single cell sales deck v2 - sites.cns.utexas.edu · the chromium™ single cell 3’ solution...
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FORRESEARCHUSEONLY.Notforuseindiagnosticprocedures.©10xGenomics,Inc.2016
Chromium™SingleCell3’Solutionv2
ANewStandardforSingleCellRNA-Seq
RevisionA,November2016
2
TheChromium™SingleCell3’Solution
• GemCode™Technology
• Automated
• Flexiblethroughput:fromahundredtoamillioncells
Chromium™ Controller
• ChipforsinglecellpartitioninginGEMs
• ReagentsforRT,amplificationandlibraryconstruction
ChromiumSingleCell3’v2Consumables
• Informaticssolutionforsinglecellexpressionprofiling
• Pre-processing,QCandanalytics
CellRanger™Pipelines
I
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SingleCellAnalysis:BiologyinHD
“Tumor”
“Blood”
• Cancerstemcells
• Stromalcells
• Tumor-infiltratinglymphocytes
• Plateletsandredbloodcells
• Immunecells
• Circulatingtumorcells
TumorimageattributedtoWikimediaCommons
Bulk SingleCellResolution
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SingleCellPartitioninginGEMs
Cell
GelBeadwithBarcodedRTPrimers
RTReagentsinSolution
PartitioningOil
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High-diversityLibrary
HighDiversityBarcodeLibrary
FunctionalOligowithBarcodeGelBeadScaffold
• ~750,000DiscreteReagentsinOneTube• Definedbarcodesequences• Highlyuniformsizeandbarcoderepresentation• Built-insequencingadapter,barcodeandprimer
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SingleCellPartitioning,LysisandBarcoding
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SingleCell3’DigitalGeneExpression
• Rapidpartitioningandlysisofcellsin<7minutes
• Lowcellloss• Nolowerlimitoncellsize
• Output:Digitalgeneexpressionprofilesfromeverypartitionedcell
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•Partitions100- 10,000+cellsperchannelin<7minutes
•Recovers~65%ofallloadedcells
•Lowdoubletrate:0.9%per1,000cells
RapidandEfficientMicrofluidics
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Human-MouseMixturesConfirmPredictedDoubletRates
1:1MixtureofHuman(293T)andMouse(NIH/3T3)Cells,sequencedto~30-60Kreads/cell
~150loadedcells
100recoveredcells
0doublets*(0.0%)
~1,530loadedcells
1,015recoveredcells
6 doublets*(0.6%)
~10,000loadedcells
6,806recoveredcells
345doublets*(5.1%)
~19,000loadedcells
13,096recoveredcells
1,370doublets*(10.5%)
*Includesobserved(human-mouse)andinferred(human-human,mouse-mouse)doublets
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•Standardsequencingconfigurations
•Easiertomultiplexwithnon-SClibraries
•HighqualityUMIandCellBarcodereads
•Highperformanceonpatternedflowcells
3’AssayScheme- GelBeadRTPrimersforInlineBarcoding
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•OptimizedreversetranscriptionandcDNAclean-up
•Enzymaticfragmentation
•Cellsuspensiontolibraryin1day
OneDayWorkflow
Cell prep
GEMGeneration&Barcoding(GEM-RT)
PostGEM-RTRecovery
Dynabead
cDNAamplification
SPRI
QCBioA
GEM-RT&cDNAAmp LibraryConstruction
DSSPRI
FragmentaseEndRepairA-Tailing
Ligation
SPRI
SI-PCR
Libraryquant&sequencing
DSSPRI
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293THumanEmbryonicKidneyCells NIH/3T3MouseFibroblasts
v2ReagentsandWorkflowBoostsSensitivity
v2
v1
v2
v1
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•Choosetheoptimalsequencerforthescaleofyourexperiments
•HiSeq 4000enablessequencingof10,000s-100,000+cellsperflowcell
CompatibilitywithAllIlluminaSequencers
