(12.7.2.1)-2010 基因芯片司法鉴定的奥秘

MicroarrayandBioinformatics

基因芯片的生物信息学

AimsfortheMicroarrayBioinformaticsUnderstandbasicmicroarraytechnologyanditsuseingeneexpressionanalysis.基因芯片技术与表达谱分析中的应用Learnbasicdataanalysismethodsandhowtoapplythemintheanalysisofgeneexpressiondata基因芯片的数据分析Dataacquisition数据获得Datanormalization数据归一化Dataanalysis数据分析DataClustering数据聚类Vocabulary-Review回顾Gene基因:

hereditaryDNAsequenceataspecificlocationonchromosome.Genetics遗传学:

studyofheredity&variationinorganisms.Genome基因组:anorgan'stotalcontent(fullDNAsequence)Genomics基因组学:studyoforganismsintermsoftheirgenome.2002年2月12日,历时10载耗资20亿美元的人类基因组计划最终完成,并报道了99%的人类基因组序列.Vocabulary-Review回顾Protein蛋白质

:sequenceofaminoacidsthat“doessomething”Proteomics蛋白质组学

:

studyofalloftheproteinsthatcancomefromanorganismsgenomeBioinformatics生物信息学

:thecollection,organization&analysisoflarge-scale,complexbiologicaldata.FunctionalGenomics功能基因组学:studyofobtaininganoverallpictureofgenomefunctions,includingtheexpressionprofilesatthemRNAlevelandtheproteinlevelMicroarrayTechnology基因芯片技术BasicIdeaofMicroarrays

芯片基本原理TypesofMicroarraytechnologiesandhowtheywork芯片技术种类与原理OutputsofMicroarrays

芯片的制备ImageAnalysisrequiredtotransformoutputtogeneexpressionmatrices芯片数据采集与分析Microarray

基因芯片–AhighthroughputtechnologythatallowsdetectionofthousandsofgenesSimultaneously是指通过微阵列技术将高密度DNA片段阵列通过高速机器人或原位合成方式以一定的顺序或排列方式使其附着在如玻璃片等固相表面，以荧光标记的DNA探针，借助碱基互补杂交原理，进行大量的基因表达及监测等方面研究的最新革命性技术。–genechip,biochip,array–Muchrelyoncomputeraids–CentralplatformforfunctionalgenomicsHistory历史HGP(humangenomeproject):suggestedbyDelbeccoonMar.7,1986，startedinOct.1990,rapidandsensitivetechniquesforhumangenomeinformationanalysis80S:

suggestionbasedoncomputerchip,WBrainstrieditfirstly.90S:

StephenFodor(PresentofAffymetrixnow)madeitsuccessfully.1995:QuantitativemonitoringofgeneexpressionpatternswithacomplementaryDNAmicroarray

Endof1996:

thefirstDNAchip1998年底美国科学促进会将基因芯片技术列为1998年度自然科学领域十大进展之一

1997年《财富》杂志：在20世纪科技史上有两件事影响深远：一是微电子芯片，它是计算机和许多家电的‘心脏’，改变了我们的经济和文化生活，并已进入每一个家庭；另一就是生物芯片，它将改变生命科学的研究方式，革新医学诊断和治疗，极大地提高人口素质和健康水平。”History历史MicroarraysarePopular芯片技术的普及AtNYUMedCenternowcollectingabout3GBofmicroarraydataperweek(60chips,6-10differentexperiments)PubMedsearch“microarray”=24,431papers（2008）38,467(2010)MicroarraysarePopular芯片技术的普及NSFCHistory芯片中国——博奥生物博奥生物有限公司暨生物芯片北京国家工程研究中心成立于2000年9月30日，现已迅速发展成为国际领先的生物高技术公司2002年，博奥生物作为亚洲唯一入选的公司，被美国《财富》杂志评为2002年度“全球最有发展前景的生物技术公司”；

2005年，博奥生物成为美国RedHerring杂志评选的“亚洲非上市技术100强公司”中的八家生物技术公司之一

2007年，博奥生物申报的“系统化生物芯片和相关仪器设备的研制及应用项目”荣获国家技术发明奖二等奖。

2008年，全国生物芯片标准化技术委员会经国务院标准化主管部门批准成立，生物芯片北京国家工程研究中心作为秘书处单位负责日常管理，中心主任程京博士任标委会主任委员。

2009年，程京博士当选工程院院士2009年12月28日，博奥生物研发生产和服务基地正式签约入驻位于四川温江的成都国际医学城，同一天，成都博奥独立医学实验室也正式开始运行

