1、IlluminaIllumina SentrixSentrixTMTM Arrays Arrays 预制的基因芯片预制的基因芯片 芯片滚动杂交仪芯片滚动杂交仪 全自动芯片洗涤工作站全自动芯片洗涤工作站 智能化的分析软件智能化的分析软件高分辨率的芯片扫描仪高分辨率的芯片扫描仪光蚀刻原位合成技术光蚀刻原位合成技术高密度的点阵技术高密度的点阵技术 基因序列基因序列11112020对对25mer25mer探针探针Perfect MatchPerfect MatchMismatchMismatchPerfect Match MM Mismatch 单点突变单点突变Oligo Oligo 探针探针基因基因
2、1 1的的OligoOligo探针组探针组基因基因2 2的的OligoOligo探针组探针组基因基因1 1基因基因2 2基因基因1 1cDNAcDNA基因基因2 2cDNAcDNA用于用于cDNAcDNA芯片的探针芯片的探针（1620)特异性高特异性高基因表达基因表达开放开放基因表达基因表达关闭关闭多个检测结果可以参考多个检测结果可以参考只有一个点的信号只有一个点的信号PM探针探针MM探针探针重复性高重复性高13,400Signal Intensities:13,090Signal Intensities:Signal Intensities:13,400Signal Intensities:
3、11,670GeneChipGeneChip 的操作流程的操作流程以真核生物为例以真核生物为例总总RNA的制备的制备反转录反转录体外体外转录转录生物素标记的生物素标记的cRNA片段化处理片段化处理带标记的带标记的cRNA片断片断35-200 bases0.5-2 ug/ul起始用量起始用量5-10ug（IVT）杂交混合液的制备杂交混合液的制备EukaryoticHyb.ControlControlOligo B2数据分析数据分析GeneChipGeneChip 绿脓杆菌绿脓杆菌枯草杆菌枯草杆菌人人果蝇果蝇大鼠大鼠小鼠小鼠拟南芥拟南芥酵母酵母大肠杆菌大肠杆菌表达谱芯片表达谱芯片 秀丽线虫秀丽线虫
4、斑马鱼斑马鱼爪蟾爪蟾金葡菌金葡菌大麦大麦目录产品研发中的产品蚊子和疟原虫蚊子和疟原虫狗狗牛牛大豆大豆葡萄葡萄 Agilent Gene Expression PlateformTotal Solutions for Gene ExpressionOptimized Experiment ProtocolTotal Solutions Provide quality tools for gene expression analysis:Microarray,Labeling,Scanner,Analysis Software.Open PlatformCan be used for existe
5、nt system.Or,can be used as a whole system.Sample CheckLabelingHybridization&WashScannerSpot AnalysisSoftwareData AnalysisData BasecDNA ArrayOligo Array Custom ArrayPin SpottingcDNA(PCR products)onto slide glassInkjet SpottingcDNA(PCR products)onto slide glassInkjet PrintingPin SpotterAgilent Techno
6、logiesAgilents DNA MicroarrayMicroarray(DNA Chip)Development EraAs of June 2003AffymetrixIn Situ oligo synthesisonto wafer using photolithography“Pat-Brown”cDNA(PCR products)onto 1x3 slide glassusing Pin-SpotterClosed SystemWafer1 x3 Slide GlassOpen SystemAffymetrixIn Situ oligo synthesisonto wafer
7、using photolithographyInkjet In Situ SynthesisOligo(60mer)onto slide glassPin SpottingOligoonto slide glassInkjet SpottingOligo(60mer)onto slide glassIn Situ Oligo SynthesisGlass SubstratePhosphoamidite ReactionIn Situ Microarray:Microarrays&FormatsIn situ Oligonucleotide-60 merThree density formats
8、:-8.4 k array-22.5k array-44k arrayTwo Color Labeling Differentially expressed genes from two different biological samples are assayed on a single array in the same hybridization reaction.Direct measurement of gene expression differences No variability introduced from differences between arrays or h
