DNA TO SURFACEX BRIDGE AMPURCAT1ONOww turf ion ilng*trancidtcmptotes enchoced to the 9ubttrt.Sewral ml Bon dnM dust an of double branded DMA m g*wrted In wdi charawl th*flow csfl.Afiar UMrodtvtion.captm the Imagv of mnd flutxvMance from each duster on fho Bow caft.Record tfw idMHy of tfw flHt iorwchduataca.IMAG FIRST BASE11.SEQUENCE RADS OVEA MULTFt CHEMISTRY CYCLES12.AUGN DATASMMtd chamtfy cydc to kthfarta the rant wqiMndng cyd add al four labW tvw-dbk tarmAfwtort and nynw to the floweLAfter ter evcftMion,co fy the dt b*oRpt cyciM of wqvandng to detormArt*the eqywwa of betw In Wgmem ahngie be at timetCAC1CCTQ1GCCTCACTCCTG1GGGCTGATGrGCCXCCTCA GATGTGCCAC ctcactc G5；CTvCTGTGGABgn dM*.comper*to.w Illumina Solexa Base Calling Solexa的特点与主要应用读长较短，100150bp通量高，25G每天，120-150G每Run主要应用：RNA测序、表观遗传学研究 ABI SOLiD 简介 SOLiDSequencing by Oligo Ligation/Detection Olig。连接测序：通过连接酶连接，再对 olig。上荧光基团进行检测J()2B anPr mfirTTTTTrill.Adaptor Sequonco3,-I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I,Tcmplsto SoquencoS_SNV A9xyzDNmuGi :|dxfAdapter Sequence Template Sequence3(!3B山括dPrmer 2.211 1121111 山山 1111 HI I“1111111 3.Adapter Sequanco Tcmplsto SoquencsSOLiD 5500 x1ON 照ABI SOLiD测序前期制备B乳化PCR3,末端修饰C磁珠富集 转到测序玻片A样品片段化 磁珠连接 ABI SOLiD测序原理1.Prime and Ligate5.Repeat steps 1-4 to Extend SequencePRIMER ROMMO 1XHWILi Option cycle567.(n cycles)6.Primer Reset2.Image3JL-3*fluo rescence 口Lein-1ta3.Cap Unextended Strands1.Me”口疗 sx:enaea sequence3A13JL133 37.Repeat steps 1-5 with new primer4.Cleave off FluorCleavage Agent v HOttTz cY primersRood Po sitio n 1 0Univorsal soq primer rr)2Universal doq primer(n-t 凭-a Urvgq 管a pr mor S-：)U W W/.q pr?mws KJn vo eqi exoo prEer mN)；AA TA CCTT AT GG 2PRJMER ROUND 2Universal saq m-1 3 TTTTTTVTTTTTTT巴叫27户2930pANGON ABI SOLiD荧光结合和结果示例Possible Dinucleotides Encoded By Each Color2na BaseTemplat e Sequenc eA c G T 一 G T A c A c G T AJC G T c A T G,A c G TTACGGCTADouble InterrogationWit h 2 base enc oding each base is defined t wic eA.SOLiD Olig。荧光基团模式图0SRRO29969.1 VAB_5551_12_381_F3 1ngth=35 Til.0203.3.11132110103321113023302 01+SRR029969.1 VAB_5551_12_381_F3 length=35!36!8/8:!:!4 626=(8.)43),(95,B.SOLiD 测序结果示例(Color Spac e)SOLiD的特点与主要应用 读长较短，50-75bAi_ 二二 精度高，可达Q40 通量高，20-30G每天，IRun可达120G 主要应用：基因组重测序、SNP检测等三种平台的技术差异平台454So 1exaSOLiDPCR磁珠乳化PCR桥式PCR磁珠乳化PCR测序载体磁珠玻片玻片测序方式焦磷酸、荧光可逆终止物、荧光连接酶、荧光结果序列FastQFastQCSFastQ三种平台的效能参数差异平台读长通重周期精度Solexa HiSeq 2000Single-end:1 x 35 bpPaired-end:2 x 50 bpPaired-end:2 x 100 bp25 Gb/d1.5d4cl 8d50 bp 85%以上 Q30100 bp 80%以上Q30SOLiD 5500 x1Single-end:75 bp Paired-end:75 x 35 bp Mate-pair:60 x 60 bp20-30 Gb/d1d/1lane7d/12 lane7d/12 laneQ40454 GS FLX400-600 bp400-600 Mb/Run10hQ20报告纲要 高通量测序简介、高通量测序平台 高通量测序的应用范围及案例分析 相关生物信息学分析软件介绍ANGON高通量测序应用范围 DNA测序、.