芯片中国——中国生物芯片中心世界上第一张遗传性耳聋基因检测芯片；世界上第一张17种分枝杆菌菌种鉴定芯片；世界上第一张结核耐药检测芯片；世界上第一张乙肝耐药基因检测芯片；世界上第一张SARS病毒检测芯片；世界上第一张HLA分型检测芯片；世界上第一张转录因子活性谱检测芯片；世界上第一张细胞电旋转检测芯片；世界上第一张家蚕全基因组表达谱芯片；2007年开始局部赢利，2009年销售过亿并全面赢利，销售收入平均每年增长75%。预计公司2015年销售收入将达到30亿元以上。863计划与973计划863计划标志863计划即国家高技术研究发展计划，是中华人民共和国的一项高技术发展计划。这个计划是以政府为主导，以一些有限的领域为研究目标的一个基础研究的国家性计划。中国根据本身的经济实力，以“有限目标，突出重点”为方针，主要的科学研究集中在生物技术、航天技术、信息技术、激光技术、自动化技术、能源技术、新材料领域以及1996年新加的海洋高技术。1997年由国家科技领导小组第三次会议决定实施的一项计划。实施“973计划”的战略目标是加强原始性创新，在更深的层面和更广泛的领域解决国家经济与社会发展中的重大科学问题，以提高我国自主创新能力和解决重大问题的能力，为国家未来发展提供科学支撑。战略目标:加强原始性创新，在更深的层面和更广泛的领域解决国家经济与社会发展中的重大科学问题，以提高我国自主创新能力和解决重大问题的能力，为国家未来发展提供科学支撑。Features特点–Parallelism高平行

•

Thousandsofgenessimultaneously–Miniaturization小型化

•

Smallchipsize–Multiplexing高通量

•

Multiplesamplesatthesametime–Automation自动化

•

Chipmanufacturing

•

ReagentsWhatproblemscanitsolve?

基因芯片的应用–Differingexpressionofgenesovertime,betweentissues,anddiseasestates

基因表达差异–Identificationofcomplexgeneticdiseases

复杂性基因疾病的诊断–Drugdiscoveryandtoxicologystudies

药理与毒理学研究–Mutation/polymorphismdetection(SNP’s)

SNP检测–Pathogenanalysis

诊断病原DifferentialGeneExpression

基因表达差异AFewExamples:Celltypespecific

-e.g.skincellvs.braincell

Developmentalstage

-e.g.embryonicskincellvs.adultskincellDiseasestate

-e.g.normalskincellvs.skintumorcellEnvironment-specific -e.g.skincelluntreatedvs.treated drugs,toxinsWhatisitspitfall缺陷与不足?–DetecttranscriptionmRNAlevel,nottranslationproteinlevel–Manyfactors(variations)canaffecttheresult：影响因素众多

•Chipandprobedesign

•Experimentdesign

•Samplepreparation

•Imageacquisition

•Datanormalization

•Dataanalysis

•

….–Successcrucial成功关键:

•Youknowboththebiologyproblemandthecomputeraids(software,statistics).RequrimentsArrayspotter点样仪Arrayscanner扫描仪Chemistrysystems杂交体系Softwares

软件Marketpredict市场预期

At1999：1billionUSDLessthan5yrs:20billions2005：5billions(USA)2010：40billions(USA)Don’tincludediseasediagnosticThelargestindustryinsteadofmicroelectricsPrinciple原理•

SimilartoNorthern–Base-Pairing,hybridizationbetweennucleiccids

•

MajordifferencesfromNorthern–Detectsthousandsofgenessimultaneously/individual–Probesfixationonglassslide/nylonmembrane–Targetsampleslabelingwithfluorescent/radioactivedNTPTypesofMicroarrays

基因芯片的种类

1.cDNAchip(DNAmicroarray,two-channelarray)cDNA芯片

:–ProbecDNA(500~5,000baseslong)isimmobilizedtoasolidsurfacesuchasglass–Usingrobotspotting–TraditionallycalledDNAmicroarray–FirstlydevelopedatStanfordUniversity2.Genechip(DNAchip,Affymetrixchip)基因芯片:–Oligonucleotide(20~80-meroligos)issynthesizedeitherinsitu(on-chip)orbyconventionalsynthesisfollowedbyon-chipimmobilization–HistoricallycalledDNAchips–DevelopedatAffymetrix,Inc.,undertheGeneChip®trademark–ManycompaniesaremanufacturingoligonucleotidebasedchipsusingalternativetechnologiesSpottingProcess点样过程