9、ybridization conditionsHigh Sensitivity in 60mer OligoAverage LLD*60mers:0.004pM25mers:0.032pM*LLD:Lower Limit of DetectionSpike-in ConcentrationBG Substracted SignalLog ratio values from two halves of dye swap experiment Log ratio values from two halves of dye swap experiment plotted against one an
10、otherplotted against one anotherAnticorrelated values=0.3%Anticorrelated values=0.3%Oligo Array Performance:System ReproducibilityData Comparison vs.Q-PCRCorrelation=0.91Validate 71 genes microarray results with Q-PCR.-37 genes expressed in placenta.-34 genes randomly selected from high-to-low Inten
11、sity&high-to-low LogRatio.M.Ko et.al,Genome Research,13,1011(2003)Feature Extraction Software Rapid Automated feature finding Intra-feature pixel statistics Inter-feature statistics Flexible dye normalization Statistical significance of signals and ratiosAutomated Analysis and Feature ExtractionAgil
12、ents Gene Expression Workflow Rosetta bioinformatics solutions both Resolver and Luminator for gene expression analysis Most high-throughput and sensitive Microarray scanner on the market Sure Hybe chamber for easy manipulation Superior Sensitivity of 60mer oligo microarray 50ng of total RNA for sen
13、sitive labeling solutions QA/QC of RNA Integrity Total Solution for Gene Expression30 min to run 12 RNA Samples 6 hr labeling rxn17 hr hybridization7 Min Scan1 Min for Feature Extraction 30 Min Frag RXN and Clean-up BeadArrayTM 96 arrays configured to standard format to enable high throughput Parall
14、el processing of 96 samples Up to 150,000 assays per array matrix One arrayan optical fiber bundle 1.5 mm in diameter Containing 50,000 individual optical fibers Beads on each bundlean optical fiber bundle Each bead is positioned to the end of an optical fiber Bead diameter 3 mm Bead center-to-cente
15、r distance 5 mmhighest feature density Bead surface is derivatized with DNA probesIllumina SentrixTM ArraysReadoutAGAddressAllele Specific ExtensionAllele Specific Extension AssayPCR with common primersP1P2P1P2Product captureby hybridizationto arrayP3P3/AddressAGA/AAGA/GAGG/GConfocal Scanning Produc
16、es Superior Image QualityImage of one BeadArrayTM bundleEnlarged vieweach bright spot is a bead with fluorescent signalBeadLabTMIntegrated Genotyping LaboratoryLab Automation&Lab Management基因芯片的实验设计Biological verification and interpretationMicroarray experimentExperimental designImage analysisNormal
17、izationBiological questionTestingEstimationDiscriminationAnalysisClustering实验周期Quality MeasurementFailedPassPreprocessingExperimental designProper experimental design is needed to ensure that questions of interest can be answered and that this can be done accurately,given experimental constraints,su
