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MiSeq高通量测序在临床的应用介绍IlluminaCOMPANYCONFIDENTIAL–FORINTERNALUSEONLYLibraryPreparationDataAnalysisClusterGenerationSequencing•文库制

备：处理样品，加接头和样本特异性标签，为上机做准备•簇生成：信号放大的过程2•测序•数据处理Illumina新一代测序流程COMPANYCONFIDENTIAL–FORINTERNALUSEONLY文库制备---决定测序成功与否的关键步骤dualIndexLibraryshown文库制备的目

的是在需要测序的DNA片段两端加上能够与测序仪配合的接头序列（Multiplexed,SR,PE）①①②②③④3④双端标签文库①与流动槽（FlowCell）结合的区域②Read1和Read2测序引物结合的区域③插入片段④标签序列区域（Index）COMPANYCONFIDENTIAL–FO

RINTERNALUSEONLYWhatisaFlowCell?流动槽(FlowCell)是什么?Aflowcellisathickglassslidewithchannelsorlanes一种含有通道的厚玻璃片Clustergenerationoccursonaflowcell簇生

成在流动槽（flowcell）上完成Eachlaneisrandomlycoatedwithalawnofoligosthatarecomplementarytolibraryadapters每条通道中都随机植入了能与文库接头互补结合

的大量短DNA片段4COMPANYCONFIDENTIAL–FORINTERNALUSEONLY簇生成过程接头序列3’extension3’延伸流动槽表面随机植入了大量引物链片段杂交与延伸新合成链双链DN

A变性原始模板链Discard洗去丢弃单链DNA桥式PCR扩增双链DNA桥式结构变性桥式PCR扩增56COMPANYCONFIDENTIAL–FORINTERNALUSEONLYIllumina高通量测序原理——准确的

边合成边测序技术Add4Fl-NTP’s+Polymerase加入4种不同荧光标记的dNTP和DNA合成酶IncorporatedFI-NTPimaged对结合上的荧光dNTP进行照相Terminator&fluorescentdyecleavedfromFI-N

TP结合在DNA链上的荧光NTP中的荧光标记和阻断基团被切除，可以继续下一轮反应X36-301COMPANYCONFIDENTIAL–FORINTERNALUSEONLY100Microns7ClustersDNA簇COMPANYCONF

IDENTIAL–FORINTERNALUSEONLY123789456TTTTTTTGT…TGCTACGAT…Theidentityofeachbaseofaclusterisreadofffrom

sequentialimages根据每个点每轮反应读取的荧光信号序列，转换成相应的DNA序列8照相读取序列9COMPANYCONFIDENTIAL–FORINTERNALUSEONLYi5IndexSeqPrimer(SBS3rev/

BP13)i5IndexIndexRead2(i5)i7Index123PairedEndaroundTurn4123Paired-EndTurnaround4双index文库MiSeq,HiSeq2500,NovaSeqMiniSeq,NextSeq550,HiSeq3k/4

k/XtenDualindexedsequencingutilizes4sequencingreadsCOMPANYCONFIDENTIAL–FORINTERNALUSEONLYBaseSpace提供完整的数据存储和分析功能Step1:ObtainResults数据可以

实时上传Step3:Annotate/Filter注释及时更新Step2:Align/CallVariants分析即点即用BWAEnrichment•ProcessesWGSreadsusingBWAforalig

nment•UsesGATKforvariantdetectionEnrichment•PerformsreadmappingusingIsaac•Offers4xfasteralignmentwithsameaccuracyasBWA;DesignedbyIllumina•Enable

sextractionofbiologicalknowledgefromvariantdatabyprovidingarichcollectionofannotationdatabases,flexiblefiltering,andastreamlinedvariantclassif

icationandreportingtool10COMPANYCONFIDENTIAL–FORINTERNALUSEONLYIllumina主要测序平台介绍DecreasingpriceperGBIncreasingsyst

emoutputForResearchUseOnly.Notforuseindiagnosticprocedures.15Gb|25M2x300MiSeq7.5Gb|25M2x150MiniSeq120Gb|40

0M2x150NextSeq550167–6000Gb|1.6–20B2x150Mid,HighMicro,NanoV2,V3MidHighS1,S2,S4NovaSeq6000桌面式测序仪HiSeqX1300-1800Gb|5.3-6B2x150生产级测序仪1.2Gb|4M2x15011iS

eq12COMPANYCONFIDENTIAL–FORINTERNALUSEONLY.最新型台式测序平台–iSeq靶向测序ChoosefromfixedorcustompanelsAmpliconDNAandRNAavailable小基因组测序Sequencesmallgenom

eswithease辅助性应用Off-the-shelf,optimizedsolutionsIntuitiveworkflowsforfastimplementation2x150basepairs1.2gigabases4Mreads9

