基因组测序的原理与方法课件

1、大规模基因组测序的原理与方法胡松年husn 元素周期表的发现奠定了二十世纪物理、化学研究和发展的基础元素周期表“基因组序列图”将奠定二十一世纪生命科学研究和生物产业发展的基础！ “基因组”-生命科学的“元素周期表”人体解剖图奠定了现代医学发展的基础生命的奥秘蕴藏于 “四字天书”之中GCTTCTTCCTCATTTTCTCTTGCCGCCACCATGCCGCCACCA TCATTTTCTCTTGCCGCCACCATGCTTCTTCCTCATTTTCTCT CCACCATGCCGCCACCACGCCACCATGCTTCTTCCTCATCTC GCTTTCTTGCCGCCACCATGCCGCCACCG

2、CTTCTTCCtTCTCT基因组学的基础理论研究基因组学是要揭示下述四种整合体系的相互关系:基因组作为信息载体 （碱基对、重复序列的整体守恒与局部不平衡的关系）基因组作为遗传物质的整合体 (基因作为功能和结构单位与遗传学机制的关系) 基因组作为生物化学分子的整合体 (基因产物作为功能分子与分子、细胞机制的关系）物种进化的整合体 (物种在地理与大气环境中的自然选择） 基因组学是一个大学科“界门纲目科属种”，地球上现存物种近亿，所有生生灭灭的生物，无一例外，都有个基因组。基因组作为信息载体，它所储存的信息是最基本的生物学信息之一；既是生命本质研究的出发点之一，又是生物信息的归宿。基因组学研究包括

3、对基因产物（转录子组和蛋白质组）的系统生物学研究。基因多态性的规模化研究就是基因组多态性的研究。基因组学的研究必然要上升到细胞机制、分子机制和系统生物学的水平。基因组的起源与进化和物种的起源与进化一样是一个新的科学领域。基因组信息正在以天文数字计算，规模化地积累，它的深入研究必将形成一个崭新的学科。基因组与生命之谜基因组的产生与进化。基因组DNA组分的变化、GC百分比、嘌呤：嘧啶守恒。遗传密码的发生、发展和进化。内含子（尤其是大于100，000 核苷酸的大内含子）剪出后的运输和降解。最小内含子的生物学意义。动物基因组与植物基因组在基因分布上的共性和个性。物种衍变过程中基因组水平的变化。基因组大

4、小变化与遗传、分子、细胞机制的关系。“JUNK DNA”的发生、分类、进化与功能。大规模基因组测序的几个支撑技术 Sanger双脱氧末端终止法 PCR 技术 DNA 自动测序仪的发展 生物信息学分析软硬件设施 PCR（聚合酶链式反应）原理反应所需物质：DNA模板、引物、DNA聚合 酶、dNTP、缓冲液每个循环包括：变性（90）、退火（54 ）、延伸（72 ）Sanger 双脱氧末端终止法测序原理大 规 模基因组 测 序 的 两 种 策 略逐步克隆法（Clone by Clone）全基因组霰弹法 （Whole Genome Shot-gun) 两种大规模基因组测序策略的比较 项 目 策 略全基因

5、组霰弹法逐步克隆法 遗传背景不需要需要（需构建精确的物理图谱）速度快慢费用低高计算机性能高（以全基因组为单位进行拼接）低（以BAC为单位进行拼接）适用范围工作框架图精细图代表测序物种果蝇、水稻人、线虫BAC by BACWhole Genome Shotgun the sequencing of the human genome is likely to be the only large sequencing project carried to completion by the methods described in this issue. Maynard V. Olson , The

6、 maps: Clone by clone by clone , Nature 409, 816 - 818 (2001) BAC by BAC The sequence of the human genomeC. Venter et al.Science 16 Feb. 291: 1304 1351, 2001人类基因组计划研究的主要成果和进展表现在这“四张图”上 遗传图谱 又称为连锁图谱（linkage map），指基因或DNA标志在染色体上的相对位置与遗传距离物理图谱 以定位的DNA标记序列如STS作为路标，以DNA实际长度即bp、kb、Mb为图距的基因组图谱。转录图谱 利用EST（ex