18、ch as cost of reagents and availability of mRNA.Data Analysis is Fundamentally Tied to Experimental Design Data analysis strategy should be determined as part of the design The experimental design,whether good or bad,is going to restrict how the data can be analyzed A planned data analysis strategy
19、provides a path forward for dealing efficiently and rationally with the dataATypes of SamplesTissueCellFFPE tissueLCM cellPooled vs individual samples.Pooled vs amplification samples.B Replication technical.biological.Taking physical limitations or cost into consideration:the number of slides.the am
20、ount of material.Some aspects of design Allocation of samples to the slides Three Sources of Variabilility Biological variation:Differences between individuals Differences between specimens from the same individual The ultimate goal of the research Technical variation:Sample preparation Extraction,l
21、abeling and hybridization System variation:Probe array analysis Arrays,instruments,reagentsExamples of Biological Variability Gender Sex-related expression patterns Cell cycle patterns What time of day were the samples isolated?Tissue Each cell type has different expression patterns Diet Eating habi
22、ts or media types will affect expression levelsControlling biological Variability Biological variability contributes more to overall experimental variation than technical variability To mitigate biological variability:Consider all potential variables as part of the experimental design Increase the n
23、umber of biological replicates until coefficient of variation(CV)stabilizesControlling Technical Variability Define processes and boundaries Protocol responsibilities Sample quality control Running gels,260/280 ratios Validation of technical personnel Reduce process variability Technician-to-technic
24、ian variability Calibration of instrumentation Control reagent variabilityExperimental Replicates Technical replicates from the same sample reproduce the contribution of the bench effects to the overall variability Biological replicates:“True”replicates that reproduce biological conditions explored
25、in the experimental design Permit the use of formal statistical tests Also allows interrogation of technical variabilityBiological replication is essential Technical replicates estimate measurement variability Biological replicates estimate both biological differences between cases and measurement v
