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Chromium™SingleCellV(D)JSolution
RevolutionizingAppliedImmunologyWithImmuneRepertoireProfilingatSingleCellResolution
May,2017
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•TheV(D)JSolutionwillRevolutionizeBiology:–BasicandTranslationalResearch– HumanHealthandDisease– Immunotherapy–VaccineDevelopment
•TheV(D)JSolutionsupportscriticalimmunerepertoireprofilingapplications:–Assessthediversity/clonality oftheimmunerepertoire–TrackspecificT-cellclonesovertimeand/orbetweensamples– Identifyimmunereceptors(antigenspecificity)sharedbetweenpeople
•Full-lengthpairedsingle-cellsequencingofTCRα/βtranscriptsiscriticalfor:–understandingthetrue biologicaldiversityofT-cell(antigen)receptors–elucidatingthemoleculargeneticdeterminantsofantigenspecificity
TheOpportunityforV(D)J
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T-CellsCarryOutCellularImmuneResponses
Tcellsattackingatumorcell
http://www.diamond.ac.uk/Home/News/LatestFeatures/02_09_15.html
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T-CellsareSelectedforSpecificityofResponse
• PositiveselectionforMHC(HLAtype)
• NegativeselectionforfailingMHCrecognition
• Negativeselectionforspecificityagainstself
• ThisprocessresultsinaT-cellrepertoireselectedforspecificityfor“non-self”antigenspresentedonMHC.
https://www.slideshare.net/jugafoce/immune-15100391
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T-CellSelectionandFunction
• T-cellselectionrequirestwofeatures:
• Heterodimericcell-surfaceT-cellreceptors
(TCRs:TCRa andTCRb)
• antigenspresentedbyMHC
• Inhealthyindividuals,TCRsarehighlydiverse
• Estimated>1015 possiblecombinations
• “True”diversityisacurrentresearchquestion
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T-CellDiversityisCreatedByV(D)JRecombination
• TCRb hasVariable(V),Diversity(D)andJoining(J)genesegments
• TCRa hasVariable(V)andJoining(J)genesegments
• V(D)JreferstocombinedTCRa/b recombination
• TCRb has~50Vx2Dx12Jsegments
=~1,200possiblecombinations(103)
• So…wheredoes1015 diversitycomefrom?
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T-CellDiversityisCreatedByV(D)JRecombination
• Untemplated diversity(indels) notencoded bythegermlineisgeneratedatrecombinationjunctions(N)betweenVaja andVbDb /DbJb
• Thisuntemplated nucleotidediversitycreatesthemassivepotentialcombinatorialdiversityoftheadaptiveimmunerepertoire
• T-cellreceptordiversityisencodedbyComplementarity-DeterminingRegion3
(CDR3)
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V(D)JRecombinationCreatesClonal“Barcodes”V(D)JRecombinationCreatesClonal“Barcodes”
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TheAdaptiveImmuneSystem
• V(D)JRecombinationcreatesbothT-cellandB-cellreceptor(antibody)diversity
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MethodsforImmuneRepertoireProfiling– SingleChain
•Spectratyping–Multiplexsingle-chain(TCRb)PCR–Bands-on-gelvisualization–Lowcostandlowresolution–Verypoorgaugeofdiversity
•RT-PCRandNGS–Usesuniversalsequencesforamplification–Typicallymeasuresexpressionratherthantrueclonality–Single-chain(e.g.TCRb)
•MultiplexPCRandNGS–Flexiblesampletypes,noenrichmentrequired–Canenablequantitativemeasurements–Single-chain(e.g.TCRb)
Characterizingimmunerepertoiresbyhighthroughputsequencing:strategiesandapplicationshttp://dx.doi.org/10.1016/j.it.2014.09.004
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MethodsforImmuneRepertoireProfiling– PairedChains
• AdvancedMethodologiesinHigh-ThroughputSequencingofImmuneRepertoiresSimonFriedensohn etalTrendsMarch2107
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TheChromium™SingleCellV(D)JSolution
•Comprehensive,scalable solution forprofilingfull-length,pairedV(D)Jtranscriptsfrom100to100,000HumanT-cells–HumanB-cellstobesupportedlaterin2017
•Builtfromprovencomponents–ChromiumSingleCellController–RobustconsumablesandreagentsadaptedfromtheChromiumSingleCell3’Solution–CellRangersoftwarewithnewV(D)Janalysismodule–LoupeVDJvisualizationsoftware
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SingleT-CellPartitioning,LysisandBarcoding
• RapidpartitioningandlysisofT-cellsin<7minutes• Lowestcellloss
• Output:DigitalV(D)JprofilesfrompartitionedT-cells
V(D)J Profiling of Individual T-cells
TCRα TCRβ
TCRα TCRβ
Cell 1...