7、pressed sequence tags 表达序列标签）作为标记所构建的分子遗传图谱序列图谱 通过基因组测序得到的，以A、T、G、C为标记单位的基因组DNA序列 逐步克隆法（Clone by Clone)物理图谱的构建大片段克隆的筛选霰弹法测序与“工作框架图”的构建序列的全组装与“完成图”构建物理图谱的制作 物理图谱的制作序列标签位点（STS）作图 物理图谱是以特异的DNA序列为标志所展示的染色体图。标志之间的距离或图距以物理距离如碱基对（base pair；bp，Kb , Mb)表示。最精细的物理图是核苷酸顺序图，最粗略的物理图是染色体组型图。 STS图谱是最基本和最为有用的染色体物理图谱

8、之一，STS（Sequence Tagged Site)本身是随机地从人类基因组上选择出来的长度在200300bp左右的特异性短序列（每个STS在基因组中是唯一的，STS图谱就是以STS为路标（平均每100Kb一个），将DNA克隆片段有序地定位到基因组上。 STS的来源随机基因组序列表达基因序列，如EST遗传标记序列，如微卫星标记有关STS的信息可在基因组数据库GDB中找到 http:/gdbwww. gdb. org关 于 文 库作为载体的基本要求 能在宿主细胞中进行独立的复制 具有多克隆位点，可插入外源 DNA片段 有合适的筛选标记，如抗药性 大小合适，易于分离纯化 拷贝数多 文库的概念

9、含有某种生物体全部基因的随机片段的重组DNA克隆群体 载体：能携带外源DNA进入宿主细胞的工具，常用的载体有质粒载体、噬菌体载体、细菌人工染色体等宿主：能容纳外源DNA片段的生物体，常用的有大肠杆菌、酵母等BAC文库的构建NotI、SacI脉冲场凝胶电泳得200Kb左右的大片段DNA 纯化后与载体连接 电转化，将连接产物导入大肠杆菌感受态细胞插有外源DNA片段的BAC载体在含有氯霉素的固体培养基中培养每一个菌落为带有相同外源DNA片段的单克隆Regional mappingRegional mappingMinimal tiling path selected for sequencing.R

10、egional mappingBeijing Map共48个每组 8 个每8个96孔板组成1个superpool，384个96孔板组成48个superpools 48 superpoolsColumn pools Row pools 1 2 3 4 5 6 7 8 9 10 11 12第八板第二板Plate pools第一板 plate pools，row pools，column pools的构成 “STS-PCR反应池”方案（Pooling Protocol） 1 2 3 4 5 6 7 8 9 10 11 12超级池（8个96孔板，共768个克隆）板池（96个克隆）行池（12个克隆）列池

11、（8个克隆）大大减少筛选的工作量，降低成本，所得筛选结果准确可靠 28 VS 768sheet of superpools, plate pools, row pools, column pools 一 BAC Screening前48个样品为引物OGG1.51对superpool(sp)的筛选结果后48个样品为引物OGG1.52对superpool(sp)的筛选结果 引物OGG1.52对应sp#27,34,45的plate,row,column pools的筛选结果BAC clone 确定 (+为阳性克隆) 引物OGG1.52的Colony-PCR 延 伸 克 隆 的 筛 选 STS的密度尚

12、未达到绘制高精度物理图谱的要求，且在基因组中的分布不均匀，造成很多区域没有阳性克隆覆盖,形成空洞。因此需用指纹图谱（FPC法）或末端序列（Walking by End Sequence)步移等手段对种子克隆进行延伸，形成连续克隆群。利用延伸方法筛选得到的克隆称为延伸克隆。 Contig 1Contig 2重叠序列重叠序列延伸引物筛选到的延伸克隆20 kb300 bpMolecular weightmarker every 5th lane BAC clones 在96深孔 板中培养- Hind III 完全酶切- 1% 琼脂糖凝胶电泳 指 纹 图 谱 法 （Walking by Fingerp