仝基向组de novo测序 基因组重测序宏基因组测序人类外显子组捕获测序 RNA测序转录组测序小RNA测序电子表达谱测序表观基因组研究ChlP-SeqDNA甲基化测序pANGON基因组测序基因组测序是对物种的打断后进行高通量测序，根据是否有已知基因组数据主要分为de novo全基因组测序和基因组重测序。De novo基因组测序是对未知基因组序列的物种 进行基因组从头测序，利用生物信息学分析手段 对序列进行拼接、组装，从而获得该物种的基因 组图谱。全基因组重测序是对已知基因组序列的物种进行 不同个体的基因组测序，并在此基础上对个体或 群体进行差异性分析。OANGON基因组测序策略Paired-EndpANGON基因组测序流程一两种测序策略Genomic DNAF rag ment(200-50 Obp)Lig at e Adapt orsSequenc e First EndReg enerat e Clust ers and Sequenc e Paired EndMat e-End Paired-end 原理100bps 100bpsGenome rearrang ement analysisChrA Genomic DNA-Correc tChrB 3KI nversion6Kt/Transloc at ionI 3KEzr二二二 3KDelet ionAmplific at ionK 1.Paired-end基因组重排分析ANGON Paired-end和测序深度对测序效果的影响o o o o o o0 8 6 4 2S6ecs Michael James Clark,Nils Homer,Brain D.O Connor,et al.U87MG Decoded:The Genomic Sequence of a Cytogenetically Aberrant Human Cancer Cell Line.PloS Genetics,2010,6(l):el000832.3、Wei Chen,Reinhard Ullmann,Claudia Langnick,et al.Breakpoint analysis of balanced chromosome rearrangements by next-generation paired-end sequencing.European Journal of Human Genetics,2010,18:539-543.4、Van Tassell CP,Smith TP,Matukumalli LK,Taylor JF,Schnabel Rd,et al.Wholegenome sequencing and variant discovery in C.elegans.Nat Methods,2008,5(2):183-188.5、Jun Wang,Wei Wang,Ruiqiang Li,et al.The diploid genome sequence of anAsian individual.Nature 456,60-65(6 November 2008)6、Huang SW,Li RQ,Wang J,et al.The Genome of the Cucumber(Cucumis sativusLinnaeus).Nature Genetics 2009;doi:10.1038/ng.475 7、David Hernandez,et al.De novo bacterial genome sequencing:Millions of veryshort reads assembled on a desktop computer.Genome Res.2008.18:802-809pANGON33基因组重测序案例分析 Erin D.Pleasance,et al.The compendium of somatic mutations in a small-cell lung cancer genome.Nature,2010,463:184-190.此研究用高通量测序对一个小细胞肺癌细胞 系NCIH209基因组进行重测序，以探讨 吸烟引发该细胞系基因组中特定碱基及其周 围序列的突变及细胞损伤修复原理。a pa=B。SUOQsnEo(DqEnNAverag e c overag e(t umour and normal)3 2 10 b a EOU 6(Doual05-o(D6SUS d肺癌基因组变异情况统计图Qt 66基因组重排和CNV分析8 q w n u Ad。flChr 4从头基因组测序案例 David Hernandez,et aL De novo bacterial genome sequencing:Millions of very short reads assembled on a desktop computer.Genome Res.2008.18:802-809 止匕研究对Ship网/ococcvs aureus strain MW2Helicobacter acinonychis strain Sheeba两种细菌基因组进行从头测序，并 比较了几种拼接方法的效果。