26、ariability Technical replicates are NOT needed,exceptQuality-control studiesNumber of cases available is smallObtaining another case exceeds the cost of an arrayStatistical power and sample size-how many biological replicates Differential expression(not classification)Evidence indicates at least 5 b
27、iological cases per group Methods to get optimal number of replicatesStatistical Methods in Medical Research 2004;13(4):325338For classification study:Sample size determination in microarray experiments for class comparison and prognostic classification Biostatistics 2005 6:27-38Sample sizePooled vs
28、 Individual samples Pooling is seen as“biological averaging”.Trade off between Cost of performing a hybridization.Cost of the mRNA samples.Cost or mRNA samples 5 pool Measurements from pools do not necessarily correspond to mathematical averages of measurement from individuals comprising the poolSum
29、mary Statistical quantification is preferred over the qualitative description Use adequate biological and technical replication Make direct comparisons between samples Always keep the goals of the experiment in mind 蛋白质芯片是指在固相支持物上有序排列的各自独立的多肽、蛋白或相应配体，利用蛋白质与蛋白质、蛋白质与DNA、蛋白质与RNA，或与其它配体间的相互作用，同时平行分析大量蛋白
30、质的生物化学性质，在蛋白质组学基础研究以及临床诊断、药物研究、环境监测、食品卫生等方面有广阔的应用前景。蛋白质芯片原理示意图蛋白质芯片原理示意图Zhu et al.(2001)Science.293,2101Protein Array Assay ImagesControl SampleTreated SampleCapture ArraysSAMPLE ARRAYSReverse Phase ScreenANTIBODY ARRAYSSample Labelling ApproachesANTIBODY ARRAYSMutiplexed Sandwich ELISAshighly paral
31、lelhigh sensitivityexpensive equipmentlabour intenseautomation difficult 制备好的蛋白通过使用DNA芯片中成熟的机械针点样系统或是微量液体分配系统实现。也可以通过原位合成的方法制备。值得注意的是蛋白的粘稠度比较高，选择点样针的时候，需要针的孔径大一些。v高通量高通量v多组织标本多组织标本v同时、平行分析同时、平行分析 v同时研究一个或多个基因或同时研究一个或多个基因或蛋白的研究工具蛋白的研究工具v高质、高效，省材，操作便高质、高效，省材，操作便捷捷v应用广泛应用广泛 FISH FISH（荧光原位分子杂交）（荧光原位分子杂交
32、）mRNA ISHmRNA ISH（原位杂交）（原位杂交）IHCIHC（免疫组织化学）（免疫组织化学）成百甚至上千份成百甚至上千份 正常或处于疾病状态正常或处于疾病状态 疾病不同发展阶段疾病不同发展阶段基因、转录、表达水平三方面基因、转录、表达水平三方面概概 述述组织芯片的优点组织芯片的优点 v 高通量高通量短时（一次即获大量信息短时（一次即获大量信息 ）、高效（成百成千倍地提高效率）、高效（成百成千倍地提高效率 ）仅仅2CM2CM2 2 含数十甚至数百例组织样本，含数十甚至数百例组织样本，一次操作即可完成普通实验所一次操作即可完成普通实验所需的数十甚至数百次操作。需的数十甚至数百次操作。v
33、有助于减少实验误差有助于减少实验误差实验条件保持一致实验条件保持一致v 便于设置各种实验对照便于设置各种实验对照v 费用仅费用仅1/10-1/100 1/10-1/100 六个月六个月一年一年一周一周几万几万几十几十 万万一次一张玻片一次一张玻片上百个单位的珍贵试剂上百个单位的珍贵试剂一个单位的试剂一个单位的试剂几十次几十次上百张玻片上百张玻片只只需需1/101/100实验操作工作量研发费用组织芯片（意义）组织芯片（意义）对人类基因组学、后基因组学的深入研究与发展对人类基因组学、后基因组学的深入研究与发展特别表现在如下几个方面：特别表现在如下几个方面：v 研究特定基因及其所表达的蛋白质与疾病之