13、rinting database） 挑取靠近空洞的种子克隆，酶切构建其指纹图谱，在FPC数据库中进行比对，搜索含有此克隆的重叠克隆群信息，从中确定覆盖空洞区域的克隆，达到延伸目的。Hind III 完全酶切Hind III 完全酶切FPC数据库中比对Clone AClone BClone CCABcontig搭建中克隆的错位 末端序列步行法 （Walking by End Sequence） 挑取靠近空洞的种子克隆进行末端测序，然后在基因组数据库中进行比对，确定专一性的序列片段作为新的STS路标。最后设计新路标的PCR引物，按照STSPCR“反应池”方案筛选新的克隆，达到延伸的目的 。克隆35

14、0A18序列输入 end sequence database的查询结果四、Clone Identification 1、STS-PCR 2、BAC end sequencing 3、Fingerprinting 4、FISH CK2CK1CK2CK113f06267l16481o07250a15204c23340j13对15个克隆进行HindIII酶切后电泳结果 “工作框架图”绘制根据序列与STS database进行blastn比较结果，将克隆定位末端序的比较，判定延伸在contig外的一端序列。并可及时进行walking，筛选新的克隆 霰弹法测序组装与Finishing工作流程图 Shot

15、gun Sequencing I :RANDOM PHASEBac Clone: 100-200 kbSheared DNA: 1.0-2.0 kbSequencingTemplates: RandomReadsShotgun Sequencing II:ASSEMBLYConsensusSequenceGap Low Base QualitySingleStrandedRegionMis-Assembly(Inverted)ConsensusSequenceGap Low Base QualitySingleStrandedRegionMis-Assembly(Inverted)Shotgu

16、n Sequencing III: FINISHINGConsensusSequenceGap SingleStrandedRegionMis-Assembly(Inverted)Shotgun Sequencing III: FINISHINGConsensusSequenceGap Mis-Assembly(Inverted)Shotgun Sequencing III: FINISHINGConsensusMis-Assembly(Inverted)Shotgun Sequencing III: FINISHINGShotgun Sequencing III: FINISHINGHigh

17、 Accuracy Sequence:Sp6Sp6Sp6T7Completed sequenceSequenced cloneBAC selected by end-sequence113L10324K11173F11101A4167P17586C2116K5572B22544N5R-155E142006P232306M15R-149E1560K?Gap filling by end sequences2、Filling “interclone gaps” The actual and predicted fingerprint of R-260J13 digested with HindII

18、I Lane 1: marker, Lane 2: R-260J13 digested with HindIII, 3 : the predicted 克隆211B19组装后的序列的错误率为零 Whole Genome Shotgun This bacterium has a circular genome structure with 2,689,445 base pairs, the second largest one of thermophiles decoded completely to date.Circular representation of the genome of T

19、. tengcongensis What is under heaven is for all. Sun Yat-sen, the father of modern China 天下为公 /riceDDBJ/EMBL/GenBank:AAAA01000000国际一流测序生产线7万克隆，3000万碱基/天高产出、低成本：$/bp￥/bp美分/bp分/bp基因组学：数据导向的大科学有数据才是硬道理世上无难事只要肯登攀Contigs:127,550 (N50=6,688 bp)Scaffolds: 102,444(N50=11,764 bp)Quality: 546 bp at Q20测序平台的构成

20、 大 规 模 测 序 平 台 的 构 成 文库组 培养组 模板组 电泳组 反应组 MegaBACE 上机组 数据处理组 基因组随机文库的构建 测序模板的制备 测序反应与纯化反应产物上机测序 序列数据的接收、质量评估、 组装等 含有质粒载体的大肠杆菌培养检测模板质量与测序定量文库组 各种物种DNA的提取载体的制备大片段文库(BAC、Cosmid) 的构建霰弹法文库(pUC18)的构建文库质量的检测转化文库的保存 霰弹法文库与BAC文库的区别项 目 两种文库BAC 文库霰弹法文库文库的直接用途大片段DNA在基因组上定位测序基因组片段化的方法NotI / SacI不完全消化酶切超声波打断插入的DNA

21、片段长度大 100300 Kb小 23 Kb载体细菌人工染色体 Bac 质粒载体 pUC18 霰弹法文库构建过程 超声：打断基因组DNA，电泳检测超声效果 纯化、末端补平：片段化的DNA纯化，并用T4 DNA聚合酶补平末端 电泳：1%琼脂糖凝胶电泳，切下含1.6-2.0 kb、2.0-2.5 kb及2.5-3.0kb 的DNA片段的凝胶 回收：用QIAEXII GEL Extraction Kit 回收试剂盒从胶中回收DNA片段 连接：用T4 DNA连接酶连接DNA片段和用SmaI酶切后的pUC18载体 电转化：连接产物用细胞导入仪电转化入感受态大肠杆菌 涂平板、重组子筛选：培养18小时候观察