ANGON多种拼接软件拼接结果比较Table L Comparison of m於mbly result s of SSphyloetcus 010,st rain MW2 m obt ained by Ed啊 Velvet,SHARCCS,and SSAKEAssembly soft wareNo.of correct cont igs(t ot al size)No.of ml$ds$embled cont igs(t ot al size)Correct cont igsNSOAverage lengt hMax lengt hTot al no.of mismat chesGenome coverageEdena st rict1122(2762 kbp)0(0 bp)6.0 kbp2.5 kbp25.7 kbp198%Edena nonst rict733(2737kbp)14(45.1 kbp)9.4 kbp17 kbp51.8 kbp9097%Velvet1093(2768 kbp)2(362 bp)5.4 kbp2.5 kbp22.9 kbp26098%SSAKE2334(2782 kbp)99(85.1 kbp)2.0 kbp1.2 kbp12.6 kbp242797%SHARCGS3632(2760 kbp)3(1.5 kbp)12 kbp760 bp8.6 kbp4497%多种拼接软件拼接结果比较五种拼接方法的拼接结果比对宏基因组测序宏基因组测序是对某一特定环境，如肠道、土壤、海水等中的所有微生物进行基因组测 序。通过此方法可对该环境中的微生物种类 和优势物种进行检测，揭示微生物群落多样 性、种群结构、进化关系、功能活性、相互 协作关系及与环境之间的关系。自然环境 中很多微生物无法分离培养，而此方法无需 对微生物进行分离培养。宏基因组测序方法现在有全基因组的宏基因 组测序和16S/18SrRNA宏基因组测序。pANGON全基因组的宏基因组测序通过高通量测序技术，对环境样品的总 DNA直接进行全基因组的宏基因组测序，能够实现微生物群落的物种分类研究、群落 结构、系统进化、功能注释以及物种间的代 谢网络研究，挖掘具有应用价值的基因资源,开发新的微生物活性物质。与传统的 Sanger法相比，速度快，性价比高，周期短,单个样品的测序量可以接近饱和。ANGON宏基因组测序信息分析主要内容 拼接组装 物种分类组成分析 基因预测和功能注释 生成Profiling table 主成分分析(PCA)筛选与样品分组显著相关的因子 多样品间比较分析pANGON16S/18S rRNA宏基因组测序 16S/18SrRNA是微生物群落分析和细菌进 化研究以及分类研究最常用的靶分子，采用 新一代测序技术，对16S/18SrDNA的可变 区进行测序分析，不需进行克隆筛选，能全 面的反映微生物群体的物种组成，真实的物 种分布及丰度信息。ANGON16S/18S rRNA测序信息分析内容 物种分类、物种丰度分析 OTU(Operational Taxonomic Units)分 析 多样性分析 系统进化分析 多样品间的比较分析 ReferencesMeyer,F;Paarmann D,DSouza M,Olson R,Glass EM,Kubal M,(2008).The metagenomics RAST server-a public resource for the automatic phylogenetic and functional analysis of metagenomes.BMC Bioinformatics 9:0.doi:10.1186/1471-2105-9-386.George I et al.(2010).Application of Metagenomics toBioremediation.Metagenomics:Theory,Methods and Applications.Caister Academic Press.Wong D(2010).Applications of Metagenomics for IndustrialBioproducts.Metagenomics:Theory,Methods and Applications.Caister Academic Press.Nelson KE and White BA(2010).Metagenomics and Its Applications to the Study of the Human Microbiome.Metagenomics:Theory,Methods and Applications.Caister Academic Press.CharlesT(2010).The Potential for Investigation of Plant-microbe Interactions Using Metagenomics Methods.Metagenomics:Theory,Methods and Applications.Caister Academic Press.Allen,EE;Banfield,JF(2005).Community genomics in microbial ecology and evolution.Nature Reviews Microbiology 3(6):489-498.Zheng,Hao;Wuz Hongwei(2010).Short prokaryotic DNA fragment binning using a hierarchical classifier based on linear discriminant analysis and principal component analysis.J Bioinform Comput Biol.8(6):995-1011.X3/I JC)OI J人类外显子组捕获测序外显子组是指全部外显子区域的集合，该区 域包含合成蛋白质所需要的重要信息，涵盖 了与个体表型相关的大部分功能性变异。与全基因组重测序相比，外显子组测序只 需针对外显子区域的DNA,覆盖度更深、数据准确性更高，更加简便、经济、高效。