34、间的相互关系研究特定基因及其所表达的蛋白质与疾病之间的相互关系v 疾病的分子诊断疾病的分子诊断v 预后指标的确定预后指标的确定v 治疗靶点的定位治疗靶点的定位v 治疗过程的追踪与预测治疗过程的追踪与预测v 抗体和新药物的开发和筛选抗体和新药物的开发和筛选v 基因治疗的研发等方面基因治疗的研发等方面广 阔 的 应 用 前 景广 阔 的 应 用 前 景 高通量研究高通量研究DNA、RNA和和PROTEIN前列腺癌前列腺癌乳腺癌乳腺癌组织库组织库芯片设计芯片设计组织取样组织取样排列排列组织阵列组织阵列制片、染色制片、染色芯片诊断芯片诊断芯片查询管理芯片查询管理相相关关数数据据信信息息组组织织芯芯片片
35、库库组织选择组织选择组织阵列组织阵列组织芯片组织芯片芯片编码存储芯片编码存储outdooutdo组织芯片组织芯片脱蜡水化脱蜡水化抗原修复抗原修复内酶封闭内酶封闭位点封闭位点封闭加入一抗加入一抗加入二抗加入二抗DAB显色显色二甲苯、酒精二甲苯、酒精高压、微波、煮沸、酶消化高压、微波、煮沸、酶消化内源性过氧化物酶内源性过氧化物酶非特异性位点非特异性位点内源性生物素内源性生物素洗涤洗涤洗涤洗涤复染、脱水、透明、封片、阅片复染、脱水、透明、封片、阅片IHC组织芯片组织芯片脱蜡水化脱蜡水化微波处理微波处理胃酶消化胃酶消化加预杂交液加预杂交液进行杂交进行杂交封闭封闭显色显色二甲苯、酒精二甲苯、酒精非特异性
36、位点非特异性位点洗涤洗涤洗涤洗涤脱水、透明、封片、阅片脱水、透明、封片、阅片ISH组织芯片组织芯片脱蜡水化脱蜡水化蛋白酶蛋白酶K消化消化变性变性D标探针杂交标探针杂交FITC抗地高辛抗地高辛显色显色二甲苯、酒精二甲苯、酒精洗涤洗涤洗涤洗涤封片、阅片封片、阅片洗涤洗涤FISHIS-PCR流程图流程图组织芯片组织芯片脱蜡水化脱蜡水化蛋白酶蛋白酶K消化消化RNase处理处理原位扩增原位扩增原位杂交原位杂交NBT-BCIP显色显色二甲苯、酒精二甲苯、酒精洗涤洗涤洗涤洗涤脱水、透明、封片、阅片脱水、透明、封片、阅片Primer底物底物DNA聚合酶聚合酶碱磷酶标抗碱磷酶标抗D洗涤洗涤肝癌组织芯片（肝癌组织
37、芯片（OD-CT-DgLiv03-001）v高通量高通量478个样本个样本v高质量高质量最新鲜的标本材料和最敏感的生物学活性最新鲜的标本材料和最敏感的生物学活性v高价值高价值203例肝细胞癌例肝细胞癌 18例肝硬化例肝硬化 10例正常肝例正常肝 14例其他组织对照例其他组织对照Tissue Microaray of HCC（2001/10/03）1 12 23 34 45 56 67 78 89 91010111112121313141415151616171718181 1QD-01KQD-01NQD-02KQD-02N QD-03K QD-03N QD-04K QD-04N QD-05K
38、QD-05N QD-06KQD-06NQD-07K QD-07N QD-08K QD-08N QD-09K QD-09N2 2QD-10KQD-10NQD-11KQD-11N QD-12K QD-12N QD-13K QD-13N QD-14K QD-14N QD-15KQD-15NQD-16K QD-16N QD-17K QD-17N QD-18K QD-18N3 3QD-19KQD-19NQD-20KQD-20N QD-21K QD-21N QD-22K QD-22N QD-23K QD-23N QD-24KQD-24NZS-01KZS-01NZS-02KZS-02NZS-03KZS-03
39、N4 4ZS-04NZS-04NZS-05NZS-05NSY-01KSY-01NSY-02KSY-02NSY-03KSY-03NSY-04KSY-04NSY-05KSY-05NGX-01K GX-01N GX-02K GX-02N5 5GX-03KGX-03NGX-04KGX-04NGX-05K GX-05N GX-06K GX-06N GX-07K GX-07N GX-08KGX-08NGX-09K GX-09N GX-10K GX-10N GX-11K GX-11N6 6GX-12KGX-12NGX-13KGX-13NGX-14K GX-14N GX-15K GX-15N GX-16K G
40、X-16N GX-17KGX-17NGX-18K1 GX-18N1 GX-18K2 GX-18N2 GX-19K GX-19N7 7LIVER1LIVER2LIVER3LIVER4LIVER5LIVER6LIVER7LIVER8LIVER9中山（中山（M）中山（中山（H）军军HCC浙浙1号号K浙浙1号号NQ01-06K Q01-06N8 8WM-LBALB/c-LSCID-L-T 7402-1 7402-277217721 Hep3B-WHep3B-M Hep3B-PHEIHC肝癌组织芯片的应用肝癌组织芯片的应用p53、COX-2、AFP蛋白表达蛋白表达COX-2AFPp53人胰腺癌组织芯片人
41、胰腺癌组织芯片 高通量高通量289个样本个样本 高质量高质量最新鲜的标本材料和最敏感的生物学活性最新鲜的标本材料和最敏感的生物学活性 高价值高价值167 胰腺癌（胰腺导管腺癌胰腺癌（胰腺导管腺癌133例，粘液性囊腺癌例，粘液性囊腺癌16例，腺鳞癌例，腺鳞癌7例，其他例，其他11例）例）01 癌旁胰腺组织、癌旁胰腺组织、7例粘液性囊腺瘤、胰腺炎组织例粘液性囊腺瘤、胰腺炎组织4例例与与10正常胰腺正常胰腺 P21P33P14Shanghai Outdo BiotechCo.,Ltd.CT-Dig-Pan01-001Shanghai Outdo BiotechCo.,Ltd.CT-Dig-Pan01
42、-001胰腺癌组织芯片的应用胰腺癌组织芯片的应用P53ATMATM、p53、p21、p14、p33 AFP蛋白表达蛋白表达Next Generation SequencingDNA片段化文库构建测序反应数据分析最近最近RocheRoche宣布停止宣布停止454454测序业测序业务，务，20142014年停止仪器年停止仪器Roche 454 焦磷酸测序Pyrophosphate Sequencing基本原理454 sequencing：Emulsion PCR(emPCR)Mix DNA Library&capture beads(limited dilution)“Break micro-re