22、蓝白斑，白斑为重组克隆培 养 组 96孔板培养基的分装挑取克隆过夜培养菌液生长的检测离心4度贮存 培养的目的是通过大肠杆菌扩增系统， 为 测序反应提供充足的模板国产96自动分液器培养用96孔深孔板培养组 分装培养基：8道瓶口分液器1分钟/板出错率高工作强度高国产96自动分液器30秒/板杜绝错误轻松一按VSVS模 板 组 负责测序反应模板DNA的制备。采用碱裂解过膜法从转化获得的目的重组子大肠杆菌中提取目标产物插入外源DNA片段的PUC18质粒 步骤：培养好的克隆加 I 液（ 含Tris 、EDTA ）悬浮大肠杆菌加 II液（ 含NaOH、SDS ）裂解大肠杆菌，并使DNA、蛋白质变性加 III

23、液（ 含乙酸钾、乙酸 ）中和，并使DNA复性反应液过膜 收集滤液，弃膜质粒DNA的纯化模板组 96孔醋酸钾法(1999年)96孔亚精胺法(2000年)96孔Millipore过滤膜法(2001年) 96孔自制醋酸纤维吸附膜法(2002年)384孔过膜法(2002年?)全部试剂及耗材的国产化 电 泳 组 检测模板质量： 条带超过Marker的为小片段，空孔、弱孔和小片段均为不合格模板 测序定量： 与Marker比较条带亮度，建议测序反应模板用量反应组 测序模板检测反应试剂与引物检测和分装反应及纯化样品溶解96孔板反应(1999年)384孔板反应(2000年)MegaBACE上机组 上机样品的分装

24、上机试剂的分装上机数据传送设备的保养与维护数据处理组数据接收序列质量评估序列组装序列Finishing数据备份自动化程序的编制优化组新技术的资料收集与分析新方法推广的技术支持与监督大片段文库技术的建立模板制备方法的改进反应体系的优化上机参数与试剂使用的优化384孔板的全线使用配液组 各组配制各种试剂提高工作效率降低溶液配制的出错率减少浪费生产辅助组 挑菌卫生清洁器皿清洗孔板回收再利用 滤板制备测序模板保存Solid reads assembly strategy- Assembly & scaffolding of rice blast fungus genomeSongnian HuBeij

25、ing Institute of GenomicsChinese Academy of Sciences Applications of SOLiD System in BIGWhole genome re-sequencing: Fragment: SNP, Mate-paired: SNP, structure variation Drosophila, wild rice, Magnaporthe grisea, bifidobacteriumRegional re-sequencing: Chip-seq (human stem cell), Nimblegen-SOLiD (inve

26、rsion)Whole transcriptome expression (barcoding): Human stem cell, human cancer tissue, rice, dynamic metagenome (non-coding RNA, small RNA & mRNA)de novo sequencing (CHALLENGING): Sequencing data & referenceNCBI reference3730&454 reads/contigsSolid mate-pair readsReference3730&454 ContigsNumber176

27、5,856 Max4,853,688 86,597 Min3,126 99 Mean234,537 6,403 Median8,733 2,792 N502,508,602 15,406 Total41,513,201 37,503,638 3730 Reads 454 ReadsSolid ReadsReads#147,386 1,328,014 63,086,699 Total77,759,992 314,407,091 1,577,167,475 Coverage2X8X40XScaffolding methods3730&454 contigsSolid MP readsContig

28、LinksContigIDContigID#LinksInsert Length(bp)ContigAContigB10005000ContigAContigC2007000Scaffolding methods3730&454contigsreferenceA +B C D +A +C D +ScaffoldframeRules: 1. Contigs in a scaffold must have links with neighbors2. Insert length must be validScaffolding methodsA +C D +ScaffoldframeX A +C