Spotrobot点样仪Cheungetal.1999点样针Affymetrix

基因芯片表达差异检测ComparisonofProbeTypes

两种探针比较

AdvantagesNoneedtoisolateandpurifycDNAsbecauseoligonucleotidescanbesynthesized.Shortoligonucleotidesarelesslikelytohavecross-reactivitywithothersequencesinthetargetDNA.DensityofchipsishigherthanwithcDNAs.LimitationsThesequencehastobeknown.Synthesiscanbeexpensiveandtime-consuming.TheshortsequencesarenotasspecificfortargetDNA,soappropriatecontrolsmustbeadded.In-situSynthesis/OligosPCRProducts/cDNAProbesAdvantagesFlexibilitytostudycDNAsfromanysource.cDNAsdonotrequireanyaprioriinformationaboutthecorrespondinggenes.Longersequencesincreasehybridizationspecificity,whichreducesfalsepositives.LimitationsIsolationofindividualcDNAstoimmobilizeoneachspotcanbecumbersome.Densityislowerthansynthesizingoligonucleotidesonthesurfaceofthechip.cDNAsarelongersequencesandaremorelikelytorandomlycontainsequencesfoundintargetDNA,whichresultsincross-reactivity.Manyothervariationsofthetechnologyexist,suchastheuseoflongeroligos,theuseoffibreoptics,etc.HomemadeTailoredCheaper?????Maximum24,000featuresperarrayPronetovariabilityCommerciallyavailable“Offtherack”Moreexpensive?????Maximum500,000featuresperarrayLessvariabilitySpottedArraysAffymetrixArraysProcessofmanufactureamicroarray

芯片制备流程Startwithindividualgenes,e.g.the~6,200genesofthegenomeorY.pestisAmplifyallofthemusingpolymerasechainreaction(PCR)“Spot”themonamedium,e.g.anordinaryglassmicroscopeslideEachspotisabout100µmindiameterSpottingisdonebyarobotComplexandpotentiallyexpensivetaskB21B22B23B24B25B26B27B28B29B30B31B32B17B18B19B20B5B6B7B8B9B10B11B12B13B14B15B16B1B2B3B44×8矩阵17×17点阵一共8448个点；4005条鼠疫菌基因+若干对照DNA；每样品相邻重复两个点。基因选择4015条芯片点样基因的PCR扩增产物纯化和浓缩4005条基因全基因组芯片研制引物设计MicroarraySteps基因芯片分析过程•

ExperimentandDataAcquisition实验过程与数据获得–Chipmanufacturing芯片制备–Samplingandlabeling点样–Hybridization杂交–Imagescaling图像扫描–Dataacquisition数据获得

•

Datanormalization数据归一化

•

Dataanalysis数据分析

•

Biologicalinterpretation生物学解释Readinganarray(cont.)BlockColumnRowGeneNameRedGreenRed:GreenRatio111tub12,3452,4670.95112tub23,5892,1581.66113sec14,1091,4692.80114sec21,5003,5890.42115sec31,2461,2580.99116act11,9372,1040.92117act22,5611,5621.64118fus12,9623,0120.98119idp23,5851,2092.971110idp12,7961,0052.781111idh12,1704,2450.511112idh21,8962,9960.631113erd11,0233,3540.311114erd21,6982,8960.59ColorCoding

扫描结果TablesaredifficulttoreadDataispresentedwithacolorscaleCodingscheme:Green=repressed(lessmRNA)geneinexperimentRed=induced(moremRNA)geneinexperimentBlack=nochange(1:1ratio)OrGreen=controlcondition(e.g.aerobic)Red=experimentalcondition(e.g.anaerobic)WeonlyuseratioMicroarrayDataAnalysis

芯片数据分析

•Imageacquisition图像获得

•Datanormalization数据归一化

•Dataanalysis数据分析-Clusteranalysis

聚类分析Noise干扰Noisesources干扰来源:Samplepreparation,labeling,amplificationReactionvariationsEnvironmentTargetvolumeHybridizationparameters(temperature,time,...)AspecifichybridizationDustScannersettingsQuantizationOtherImageProcessingProblems

SpotQualityProblemsUnevengridpositionsCurveswithinagridVariableSpotsizeorshapeVariableDistancebetweenspotsTypicalProblemsofRawOutputTwoslidesP04vs.P01(pg2)A1vs.P01(pg2)Noisefiltering干扰过滤Gridding:identifyspotlocationsSegmentation:distinguishforegroundfrombackgroundFixedCircle:putacirclearoundtheforegroundareaSeededregiongrowing:identifyinitialspot“seeds”andgrowhighintensityregionsEdgedetectionalgorithmsBackgroundcancellationIntensity=FGintensity-BGintensityNoisefiltering干扰过滤Normalization

