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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