Cell 5,000
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ChromiumSingleCell3’AssayScheme
•UMIandCellBarcodeadjacenttopoly(dT)VNRTprimer
•TSOat5’endofRTproduct
•TSOandRTprimercontainsequencesforuniversalPCRamplification
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ChromiumSingleCellV(D)JAssayScheme
• RTenzymeandpoly(dT)primerdeliveredtoallGEMsaspartofmastermix
• Barcoded5’templateswitcholigodeliveredtoGEMsfromGelBeads
• RTreactiongeneratesunbiasedcDNAwithasequencingadapter,acellbarcodeandaUMIonthe5’end
• EnrichmentPCRwithauniversalprimerforthe5’adapterandsuccessivenestedprimersfortheTCRconstantregions.
• FragmentationandsequencingoptimizedforassemblyofthefullV(D)Jsequence(5’UTRtoconstantregions)fromshortpaired-endreadsonacell-by-cellbasis.
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AdvantagesofChromiumVDJSolution• LessBias–NoV- orJ-specificprimers,avoidsbiasassociatedwithcomplexmultiplexPCR–UMIintemplateswitcholigofurtherreducessequencingerrorandenablestranscriptquantitation
• MoreDiscovery–DetectgermlineandsomaticvariantsacrosstheentireV(D)Jsegment–Seebothproductiveandexpressednon-productiverecombinationevents–SeemultipleTCRalphagenesexpressedpercell–Maptranscriptionstartsites
• FlexibleReagents–Onegelbeadforanyspecies,celltypeandisotype–Enablesfuturecapabilitytoassayadditionaltranscriptsforcelltypeclassificationorphenotyping
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NewSingleCellAChipKit- RapidandEfficientMicrofluidics
•Partition100-10,000+cellsperchannelin<7minutes
•Run1to8channelsinparallel
•Nolowersizelimitoncells
•Recoversupto65%ofallloadedcells,including:–Tcells,Bcells,PBMCsandcelllines–FACS-isolatedcells–MACSMicroBead-enrichedcells–Tumor-infiltratinglymphocytes
•Lowdoubletrate:0.9%per1,000cells
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CompatibilitywithallIlluminaSequencers
•Choosetheoptimalsequencerforthescaleofyourexperiments*
•2x150bpsequencereads,8bpi7Index
•5,000readpairs/cellforV(D)Jassembly**
*SingleCellV(D)JlibrariesontheNextSeq 500/550platformmayyieldreducedsequencequalityandsensitivityrelativetotheMiSeq andHiSeq platforms.