46pANGON人类外显子组捕获测序原理S ureSelect Tarflet Enrich nient System Capture Pro cessLMROuXD FRACTION DSCAnDDS equencing人类外显子组捕获测序分析流程iAI MGON检测序列变异分析示例SureSelect Target Enrichment System Kit Efficiently Captures 5 bp Mutant Readout on Illumina GAHeterozygous SNP callshg18_ChrX_77131408J7131467_+:Wildtype Bait Design/CTATTGTnATCAACCTCATCTT/ATCTCGTAGAGGAAATGAAAAAGCAGAnGAAGCTCTATTGTnATCAACCTCATCjTT-ATAGAGGAAATGAAAAATTGTTTATCAACCTCATQTT-AGjTAGAGGAAATGAAAAAG nGTnATCAACCTCATUTTAGjTAGAGGAAATGAAAAAGC GTTTATCAACCTCATtTT-AGfAGAGGAAATGAAAAAGCAG TATCAACCTCATtTT-AGjTAGAGGAAATGAAAAAGCAGATT ATCAACCTCATiTT一AGjTAGAGGAAATGAAAAAGCAGATTG ATCAACCTCATiTT-AtiTAGAGGAAATGAAAAAGCAGATTG ATCAACCTCATChTA8TAGAGGAAATGAAAAAGCAGATTGCAACCTCATCh-一一ACTAGAGGAAATGAAAAAGCAGATTGAA CCTCATCl-AGTAGAGGAAATGAAAAAGCAGATTGAAGCT检测到SNP数统计序歹UlnDel检测 References 1、Wei X,Walia V,et al.Exome sequencing identifies GRIN2A as frequently mutatedin melanoma.Nat Genet.2011 Apr 15.Epub ahead of print 2、Janel O.Johnson,J.Raphael Gibbszet al.Exome Sequencing in Brown-Vialetto-VanLaere Syndrome.Am J Hum Genet.2010 October 8;87(4):567-569.3、Teer JK,Mullikin JC.Exome sequencing:the sweet spot before whole genomes.Hum Mol Genet.2010 Oct 15;19(R2):R145-51.Epub 2010 Aug 12.4、Ley TJ,Mardis ER,Ding L,et al.DNA sequencing of a cytogenetically normalacute myeloid leukaemia genome.Nature 2008;456(7218):66-72 5、Gnirke A,Melnikov A,Maguire J,et al.Solution hybrid selection with ultra-longoligonucleotides for massively parallel targeted sequencing.Nat Biotechnology 2009;27(2):182-9.6、Murim Choia,Ute I.Scholia,Weizhen Jia,et al.(2010)Genetic diagnosis by wholeexome capture and massively parallel DNA sequencing.PNAS.106:19096-19101.7、Sarah B Ng,Kati J Buckingham,Choli Lee,et al.(2010)Exome sequencingidentifies the cause of a mendelian disorder.Nature Genetics 42,30-50人类外显子组捕获测序案例 Wei X,Walia Vz et al.Exome sequencing identifies GRIN2A as frequently mutated in melanoma.Nat Genet 2011 Apr 15.Epub ahead of print黑色素瘤发生率一直在上升，此研究对黑色 素瘤细胞进行外显子组捕获测序，发现了和 其相关的高频突变基因。ANGON七个新发现的非同义高频突变位点ANGON单个基因中突变位点分析32 392 458 547 554 828 639 1.464基因GRIN2A模式图，箭头表示突变位点转录组测序简介转录组即特定细胞在某一功能状态下所能转录出 来的所有RNA的总和，包括mRNA和非编码 RNA(Non-coding RNA)。第二代测序系统可精确检测单个碱基，并且 不受到研究中先验信息的干扰，科研人员能够快 速地获得某一物种特定器官或组织在某一状态下 几乎所有mRNA转录本序列，从而能够开展：UTRs区域界定、可变剪切研究、低丰度新转录本 发现、融合基因鉴定、cSNP(编码序列单核甘酸 多态性)研究等。ANGON转录组测序测序流程转录组mRNA Non-c oding RNARNA片段化、纯化、检测产量_t_连接两端接头序列_I_逆转录生成c DNAI选择适当长度c DNA进行扩增_t_纯化扩增产物，评估产量上机进行高通量测序转录组测序测序流程TRANSCRI PTI ONeSTttnrywtth rlapmnI-j_ cDNATAAATmtGGACAEXCCAGCGGXI 抬AG 阴G AT6MleAEAAOTCAAACAATATGKAfttMmoluhon mjraiMm pcoAhiShotgun soquencing by Genome AnalyzotAssuc t ieI-Gec eNw.of de poUhrn无参考序列测序流程有参考序列测序流程乌圆转录组主要分析内容无参考序列转录组分析内容1测序数据产量统计，数据成分和 质量评估；2 Cont ig及Sc affold长度分布3 Unig ene的长度分布和功能注释，GO分类，Pat hway分析，差异表 达分析4蛋白功能预测与分类，差异表达 基因GO富集和Pat hway富集分析。有参考序列转录组分析内容1基本数据统计，比对参考序列2序列在基因组上在分布3测序深度分析、随机性评估和 基因差异表达分析4新基因预测，基因可变剪接鉴 定和基因融合鉴定等。