5-MiSeq-Sample-Prep-Overview-Customer-v2

©2013Illumina,Inc.Allrightsreserved.Illumina,IlluminaDx,BaseSpace,BeadArray,BeadXpress,cBot,CSPro,DASL,DesignStudio,Eco,GAIIx,GeneticEnergy,GenomeAnalyzer,GenomeStudio,GoldenGate,HiScan,HiSeq,Infinium,iSelect,MiSeq,Nextera,NuPCR,SeqMonitor,Solexa,TruSeq,TruSight,VeraCode,thepumpkinorangecolor,andtheGeneticEnergystreamingbasesdesignaretrademarksorregisteredtrademarksofIllumina,Inc.Allotherbrandsandnamescontainedhereinarethepropertyoftheirrespectiveowners.MiSeqSamplePrepOverview2FORRESEARCHUSEONLYBytheendofthistraining,youwillbeableto:ListthelibraryconstructionmethodsavailablefortheMiSeqSystemUnderstandtheMiSeqapplicationsthateachsampleprepkitworksbestwithObjectives3FORRESEARCHUSEONLYMiSeqSequencingWorkflowDataAnalysisSequencingClusterGenerationLibraryPreparation4FORRESEARCHUSEONLYLibrarySamplePrepOverviewTheaimofthesampleprepstepistoobtainnucleicacidfragmentswithadaptersattachedonbothendsNucleicAcidModifytoproperinsertsizeAddadapterswithsitesfor:FlowcellbindingandSequencingprimerbindingSamegeneraltemplatearchitectureregardlessofapplication5FORRESEARCHUSEONLYIlluminaSamplePrepSolutions•TruSeqNanoDNA•TruSeqChIP-Seq•TruSeqEnrichment•TruSeqAmpliconTruSeqDNASamplePrepSolutions•TruSeqRNA•TruSeqSmallRNATruSeqRNASamplePrepSolutions•NexteraDNA•NexteraXT•NexteraEnrichmentNexteraSamplePrepSolutions•TruSightEnrichment•TruSightTumorTruSightSamplePrepSolutions6FORRESEARCHUSEONLYTruSeqNanoDNA•Smallgenomeassembly&re-sequencing•IdealifuniformcoverageiscriticalTruSeqRNA•GeneexpressionanalysisforsmalltranscriptomesTruSeqsmallRNA•miRNAexpressionanalysisTruSeqAmplicon•High-throughput,Highly-targetedre-sequencing•High-throughputsomaticmutationre-sequencingTruSeq/NexteraEnrichment•Megabase-sizedregiontargetedre-sequencing•TruSightcontentre-sequencingNextera•Smallgenomeassembly&re-sequencing•PCRampliconresequencing•IdealifspeedandsimplicityisdesiredMiSeqApplicationsSelectingtheRightKitTargetedRNAComingSoon!7FORRESEARCHUSEONLYNotsurewhichkittouse?Usethe“IlluminaSamplePrepKitSelectorTool”½dayworkflow-Gel-free-PCR-free-RemovesPCRbiasTruSeqChIP-Seq-Only5ngChIP-DNAinputrequired-FollowssimilarworkflowasTruSeqNanoDNATruSeqDNASamplePrepKits9FORRESEARCHUSEONLYTruSeqDNAWorkflowDNARNA(ExceptforTruSeqPCR-free)10FORRESEARCHUSEONLYNexteraDNANexteraDNA-½dayworkflow-50nginput-Idealforwholegenomere-sequencingNexteraXT-½dayhigh-throughputworkflow-1nginput-IdealforsmallgenomeandPCRampliconre-sequencingNexteraMate-Pair-1-4uginput-2-15kbgapsize-Idealforsmallgenomede-novoassembly-Automatedmate-pairanalysis(Comingsoon!)NexteraDNASamplePrepKits11FORRESEARCHUSEONLYStep1:TagmentationoftemplateDNATransposonsGenomicDNA~300bpTagmentationNexteraDNA,NexteraXTWorkflow12FORRESEARCHUSEONLYStep2:PCRtoaddadaptersandindicesTagmentationp7Index1Read2SequencingPrimerp5Index2Read1SequencingPrimerReduced-CyclePCRAmplificationNexteraDNA,NexteraXTWorkflow13FORRESEARCHUSEONLYStep3:CleanupandSequencep7Index1Rd2SPRd1SPp5Index2Sequencing-ReadyFragmentp7Index1Read2SequencingPrimerp5Index2Read1SequencingPrimerReduced-CyclePCRAmplificationNexteraDNA,NexteraXTWorkflow14FORRESEARCHUSEONLYNexteraMatePairTagmentEnzymesimultaneouslyfragments&tagsdsDNAFragmentsarecircularizedandre-fragmented,generatingsmallerfragmentsforclusteringSub-fragmentsareenrichedviaabiotinpull-downSequencingadaptersareligatedtotheenrichedset,enablingamplificationandsequencingontheflowcellNexteraMate-PairWorkflow15FORRESEARCHUSEONLYTruSeqRNATruSeqRNAv2mRNA-0.1ugtotalRNAinput-polyA-mRNAselection-Non-strandedinformationTruSeqStrandedRNAStrandedmRNA-0.1ugtotalRNAinput-polyA-mRNAselection-Strandedinformation-Idealfordetectionofanti-sensemRNAStrandedTotalRNA-0.1ugtotalRNAinput-RibosomalRNAdepletion-Strandedinformation-IdealfordetectionofmostRNAspeciesTruSeqSmallRNASmallRNA-1ugtotalRNAinput-1dayworkflow-SmallRNAselection(miRNA,piRNA,snoRNA)TruSeqRNA16FORRESEARCHUSEONLYTruSeqRNAv2,TruSeqStrandedmRNAWorkflowRNA17FORRESEARCHUSEONLYTruSeqStrandedTotalRNAWorkflowRNArRNAprobeRibosomalRNArRNAdepletion,fragmentationandrandomprimingrRNA-depletedRNA18FORRESEARCHUSEONLYTruSeqSmallRNAWorkflow19FORRESEARCHUSEONLYTruSeqAmpliconTruSeqCustomAmplicon(TSCA)-1dayworkflow-Sequenceupto1536customamplicontargets-Highspecificitytargetedassay-Sequencekilobase-sizedregionsusingoverlappingampliconsTruSeqAmpliconCancerPanel(TSACP)-1dayworkflow-212ampliconscovering48genes(35kbcovered)-Detectssomaticmutationsingenomichotspotsassociatedwithtumordevelopment-CompatiblewithFFPEsamplesTruSightTumor-175ampliconscovering26genes(21kbcovered)-Detectssomaticmutationsinwholeexonsassociatedwithtumordevelopment-DesignedspecificallyforFFPEsamplesTruSeqAmpliconSamplePrepKits20FORRESEARCHUSEONLYTruSeqAmpliconWorkflow5ulofDNAat50ng/ulrecommendedforTSCAandTSACP10ulofDNAdilutedaccordingtoFFPEQCqPCRresultsforTruSightTumor(30-300ng)gDNARegionofInterestGenomicDNA21FO