**Recommendedstartingpoint.Optimaldepthmayvaryacrosscelltypesandapplications.
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CellRanger™- InformaticsWorkflow
• CompleteLinux-basedsoftwarepackage
• Runsanywhere:LocalModeandClusterMode
• Widelyusedforsinglecellgeneexpressionanalysis
• Version2.0+includesV(D)JanalysisandsupportforLoupeVDJvisualizationmodule
OutputFileFormats
BAM,FASTQ,HTML,CSV
BarcodeProcessing
Annotation[V(D)J]
Cell-by-CellConsensusAssembly
ClonotypeInferenceandClustering
Transcriptcountingpipeline
Readfiltering[V(D)J]
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Chromium™SingleCellV(D)JSolutionworkflow
• SequenceChromiumlibrariesto~5,000readpairspercell(2x150nt)
• CellRanger™pipelineassemblesreadsintoV(D)Jsegmentsatsinglecellresolution
• Loupe™V(D)JBrowserenablesinteractiveanalysis
CellRanger™AnalysisPipelines
Loupe™V(D)J BrowserInteractiveAnalysis
StandardInformaticsPython,R
BAM
FASTQ
CSV
VLOUPE
BCL
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Pricing
Chromium™ Controller,24Mo.Warranty 120246 1Instrument $125,000
Chromium™ SingleCellController,24Mo.Warranty 120212 1Instrument $75,000
Chromium™ SingleCellA ChipKit 120236 48samples $1,440
Chromium™SingleCell 5’Gel Bead&LibraryKit 1000006 16reactions $20,000*
Chromium™ SingleCellV(D)JEnrichmentKit,HumanTCell 1000005 96 reactions $250
Chromium™i7MultiplexKit 120262 96reactions $768
PlatformAssurancePlan(Controller/SingleCellController) 130002130004 12months $12,500
ListPriceUnitsProduct
*Pricepercell$0.13to$1.20
PN
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TheChromium™SingleCellV(D)JSolution
• Paired,full-lengthV(D)Jimmunerepertoireanalysisforeverylab• Builtonprovenmicrofluidicsandsoftware• Turn-keysolutionthatdoesn’trequireextensiveoptimization
• FlexiblePlatform• SupportforHumanB-cellsandotherspeciescomingsoon
• Highcaptureefficiencyforprecioussamples• Upto65%ofloadedcellscanberecoveredfromsamplescontainingonly100sofcells
• Highthroughputfordeeprepertoireprofiling• Capture80,000+cellsin<7minutes
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TheChromiumSingleCellV(D)JSolution
Data
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HumanTcellsselectedforTetanusToxoidSpecificityinvitro
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
469 81% 96% 78% 1.2
TTTCTCATCTGGGGCTCTCTCGACAAGAAGGTTCTGGGGACCAGGCAGAGAGAATGAGGTCTCAGGATGACTTCCTTGACAGCCCTGTTCCCCTTTCATCAACACACAGACCCAGAAGACCTCTCTGTCTTGTAGCATCTGCCATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGATTGCTGGGATCACCCAGGCACCAACATCTCAGATCCTGGCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACATAATGCCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGGCTCATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAGTCCCTGATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTCCCCCTCACGTTGGCGTCTGCTGTACCCTCTCAGACATCTGTGTACTTCTGTGCCAGCAGTGACTCGGGGCTTCTCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAGATCGGAAGAGCACA
AGTTTTCTTATATGGGGAAAGCAGATTCTTTTTATGATTTTTAAAGTAGAAATATCCATTCCAGGTGCATTTTTTAAGGGTTTAAAATTTGAATCCTCAGTGAACCAGGGCAGAGAAGAATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTCCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACATCAATAACAATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAACCAAATATCCAGAAGCCTGACCCTGCCGTGTACCAGCTGAGAGACTAGATCGGAAGAGCAGATGTACCCAGGATATG
α chainconsensus(589nt)TRAV12-2:J43
β chainconsensus(610nt)TRBV6-4:D1:J2-3
Onlyoneproductiveα andoneproductiveβ chainobservedinthelibrary:
• 98.8%ofchainsfromindividual cellsofthisclonotypeareidenticaltothisV(D)Jconsensus
• Sequenceaccuracyfromindividualcellsofthisclonotype:99.98%(1.2errorsin10,000nt)
• ConsensusaccuracyacrossV(D)Jregion:100%
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HumanTcellsselectedforEpstein-BarrVirusSpecificityinvitro
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
446 83% 66% 53% 4.6