References Maher CA,Kumar-Sinha C,Cao X,et al.Transcrip tome sequencing to detect gene fusions in cancer.Nature,2009 Mar 5;458(7234):97-101.Guoiie Zhang,Guangwu Guo,Xueda Hu,et al.Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcrip tome.Genome Kes.2010 May;20(5):646-54.,Murchison EP,Tovar C,Hsu A,et al.The Tasmanian devil transcriptome reveals Schwann cell origins of a clonally transmissible cancer.Science.2010 Jan 1;327(5961):84-7,Brain B.Tuch,Rebecca R.Laborde,Xing Xu et al.Tumor Transcrip tome Sequencing Reveals Allelic Expression Imbalances Associated with Copy Number Alterations.rloS ONE,2010,5(2):e9317 Fuchou Tang,Catalin Barbacioru,Ellen Nordman et al.RNA-Seq analysis to capture the transcriptome landscape of a single cell.Nature Protocols,2010,ePub Febrary 25.Sohrab P.Shah,Ryan D.Morin,Jaswinder Khattra et al.Mutational evolution in a lobular breast tumor profiled at single nucleotide resolution.Nature,2009,461:809-813 Zhao et al.Transcriptome-guided characterization of genomic rearrangements in a breast cancer cell line.PNAS 106(6):1886-91.(2009)。、Gregory R,Darby AC,Irving H,et al.A de novo expression profiling of Anopheles funestus,malaria vector in Africa,using 454 pyrosequencing.PLoS One.2011 Feb 25;6(2):el7418.Crawford JE,Guelbeogo WM,Sanou A,Traore A,Vernick KD,et al.(2010)De NoroTranscriptome Sequencing in Anopheles funestus Using Illumina KNA-Seq Technology.PLoS ONE 5(12):el4202.doi:10.1371/journal.pone.0014202、有参考序列转录组测序案例 Maher CA,Kumar-Sinha C,Cao X,et al.Transcriptome sequencing to detect gene fusions in cancer.Nature,2009 Mar 5;458(7234):97-101.此研究使用454和Solexa两种高通量测序平 台对前列腺癌细胞系VcaP和LNCaP转录组 进行测序，以检测和研究前列腺癌细胞系中 基因融合表达情况。ANGON基因融合分析aCatego ry I Mapping ReadsCatego ry IIPartially Aligned ReadsE Contaminatio nViralOthersCategory III No n Mapping ReadsChr 14q13.3-14q21.1翳蠹rEr llfisrww 嚣懑隙皆针篦T t爨鬟 一 簧-GCGS 0 bSII45000 MIPOL1-DGKB40000 I 35000 30000|25000.Z 20000至 15000s 1000050000 VCaP LNCaP RWPE PREC VCaP Met2 MetMIPOL1 DGKBTCCCXkAGTGGCC/KATGAAAAAGTTCAAAA GCCAATGAAAAAGTTCAAAA GAAAAAGTTCAAAA Sho rt reads aaagttcaaaaAAAGTTCAAAAATAAAAAATZVKAAATTACACACAATAAAAATTACACACAAGAACCATAAAAATTACACACAAGAACCAAG ATAAAAATTACACACAAGAACCAAG基因嵌合分析流程M/P0UDGKB基因融合模式NMW无参考序列转录组测序案例 Crawford JE,Guelbeogo WM,Sanou A,Traore A,Vernick KD,et al.(2010)De NoroTranscriptome Sequencing in Anopheles funestus Using Illumina RNA-Seq Technology.PLoS ONE 5(12):el4202.doi:10.1371/journal.pone.0014202此研究通过对3个按蚊样品进行高通量测序,通过拼接组装后，和相近物种进行比较基 因组学分析。De novo序列拼接、组装和比对流程/)Alh redsTranMrriptomc AnahshHnal conli|!vclSNDdet cvt innSNPs and md*l caBeduwig SAM哂Rad nupping uiih BWAThnio pal/d4Cont ig Size(kilobases)拼接和变异检测分析流程图拼接结果统计pANGON比较基因组分析Transcriptomic Proportion of Protein Divergence Amo