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• CompleteLinux-basedsoftwarepackageforsinglecellanalysis
• BundledwithSTARforefficienttranscriptomealignment
• OutputsarestandardformatsplusLoupevisualization
• Runsanywhere:LocalModeandClusterMode
CellRanger™– InformaticsWorkflow
CellRanger™AnalysisPipelines
Loupe™CellBrowser
StandardInformaticssamtools,Python,R
BAM
HDF5
MEX
LOUPE
BCL
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•Flexibledemultiplexing features
•Built-indifferentialgeneexpresson,PCA,t-SNEandclusteringanalysis
•Newfunctionalityforaggregatingdatafrommultiplesequencingruns,librariesand/orsamples
•Highperformanceandscalability:Enablesanalysisof1,000,000+celldatasets
CellRanger™1.2Updates
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Million-scaleSingleCellAnalysis
136~10,000celllibraries
17ChromiumSCv2chips
4HiSeq 4000flowcells
E18MouseCortex,HippocampusAndVentricularZone
CellRanger1.2
1,308,421SingleCellExpressionProfiles
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3DRepresentationof1,308,421SingleCellsfromMouseBrain
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ChromiumSingleCell3’SolutionPricing
Chromium™ Controller 1Instrument $125,000
Chromium SingleCellController 1Instrument $75,000✢
ChromiumSingleCell 3’Reagent Kitv2 16reactions $20,000*
Chromium™i7MultiplexKit 96reactions $768
ChromiumSingleCell3’ ChipKitv2 48samples $1,440
PlatformAssurancePlan 12months $12,500
ListPriceUnitsProduct
*Pricepercell$0.13to$1.20
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•ASingleCellSolutionforeverylab
•SupportsChromiumSingleCell3’v1andv2
•SamethroughputanddynamicrangeasthegeneralChromiumController
•$75,000
Chromium™SingleCellController
Confidential— Donotdistribute©10xGenomics,Inc.2016 FORRESEARCHUSEONLY.Notforuseindiagnosticprocedures.
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Appendix
GemCode (v1)andChromium(v1.1)SingleCell3’Solutiondata
22Confidential— Donotdistribute
Dynamicsoflymphocyteactivationingraft-versus-host-diseaseColeTrapnell,ScottFurlan– U.ofWashington
Monocle2:Singlecelltrajectoryanalysishttp://cole-trapnell-lab.github.io/monocle-release/
NatureWebcast:www.10xgenomics.com/event/single-cell-webinar-cole-trapnell/
SingleCell3’ApplicationHighlights
Unsupervisedidentificationofimmunecelltypesfrom33,000PBMCsRahulSatija – NewYorkGenomeCenter
Seurat:RToolkitforSingleCellGenomicshttp://satijalab.org/seurat/
NatureWebcast:www.10xgenomics.com/event/single-cell-webinar-dr-rahul-satija/
23Confidential— Donotdistribute
SinglecellanalysisofleukemiabeforeandafterbonemarrowtransplantsWithJasonBielas– FredHutch
GraceX.Y.Zheng:MassivelyparalleldigitaltranscriptionalprofilingofsinglecellsASHG2016PodiumPresentation
SingleCell3’ApplicationHighlights
AML027
(post-transplant)
14%donor 86%host
24Confidential— Donotdistribute
•1:1mixtureof~1,400human(HEK293T)andmouse(NIH3T3)cells
•99.4%ofcell-occupiedGEMsyieldedreadsmappingtoonlyonespecies
•1%inferreddoubletrate*
Example1:ValidationofSingleCellBehavior
Human:Mouse
Humanonly
Mouseonly
HumanTranscriptCounts
Mou
seTranscriptC
ounts
60,00000
*includesunobservedhuman:human andmouse:mouse doubletsNumberofcellsdetected:~1400cells,Numberofrawreadspercell:~130k
25Confidential— Donotdistribute
•ProliferatingHEK293Tcellswereprofiledandscoredforexpressionofmarkersassociatedwitheachmajorcellcyclephase
•Cellsfromallphaseswereidentified
Example2:CellCyclePhases
CombinedExpressionofKnownPhaseMarkersG1/S
S
G2
G2/M
M/G1Expression
level
HEK293TCellsOrderedbyInferredCellCyclePhase