• ~55%ofcellsinferredtoshareonemajorα andβ chain(TRAV12-3:J20,TRBV9:D1:J1-4)
Top10detectedTCRs: count
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• ~9%ofcellsinferredtobeofasecond,unrelatedclonotype(TRAV5:J15,TRBV14:D2:J2-1)
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
446 83% 66% 53% 4.6
Top10detectedTCRs: count
HumanTcellsselectedforEpstein-BarrVirusSpecificityinvitro
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• ~4%ofcellsoftworelatedclonotypesthatshareacommonβ chain:
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
446 83% 66% 53% 4.6
Top10detectedTCRs: count
~3%ofcells:TRAV5:J15 (CDR3:CAESSNQAGTALIF)+TRBV29-1:D1:J1-4(CDR3:CSVGQGGTNEKLFF)~1%ofcells:TRAV5:J23 (CDR3:CAESIGKLIF)+TRBV29-1:D1:J1-4(CDR3:CSVGQGGTNEKLFF)
HumanTcellsselectedforEpstein-BarrVirusSpecificityinvitro
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PeripheralBloodMononuclearCellsfromtheSameDonor(x2)
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
3,424 55% 85% 50% 1,391.9
3,621 62% 84% 57% 1,437.6
Noclonotypemakesup>0.5%ofeithersample
Sample#1
Sample#2
912,809 2,949
ClonotypesuniquetoSample#1 ClonotypesuniquetoSample#2
Clonotypesdetectedinboth
• Tetanustoxoid- andEBV-specificclonotypesnotobserved
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RepresentationofGermlineV/JSegments
Med
ianUM
Icou
nt/con
tig
GermlineVsegments GermlineJsegments
TCRαTCRβ TCRαTCRβ
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LimitofDetection:EBV-specificTcellsspikedintoPBMCs
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
3,794 64% 86% 59% 1,223.2
2,067 50% 83% 45% 933.0
Mix#1(1%EBV:99%PBMC)
Mix#2(1%EBV:99%PBMC)
~55%ofEBV-specificcells:TRAV12-3:J20(CDR3:CATQGSNDYKLSF),TRBV9:D1:J1-4(CDR3:CASSTGQVATNEKLFF)→~0.55%ofcellsin1:99PBMCmixesshouldhavethismajorclonotype
• 16cells(0.4%)withidenticalα orβ detectedinMix#1(11paired)
• 7cells(0.3%)withidenticalα orβ detectedinMix#2(3paired)
count
count
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LimitofDetection:EBV-specificTcellsspikedintoPBMCs
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
3,794 64% 86% 59% 1,223.2
2,067 50% 83% 45% 933.0
~9%ofEBV-specificcells:TRAV5:J15(CDR3:CAESSNQAGTALIF),TRBV14:D2:J2-1(CDR3:CASSQSPGGIQFF)→~0.09%ofcellsin1:99PBMCmixesshouldhavethissecondclonotype
→LimitofDetectionforaknownclonotypeamong2,000+cellsis<1%
Mix#1(1%EBV:99%PBMC)
Mix#2(1%EBV:99%PBMC)
• 2cells(0.05%)withidenticalα orβ detectedinMix#1(2paired)
• 1cells(0.05%)withidenticalβ detectedinMix#2(0paired)
count
count
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ProfilingTcellsub-typesfromhealthyperipheralblood
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
4,827 71% 91% 65% 2,850.8
4,908 75% 94% 70% 3,290.4
4,810 64% 91% 58% 1,178.6
CD3+TCells
CD4+HelperTCells
CD8+CytotoxicTCells
Top10detectedTCRs:
• Noclonotypemakesup>0.1%ofthesample
count
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ProfilingTcellsub-typesfromhealthyperipheralblood
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
4,827 71% 91% 65% 2,850.8
4,908 75% 94% 70% 3,290.4
4,810 64% 91% 58% 1,178.6
CD3+TCells
CD4+HelperTCells
CD8+CytotoxicTCells
Top10detectedTCRs:
• EvidenceofindividualTcellsexpressingtwoproductiveαchains
count
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ProfilingTcellsub-typesfromhealthyperipheralblood
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
4,827 71% 91% 65% 2,850.8
4,908 75% 94% 70% 3,290.4
4,810 64% 91% 58% 1,178.6
CD3+TCells
CD4+HelperTCells
CD8+CytotoxicTCells
Top10detectedTCRs:
• Topclonotypemakesup~1%ofthesample
count
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TumorInfiltratingLymphocytes(TILs)Cryopreserveddissociatedtumor CD3+TILs
Deadcelldepletion
Anti-CD3beadenrichment
Deadcelldepletion
Anti-CD3beadenrichment
Clearcellrenalcellcarcinoma(CCRCC)• Primarytumor
• Stage1
• Male,white,54y.o.