归一化Thewordnormalizationdescribestechniquesusedtosuitablytransformthedatabeforetheyareanalysed.Goalistocorrectforsystematicdifferencesbetweensamplesonthesameslide,orbetweenslides, whichdonotrepresenttruebiologicalvariationbetweensamples.Normalization归一化NoralizedatatocorrectforartificialvariancesRed=FGred-BGredGreen=FGgreen–BGgreenPixelValue=log2(Red/Green)-log2(Redavg/Greenavg)Pixelcolor:Green ifpixelvalue<0Yellow ifpixelvalue=0Red

ifpixelvalue>0Normalization归一化Calibrated,redandgreenequallydetectedUncalibrated,redlightunderdetectedTheoriginofsystematicdifferences

系统误差的产生原因Systematicdifferencesdueto…Dyebiases,whichvarywithspotintensity,Locationonthearray,Plateorigin,PrintingqualitywhichmayvarybetweenPinsTimeofprintingScanningparameters,…Normalization

methods&issues

归一化方法MethodsGlobaladjustmentIntensity

dependent

normalizationWithin

print-tip

group

normalizationAnd

many

other…Selection

of

spots

for

normalizationDNAarrayDataAcquisition

DNA芯片数据的获得•ImageAnalysissoftwarepackagesexistfortheanalysisoftheoutputofcustommadechips(e.g.GenePixPro,ArrayVision,TIGRSpotFinder,etc)•Needchipdescriptionfile(CDF)–ForprobelocationIntroductionofSoftware----SAM

SAM软件介绍SignificanceAnalysisofMicroarraysTusher,TibshiraniandChu(2001):Significanceanalysisofmicroarraysappliedtotheionizingradiationresponse.PNAS200198:5116-5121,(Apr24).ExcelpluginFreeMostpublishedmethodofmicroarraydataanalysisHandlingMissingData

丢失数据的操作TherearecurrentlytwooptionsforimputingmissingvaluesinSAM.RowAverageEachvalueisimputedwiththeaverageofnon-missingvaluesforthatgene.K-NearestNeighborIntheother(default)option-missingvaluesareimputedusingak-nearestneighboraverageingenespace(defaultk=10):Clustering聚类软件Hypothesis:

GeneswithsimilarfunctionhavesimilarexpressionprofilesFindgroupofgeneswithsimilarexpressionprofilesFindgroupdofindividualswithsimilarexpressionprofileswithinapopulationClustering

=GroupidentificationBasicissuesinclustering

聚类的基本原则Issues

todecidebefore

clusteringWhichgenes/arrays

touse?Which

similarity/dissimilarity

measure?Which

clustering

algorithm?It’sanexploratorytechniqueThere’snooptimalsolutionAny

method

will

yield

groupsWhich

method

gives

good

groups?ClusteringSteps

聚类分析步骤Chooseasimilaritymetrictocomparethetranscriptionalresponseortheexpressionprofiles:PearsonCorrelationSpearmanCorrelationEuclideanDistance…Chooseaclusteringalgorithm:HierarchicalK-means…Clusteralgorithm

聚类算法 -UnsupervisedAnalysis非监督算法

-HierarchicalK-meanSelf-organizingmapsOthers-SupervisedAnalysis:classificationrules

监督算法系统聚类法步骤

1、将n个样品各作为一类;

2、计算n个样品两两之间的距离，构成距离矩阵;

3、合并距离最近的两类为一新类;

4、计算新类与当前各类的距离。再合并、计算，直至只有一类为止;

5、画聚类树形图，确定距离切点、类组，解释。

在SPSS软件中的操作步骤：

Analyze-----Classify-----HierarchicalHierarchicalClustering

分层聚类法g1g2g3g4g5g10.230.000.95-0.63g20.910.560.56g30.320.77g4-0.36g5g1g4g1g2g3g4g5g10.230.000.95-0.63g20.910.560.56g30.320.77g4-0.36g5Findlargestvalueissimilaritymatrix.Joinclusterstogether.

Recomputematrixanditerate.HierarchicalClustering

分层聚类g1,g4g2g3g5g1,g40.370.16-0.52g20.910.56g30.77g5g1g4g2g3g1,g4g2g3g5g1,g40.370.16-0.52g20.910.56g30.77g5Findlargestvalueissimilaritymatrix.Joinclusterstogether.

Recomputematrixanditerate.HierarchicalClustering

分层聚类g1,g4g2,g3g5g1,g40.27-0.52g2,g30.68g5g1g4g2g3g5g1,g4g2,g3g5g1,g40.27-0.52g2,g30.68g5Findlargestvalueissimilaritymatrix.Joinclusterstogether.

Recomputesimilaritymatrixanditerate.InterpretingtheResults