43、actors”Isolate DNA containing beads Generation of millions of clonally amplified templates on each bead No cloning and colony pickingCreate“Water-in-oil”emulsion+PCR Reagents+Emulsion Oil Perform emulsion PCR Adapter carrying library DNAABMicro-reactors Adapter complementEnrichAnneal SeqprimerCentri
44、fuge StepLoad Enzyme Beads454 sequencing：Deposition of DNA beads into the PicoTiterPlate44 m Load beads into PicoTiterPlate Illumina Solexa 合成测序Sequence by Synthesize基本原理目前使用的是Illumina Hi-Seq 2000/2500Clonal Single Molecule Arrays单分子克隆1000 molecules per 1 m cluster 1000 clusters per 100 m square40 m
45、illion clusters per experimentPrepare DNA fragmentsLigate adaptersAttach single molecules to surfaceAmplify to form clusters20 micronsSequenceReversible Terminator Chemistry可逆终止反应O PPPHNNOOcleavagesitefluor3blockNext cycleIncorporationDetectionDeblock;fluor removalODNAHNNOO3O5free 3 endXOHAll 4 labe
46、lled nucleotides in 1 reactionSequencing-by-Synthesis(SBS)5GTCAGTCAGTCAGT35CAGTCATCACCTAGCGTAFirst base incorporatedCycle 1:Add sequencing reagentsRemove unincorporated basesDetect signalCycle 2-n:Add sequencing reagents and repeat1、每轮测序反应加入四种带有荧光标记的dNTP，末端带有可以被去除的阻断基团2、每轮反应只能整合一个核苷酸，仪器读取相应的荧光信号3、信号
47、读取结束，用化学方法去除阻断基团，进行下一轮测序反应123789456T T T T T T T G T T G C T A C G A T The identity of each base of a cluster is read off from sequential images根据每个点每轮反应读取的荧光信号序列，转换成相根据每个点每轮反应读取的荧光信号序列，转换成相应的应的DNA序列序列Base calling from the raw dataABI SOLiD 连接法测序Sequence by Ligation基本原理文库制备：微珠单分子克隆1024种8碱基探针4色荧光，4种双
48、核苷酸，每色荧光有256个探针(46)SOLiD 利用探针的连接反应读取模板的利用探针的连接反应读取模板的DNA序列序列连接法测序（一）每个探针进行检测的两个碱基后面有三个匹配碱基，因此一条测序引物读取的序列是不完整的每个探针进行检测的两个碱基后面有三个匹配碱基，因此一条测序引物读取的序列是不完整的测序引物与测序引物与adapteradapter退火退火探针连接，检测荧光探针连接，检测荧光切除荧光基团切除荧光基团第二轮探针连接，检测荧光第二轮探针连接，检测荧光切除荧光基团切除荧光基团连接法测序（二）测序引物沿着测序引物沿着Adapter移动移动5次，确保每个位点都被检测次，确保每个位点都被检测
49、连接法测序（三）0位置是位置是Adapter的最后一个碱基，因此只检测一次，的最后一个碱基，因此只检测一次，该碱基是进行解码所必须的。该碱基是进行解码所必须的。三种测序平台的比较454 sequencing读取长度大，400bp可以对未知基因组进行从头测序de novo sequencing当遇到polymer时，如AAAAAA等，荧光强度和碱基个数不成线性关系，判定重复碱基个数有困难Solexa sequencing高度自动化的系统读取片段多，适合进行大量小片段的测序，如microRNA profiling基于可逆反应，随反应轮数增加，效率降低，信号衰减，读取序列较短，给de novo se
50、quencing 拼接带来困难SOLiD sequencing每个碱基读取两次非常高的准确性，特别是对于SNP的检测灵活的系统，完善的磁珠编码系统，可以进行样品的pooling，分割测序区域读取长度受连接反应的轮数限制，给de novo sequencing 拼接带来困难基因组/转录组 re-sequencing2022-7-23WGSS，基因组重测序WTSS，转录组重测序Nature October 2009 利用二代测序技术对同一个样品进行基因组和转录组的重测序工作，在人类乳腺小叶肿瘤中发现未报道过的碱基突变。81基因组/转录组 re-sequencing2022-7-23找到基因组和转录