Colorectalcancer(CRC)• Primarytumor
• StageII-A,LV-0,R-0
• Female,white,67y.o.
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TumorInfiltratingLymphocytes(Anti-CD3BeadEnrichment)
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
2,019 47% 90% 44% 445.3
755 73% 90% 67% 15.0
CRC
CCRCC
Top10detectedTCRs:
• ~0.8%ofcellsmakeupmostabundantclonotype(TRAV20:J15,TRBV29-1:*:J2-5)
• ClonotypediversityissimilartocirculatingTcells
count
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TumorInfiltratingLymphocytes(Anti-CD3BeadEnrichment)
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
2,019 47% 90% 44% 445.3
755 73% 90% 67% 15.0
CRC
CCRCC
Top10detectedTCRs:count
• ~21%ofcellsinferredtoshareonemajorα andβ chain(TRAV13-1:J5,TRBV27:D1:J1-1)• SuggestssignificantclonalexpansionofTILsinthistumor
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TumorInfiltratingLymphocytes(WithoutEnrichment)
CellswithTCR(s)detected
Productiveαdetected
Productiveβdetected
Productivepairdetected
Clonotypediversity
(InverseSimpsonindex)
755 73% 90% 67% 15.0
190 62% 72% 58% 8.0 CCRCC(Noenrichment)
CCRCC(Anti-CD3beadenriched)
• EnrichedTILstopclonotype:~21%frequency(TRAV13-1:J5,TRBV27:D1:J1-1)• UnenrichedTILstopclonotype:~20%frequency(TRAV13-1:J5,TRBV27:D1:J1-1)
TGTCTTATATGGGGATTGGTTGGCTACACAGTGTGAGAAACCCCTATGGCTGCCAGAGGAGAGAAGAGACAACCTGATGATAGAAGTAACTCTTATAACTGGAGGTTGCAGGTCAATGACTGATCTTAATTGGGAAGAACAAGGATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGAATGGAGAGAATGTGGAGCAGCATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAACTACTTCCCTTGGTATAAGCAAGAACTTGGAAAAAGACCTCAGCTTATTATAGACATTCGTTCAAATGTGGGCGAAAAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCAAACATTTCTCCCTGCACATCACAGAGACCCAACCTGAAGACTCGGCTGTCTACTTCTGTGCAGCACCCCGCCGGGGCAGGAGAGCACTTACTTTTGGGAGTGGAACAAGACTCCAAGTGCAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCCCGGATGAAGCTACAAGCGCGAGTGACGTTATCTGATTTGATGGAAGAAATCTGAAGCTGGATGAGGGCCAAGCGGAAAGAGGTCATGACTCTGCAGACAA
TATCTTATATGGGGGTTTCTGAGGCCCAAATAGCTGAAGAGGTGGAGACGTTACAGAAACCACCTGGAGCCCCCAGAACTGGCAGACACCTGCCTGATGCTGCCATGGGCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTCTCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCTGTACTTCTGTGCCAGCAGTTTAGGACAGAACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAGATCGGAAGAGCA
β chainconsensus(563nt)TRBV27:D1:J1-1
α chainconsensus(682nt)TRAV13-1:J5
→Full-length,pairedV(D)JsegmentsfromclonallyexpandedTILswithoutcloningorexvivo culture
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