29、D +X Scaffold_1Rules: 1. ContigX must have links to two other contigs at least2. Insert length must be validScaffolding methodsRules: 1. Contigs in a scaffold must have STRONG links with neighbors2. Insert length must be validM A +C D +X N A +C D +X M N Scaffold_1Statistics on scaffolding resultsSca

30、ffolds Number:187Contigs:2496(including 81contigs added)Total length:32,202,800Coverage:85%Try to fix gaps3730&454 contigsSolid readsReads between gapsde novo contigsWhole Genome Sequencing of Bifidobacterium animalis subsp. lactis V9 with the SOLiD System OutlineBackgroundsStrategiesResultsAnalysis

31、Conclusions9/18/202294BIG, CASBackgroundsWhy BAL V9?Physiological activitiesAcidity toleranceBile salt toleranceIntestinal juice toleranceProbiotic properties9/18/202295BIG, CASBackgroundsWhy SOLiD?High throughputEasy to operateError recognition (two-base encoding)Economic (much cheaper)BAL Genome P

32、rojects (good reference sequence)One draft (HN019, Apr 2008)One complete (AD011, Jan 2009)Three in progress (BB12, Bl-04 and DSM10140)9/18/202296BIG, CASStrategiesMate Pair LibraryV9 genome One circular chromosome: 2Mb One plasmid:30kbRead length: 25ntLibrary size: 3kbRead length: 25ntFive primers1/

33、4 of one of the two SOLiD slides9/18/202297BIG, CASStrategiesData ProcessingCorona Lite SOLiD data analysis softwareMappingPairing (mate pair statistics)ScaffoldingSNP AnalysisVelvet (de novo assembly)Multi-sequence alignment9/18/202298BIG, CASResultsSequencing quality assessment9/18/202299BIG, CASR

34、esults Sequencing quality assessmentProblematic base 9 has poor satay graph (good base 2 as control)9/18/2022100BIG, CASResults Sequencing quality assessmentProblematic base 9 also has poor N2S graph (good base 2 as control)9/18/2022101BIG, CASResultsSequencing quality assessmentNote: we adopted a h

35、igher beads density, so the quality is compromised compared with DH10B standard.9/18/2022102BIG, CASResultsMate Pair Span (average 3174bp)9/18/2022103BIG, CASResultsSequencing depth (V9-HN019 mapping)9/18/2022104BIG, CASResultsSequencing depth (V9-AD011 mapping)9/18/2022105BIG, CASResultsSNP calling

36、V9-HN019 (3 mismatches)SNP:50V9-AD011 (4 mismatches)SNP: 215Mismatch and error rate influence SNP numbers9/18/2022106BIG, CASAnalysis Synteny of V9 and AD0119/18/2022107BIG, CASAnalysis Velvet de novo AssemblyBases:2,255,395Contigs:73Ns:466,013Max:291,171Min:101Mean:30,896Median:7,492N50:95,8039/18/

37、2022108BIG, CASAnalysis9/18/2022109BIG, CASAnalysisScaffolding before we get AD011 complete sequenceV9 and HN019 mapping Contig:18Bases:1913436Ns:138Ms:2Rs:9 Ys:3Ss:5Max:531162Min:1399Mean:106302Median:33826N50:3212549/18/2022110BIG, CASAnalysis 9/18/2022111BIG, CASAnalysis BAL HN019 Contig Order (a

38、ligned with BAL AD011 complete genome sequence):4-2-27-23-25-28-3-1-10-26-21-9-22-20-8-7-6-5-11-12-17-18-19-14-16-13-24-15 BAL HN019 Scaffolding results (by mate pair position, cov. 800X) complement each otherThree gaps (1500, 500, 50bp) and some sequence regions with ambiguities (Watson strand or C

39、rock strand?)Use mate pair data and traditional sequencing methods to get the right sequences (going on)Sequence to get random plasmid sequences to extract the larger part from SOLiD sequencing raw data (on schedule)9/18/2022113BIG, CASConclusionsReference sequence is preferredRepeat sequences are m

40、ajor obstaclesAnalysis of such huge data require high performance workstationCoverage speaks! With SOLiD, re-sequencing is easy.9/18/2022114BIG, CASSequencing a strain of common wild rice with new generation sequencer: SOLiDAncestor of cultivated riceCommon wild rice (Oryza rufipogon Griff.)Perennia