–17hourruntimeCOMPANYCONFIDENTIAL–FORINTERNALUSEONLYMiSeq的优点长度长supportapplicationbreadth多种测序试剂盒forflexibilityinsamplesandapplications

简单易用touseworkflowForResearchUseOnly.Notforuseindiagnosticprocedures.COMPANYCONFIDENTIAL–FORINTERNALUSEONLYMiSeqS

ystemPE300|75%>Q300.3–15|gigabases1–25million|clustersForResearchUseOnly.Notforuseindiagnosticprocedures.14COMPANYCONFIDENTIAL–FORINTERNALUSEONLY

MiSeq提供多种reads数的试剂盒支持不同应用MiSeqCoreConsumablesVersion3●600cycles●150cyclesCoreConsumablesVersion2Micro●300

cycles(Micro)1Millionreads4Millionreads15Millionreads25MillionreadsCoreConsumablesVersion2Nano●500cycles(Nano)●300cycles(Nano)CoreCons

umablesVersion2●500cycles●300cycles●150cyclesForResearchUseOnly.Notforuseindiagnosticprocedures.15COMPANYCONFIDENTIAL–FORINTERNALU

SEONLYChemistryVersionNumberofReads(mil)CyclesOutput(Gb)SequencingTime(hrs)32560015563251503.8212155008.5392153005.124215500.8

55.524(Micro)3001.22821(Nano)5000.51921(Nano)3000.319MiSeq提供多种reads数的试剂盒支持不同应用ForResearchUseOnly.Notforuseindiagnosticprocedures.16COMPANYCONFIDE

NTIAL–FORINTERNALUSEONLYTargetedResequencingIllumina-AmpliSeqPanelTruSightPanelsTruSeq/NexteraExomeTruSeq/Nexte

ra-xGenExomeTruSeqSyntheticLongRead*RNA&RegulationSamplePrepTruSeqRNATruSeqStrandedmRNATruSeqStrandedTotalRNATruSeqSmallRNATr

uSeqTargetedRNATruSeqChIPDNASamplePrepTruSeqPCR-FreeTruSeqNanoNexteraNexteraXTNexteraMatePairIllumina提供完整的试剂盒

制备方案17COMPANYCONFIDENTIAL–FORINTERNALUSEONLY18靶向测序微生物检测☆AmpliseqforIllumina☆TrusightPanel系列☆16srRNA测序☆微生物宏基因组测序COMPANYCONFIDENTIAL

–FORINTERNALUSEONLYAmpliseq简单的建库+Illumina自动化高质量测序集中支持●订购简单●故障处理及时●可以选择在线下单操作快速●1.5小时手动时间●5-7小时全部时间Panel设计&下单流程简单●使用AmpliSeqdesigne

r设计功能强大而且简单●需要选择的选项减少了，但是却可以达到更好的设计效果●为客户设计提供帮助ForResearchUseOnly.Notforuseindiagnosticprocedures.19COMPANYCONFIDENTI

AL–FORINTERNALUSEONLYAmpliSeq产品组合ForResearchUseOnly.Notforuseindiagnosticprocedures.8个现成产品30个团体产品3Cellular&MolecularResearch3个细

胞&分子生物学研究1ComplexDiseaseGenomics1个复杂疾病基因组2Immunology2个免疫相关2Infectiousdisease2个传染病相关1Neuroscience1个神经性疾病相关5Oncology5个肿瘤相关16RareandInh

eritedDisease16个罕见病&遗传病相关客户定制产品•DNA•Pre-loadedgenomes•2H-2018•Customgenomes•CustomRNAComprehensiveCanc

er泛癌种CancerHotspotv2癌症热点v2Focus肿瘤位点聚焦Comprehensivev3综合性肿瘤位点v3BRCA乳腺癌ImmuneResponse免疫应答TranscriptomeHumanGex人类转录组基因研究Exome外显子组选项20COMPA

NYCONFIDENTIAL–FORINTERNALUSEONLY8个现成产品30个团体产品*可以定制<96amplicons--~750rxns>96amplicons--~3000rxns客户定制产品*可以定制<96amplicons--~750rxns>96amplicons

--~3000rxns方便的小型包装>90%Coverageuniformity>90%平均覆盖率>90%Ontarget>90%靶向率产品详情*PerformancehasnotbeenvalidatedbyIlluminabutobservedperformanceisusuallyparwi

thready-to-usepanelsgiventypicaldesignandassayconditionsForResearchUseOnly.Notforuseindiagnosticprocedures.AmpliSeq产

品组合1.5小时手动时间215–7小时总时间2.5天从DNA到结果数据22COMPANYCONFIDENTIAL–FORINTERNALUSEONLY8个现成产品和30个团体产品Panel试剂盒Genes基因数Application应用In

put样本BRCA2BRCA1/2两个基因的外显子区域和侧翼内含子区，同时支持胚系和体细胞突变检测DNACancerHotspotv250癌基因中常发生突变的热点区域DNAFocusPanel52实体瘤相

关的突变热点，包括SNV,indels,CNVs和基因融合DNA+RNAComprehensivev3161实体瘤相关的突变热点，包括SNV,indels,CNVs和基因融合，适合泛癌种检测和临床试验DNA+RNAComprehens

iveCancer409覆盖409个癌基因的外显子区域，检测SNV和indelsDNAImmuneResponsePanel395与免疫反应相关的395个基因的表达RNATranscriptomeHumanGex20,800RefSeq分

析基因表达RNAExome57.7Mb人外显子组测序DNACommunity3个细胞&分子生物学研究；2个复杂疾病基因组；1个免疫相关；2个传染病相关；1个神经性基本相关；5个肿瘤相关；16个罕见病和遗传病相关DNA30个团体产品Ca

rdiacArrhythmiasandCardiomyopathyResearchPanel(心律不齐及心肌症研究试剂盒)(92genes,2pools,2,198amplicons）DeafnessResearch

Panelv2(耳聋研究试剂盒,v2)(126genes,2pools,3,520amplicons)AutismResearchPanel(孤独症研究试剂盒)(236genes,2pools,7,194amplicons)EndocrineResearchP

anelv2(内分泌疾病研究试剂盒,v2)(340genes,2pools,6,359amplicons)CardiovascularResearchPanel(心血管疾病研究试剂盒)(404genes,2pools,10,430amplicons)DermatologyRes

earchPanelv2(皮肤病研究试剂盒v2)(222genes,2pools,4,964amplicons)Dysmorphia-DysplasiaResearchPanelv2(畸形-发育不良研究试剂盒)(519genes,2pools,12,558ampli

cons)GastrointestinalResearchPanelv2(胃肠疾病研究试剂盒,v2)(194genes,2pools,4,103amplicons)HearingLossResearchPanelv1(听力缺失研究试剂盒v1)(63genes,2pools,

2,064amplicons)HematologyResearchPanel(血液疾病研究试剂盒)(394genes,2pools,7,434amplicons)InbornErrorsofMetabol

ismResearchPanelv2(先天性代谢异常研究试剂盒,v2)(594genes,2pools,9,681amplicons)NoonanResearchPanel(Noonan综合症研究试剂盒)(14genes,2pools,268amplic

ons)OphthalmicResearchPanel(眼科疾病研究试剂盒)(316genes,2pools)PrimaryImmuneDeficiencyResearchPanelv2(原发性免疫缺陷研究试剂盒v2)(264genes,2pools

,5,241amplicons)PulmonaryResearchPanelv2(肺部疾病研究试剂盒v2)(131genes,2pools,3,365amplicons)RenalResearchPanelv2(肾脏疾病研究试剂盒v2)(154genes,2pools,4,332ampl

icons)EpilepsyResearchPanel(癫痫研究试剂盒)(385genes,2pools,8,875amplicons)BRCAPlus,ExtendedHereditaryBreasta

ndOvarianResearchPanel(BRCA及其它基因相关的遗传性乳腺癌及卵巢癌研究试剂盒)(11genes,3pools,699amplicons)InheritedCancerResearchPanel遗传性癌症研

究试剂盒)(134genes,2pools,3,340amplicons)ColonandLungResearchPanelv2(结直肠癌和肺癌研究试剂盒,v2)(22genes,1tube,92amplicons)LongNon-CodingRNAResearchPanel

(癌症相关长链非编码RNA研究试剂盒)(RNA,表达水平)438非编码RNA,1pool,418ampliconsRNAFusionLungCancerResearchPanel(肺癌相关RNA融合研究试剂

盒)(RNA)(70genes,1pool,85amplicons)EbolaResearchPanel(埃博拉病毒研究试剂盒)(埃博拉病毒基因组,2pools,150amplicons)TBResearchPanel(结核分支杆菌耐药性研究)(8genes,1pool,109amplic

ons)NeurologicalResearchPanel(神经疾病研究试剂盒)(751genes,2pools,18,077amplicons）RNAInflammationResponseResearchPanel(免疫反应RNA表达水平研

究试剂盒)(RNA)(683genes,1pool,683amplicons)DementiaResearchGenePanel(痴呆基因研究试剂盒)(17genes,2pools,214amplicons)RNAMAPKPa

thwayResearchPanel(MAPK信号通路RNA表达水平研究试剂盒)(RNA)(197genes,1pool,197amplicons)RNAStemCellResearchPanel(干细胞RNA表达水平研究试剂盒)(RNA)930genes,1pool,930a

mpliconsRNAWNTSignalingPathwayResearchPanel(WNT信号通路RNA表达水平研究试剂盒)(RNA)(169genes,1pool,169amplicons)传染病相关C神OM经PA性NY疾CO病N相FID关EN