41、l AA genome, 12 chromosomesvaried from 439 to 450 MbDomesticated about 10,000 years agoPotential gene pools for cultivated ricewild- abortive for hybrid rice breedingYield enhancingyld1.1, yld2.1, qGY2-1 Disease resistanceThe only known source of resistance to grassy stunt resistanceBacterial blight

42、 resistance: Xa-21 Stress toleranceQuality improvingDomestication of riceSome genes control key phenotypic transitionShattering, Rc, PROG1, WaxyGenome level ?Comparison of the genome sequence between wild and cultivated riceData analysis using SOLiD/Corona Lite solfwarePrimary analysis on SOLiD inst

43、rumentIf mate paired runR3/F3_QV.qualF3.fastaF3.fastaData transfer Matching pipeline on off-instrument clusterR3.ma.25.3Pairing pipelineF3_R3.mate.non-redundanta. Insert size determinationb. Pairing with insert size b.1 unique-unique b.2 unique-non-unique b.3 non-unique- -non-unique b.4 unique-rescu

44、ed (mismatch) b.5 unique-rescued (indel)F3.ma.25.3 F3.bc.txt F3.stat.txt unique.csfasta.ma.25.3individual fragment runSNP pipelinesnp.txtMAQ will be the first alternative software ！Data generation-SOLiD libraryInformation of SOLiD librariesData generation-matching resultsSummaries of matching agains

45、t the map-based rice genome The parameter selection for “Pairing” (1) 6k library insert size distribution with 6 total mismatches in pairingThe parameter selection for “Pairing” (2)Total SNPs detected in chromosome 2 with 3k mate-paired libraries: 6 total mismatches (m6) : 24509; 4 total mismatches

46、(m4): 13783Coverage analysis of 13645 common SNPs: Total coverage:37939:33197; m6m4:3744 m6=m4: 9879; m6m4: 22For the 10,864 SNPs only detected by m6 : 76.11% of SNPs in all SNPs datasets (m4) 40.64% of SNPs (distance=10 good mate-paired reads 75% of total bases is bridged by = 100 good mate-paired

47、readsSyntenic region detection using mate-paired libraries Depth-100 threshold means each base in syntenic region is bridged by at each 100 good mate-paired readsThe detected syntenic regions at two clone coverage thresholds (depth-10,depth-100)Length distribution of syntenic region in depth-100 thr

48、eshold N50=60,905 bpSmall indels detection using mate-paired reads (1)88.80% 13 bp indels are the major type of small indels Each type of small indel has comparative average indel reads coverage and +/+ (%) (Implying the comparative detection sensitivity with SOLiD mate-paired reads ?) Small indels

49、detection using mate-paired reads (2)Average coverage2.7727 coverage:28532/99.4%16058/99.0% (+/+)Small indels are located at regions with lower sequence coverage Small indels detection using mate-paired reads (3)1k+3k+6kCalling for more mate-paired reads !Small indels detection using mate-paired rea

50、ds (4)Genomic distribution of indels in non-overlapped rice fl-cDNA 1. Only 2.07% of 8621 indels are in CDS;2. CDS region has relative high percentage of 3n indels; (CDS, 48.1%; UTR, 21.2%; Intron, 16.1%) 3. 3n indels (-9,-6,-3,+3 bp indels) may have little effect on gene functionThe utilities of ma

51、te-paired library (summary)“Good” mate-paired reads: (both ends are mapped on reference with reasonable distance, order and orientation) SNPs identification ( powerful for “SNP forests”) clone coverage estimation (synteny detection)“Bad” mate-paired reads: structural variation detectionSingle end un

52、ique mapped mate-paired reads: small indels identification scaffolding target assembly in variation regions SNPs identification !High quality unmapped mate-paired reads: scaffolding target assembly in variation regions 454/3730SOLiDConsensus regions covered by the fragment and mate-paired reads SNP identification in consensus regions (1)raw SNPs : 1,390,332 (The consensus base was not heterozygotes) filter-1 SNPs: 583,013 (coverage =2 fragment reads & =1 mate-paired reads)filter-2 SNPs: 428,706