TIAL–FORINTERNALUSEONLY罕见病和遗传病相关肿瘤相关免疫相关复杂疾病基因组研究相关细胞分子生物学研究23COMPANYCONFIDENTIAL–FORINTERNALUSEONLY仪器产出数据量MiniSeqMiSeqiSeq100●FocusPanel(8s

amples)●BRCASomatic(12samples)●CancerHotspot(16samples)●BRCAGermline(96samples)24NextSeq●ImmuneResponse(24sa

mples)●FocusPanel(48samples)●BRCASomatic(80samples)●CancerHotspot(96samples)●BRCAGermline(96samples)●Exome(8samples)●Comprehensi

veCancer(12samples)●Transcriptome(40samples)●Comprehensivev3(48samples)#扩增子数1.SamplesperrunvaluesassumerecommendedcoverageAmpliseq简单的建库+

Illumina自动化高质量测序COMPANYCONFIDENTIAL–FORINTERNALUSEONLY轻松进行客户定制——DesignStudioFromDropdown,Select:CustomAssa

yDesignwithDesignStudioFromDropdown,Select:DesignStudioForResearchUseOnly.Notforuseindiagnosticprocedures.25

COMPANYCONFIDENTIAL–FORINTERNALUSEONLYDesignStudio定制主页ForResearchUseOnly.Notforuseindiagnosticprocedures.26COMPANYCONFI

DENTIAL–FORINTERNALUSEONLY选择适合用户的定制方式AmpliSeqGeneTargetsinclude:1Kb-5MbofContentGenes,Exons,and/orRegionCoordinatesA

mpliSeqHotspotTargetsinclude:1Kb–5MbofContentHotspotRegionsandSNPsNexteraRapidCaptureTargetsinclude:27>5MbofContentLargeGenePanelsand/

orExtendedExomeContentCOMPANYCONFIDENTIAL–FORINTERNALUSEONLYRNA-PanCancerInheritedDiseaseHLACancerCardioTruSight试剂盒组表现优秀的靶向测

序试剂盒MyeloidTumor28COMPANYCONFIDENTIAL–FORINTERNALUSEONLY临床全外TrusightOne/TrusightOneExpanded全面解读基因与疾病的关系NumberofGenes4,813Tar

getRegionSize12MbTargetedExons~62,000>20xMinimumCoverage目标基因捕获NumberofGenes6700+TargetRegionSize16.5MbTargetedEx

ons~86,000>20xMinimumCoverage目标基因捕获29COMPANYCONFIDENTIAL–FORINTERNALUSEONLY微生物学四大主流应用Sequencetheentiregenomeandachieveaccuratedenovoassemblyusingc

ulturesasstartingmaterial.Generateaccuratereferencegenomes,formicrobialidentification30Exploreallgenesinallo

rganismspresentinmanycomplexenvironmentalormicrobialsamples,evaluatebacterialdiversityanddetectmicrobeabundance.Identi

fyandcomparebacteriapresentwithinagivensample.ClarifyphylogeneticrelationshipsbetweenbacteriaDetectionof

low-frequencyviralparticleswithinnon-culturedsamples.Identify,characterizeandquantifyDNAandRNAviraltranscriptsofinfectiousa

gents.全基因组测序鸟枪法宏基因组测序16SrRNA测序病毒学COMPANYCONFIDENTIAL–FORINTERNALUSEONLYMiSeq®DNAWorkflowPrepSequenceAnalyzeShareBa

seSpace®Nextera®XTNextera®DNAFlexMiSeq®MSRorBaseSpace®15minuteshands-on20minuteshands-onFullyautomatedSec

ureandstoreRapidlibrarypreparation●Completelibraryprepinaslittleas90minuteswithonly15minutesofhands-ontimeOptimizedforsmallgenomes,PCRamplicon

sandplasmids●OnelibraryprepkitformanyapplicationsUltralowinput●OnlyasinglenanogramofinputDNAneededInnovativesamplenormalization●Elim

inatestheneedforlibraryquantificationbeforesamplepoolingandsequencingForResearchUseOnly.Notforuseindiagnosticprocedures.31C

OMPANYCONFIDENTIAL–FORINTERNALUSEONLYOrderampliconprimersPCRamplifyV3andV4regionsof16SrRNAgeneAdddual-indexbarcodesbyPCRLibraryQCandNormalizationLib

raryPooling3216S宏基因组测序流程Illumina提供标准化流程COMPANYCONFIDENTIAL–FORINTERNALUSEONLY人类健康遗传病感染性疾病科学研究生殖健康法医学健康筛查肿瘤生物制药农业illumina高通量检测覆盖各个领域33

COMPANYCONFIDENTIAL–FORINTERNALUSEONLYThankyouQuestions?34