0 100 200 300 400
Phase-specificgenesderivedfromWhitfieldetal.,2002Numberofcellsdetected:~400cells,Numberofrawreadspercell:~40k
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Example3:BreastCancerHeterogeneity
HCC1954
HCC38
HCC1143
t-SNEProjection
log(x+1)HER
2coun
ts
HCC1954
HCC38
HCC1143
t-SNEProjection
UnbiasedAutomaticClusteringofThreeBreastCellLines
HER2 ExpressionMatchesExpectedCellLineStatus
Numberofcellsdetected:~1000cells,Numberofrawreadspercell:~40k
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•Jurkat andRaji cellswerecombinedat9:1,99:1and199:1ratiosandthenprofiled
•TheminorityRaji populationswereidentifiedinallthreemixtures
Example4:IdentifyingRareCellTypesB-Cell (Raji) T-Cell (Jurkat)
-20 -10 0 10 20PC1
-20 -10 0 10 20PC1
-20 -10 0 10 20PC1
-20 -10 0 10 20PC1
-20 -10 0 10 20PC1
-20
-10
0
10
20
PC
2
-20
-10
0
10
20
PC
2
-20
-10
0
10
20
PC
2
-20
-10
0
10
20
PC
2
-20
-10
0
10
20
PC
2
12% 1.5% 0.6%
Numberofcellsdetected:~1000cells,Numberofrawreadspercell:~60k
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Example5:PrimaryCellPopulations
PlateletsandPlasmaComponents
Erythrocytes(RBCs),EosinophilsandNeutrophils
MyeloidCells LymphoidCells
Monocytes
MyeloidDendriticCells
WholeBlood
Macrophages
PlasmacytoidDendriticCells
BCells NKCells TCells
MononuclearCells(PBMCs)• Acomplexmixtureofdifferentcell
types• Well-studiedandreadilyavailableprimarycells
PeripheralBloodMononuclearCells(PBMC)
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Morestructureswithincreasing#ofPBMCs
4,500 PBMCs 16,000 PBMCs 68,000 PBMCs
TSNE1TS
NE2
Bulk RNA-Seq
30Confidential— Donotdistribute
Highreproducibilitybetweenchannels
68,000 PBMCs
• Librariesfrom8channelswithatargetcellloadof~8000cellseachwerecombinedintoonemetasample
• Highreproducibilitybetweenchannels
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Markershighlightdistinctsub-populationsCD3D, T Cells
TSNE1
TSN
E2
GNLY, NK Cells and T Cell subset
TSNE1
TSN
E2CD79A, B Cells
TSNE1
TSN
E2
TSNE1
TSN
E2
FTL, Monocytes
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Referencepopulationsclassifycelltypes
bead purifiedPurified cells
Single cell RNA-SeqPBMCS
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Referencepopulationsclassifycelltypes
bead purifiedPurified cells
Single cell RNA-SeqPBMCS
Can select cells from an impure population
Pure CD19+/IgD+ B Cells80% Pure CD19+/CD27+ B
Cells
TSNE1
TSN
E2
TSNE1
TSN
E2
83%
TSNE1
TSN
E2
TSNE1
TSN
E2
T cells
40% Pure CD34+ Cells
TSNE1
TSN
E2
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MajorpopulationsofPBMCsaredetected
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MajorpopulationsofPBMCsaredetected
CD4+ T (28.4%)
CD14+ Monocytes
(5.3%)
CD56+ NK (13.5%)
CD34+ Progenitors
(0.3%)
Dendritic (1.9%)
CD19+ B (5.5%)
CD8+ T (18.7%)
CD45 RA+ Naïve T (26.4%)
TSNE1
TSN
E2
36Confidential— Donotdistribute
UnbiasedprofilingoffrozenPBMCs
Frozen PBMCs, immediately thawed,
then performed single cell RNA-Seq
# of cells: 6k, # of raw reads/cell: 36k
CD4+ T
CD14+ Monocytes
CD56+ NK
Dendritic
CD19+ B CD8+ T
CD45 RA+ Naïve T
TSNE1
TSN
E2
CD34+
HealthyPBMCs,cryopreservedin2014
% c
ells
FreshFrozen
CD34+ Dendritic B Monocytes Lymphocytes
100
10
1
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MyeloidexpansioninAMLandCMLAMLPBMCs,
cryopreservedin2015CMLPBMCs,
cryopreservedin2005(10 yearsold)
FrozenPBMCsweresortedandviablecellswereprofiled
Expansionofmonocytes(45%)
#ofcells:900,#ofrawreads/cell:90k
FrozenPBMCswereimmediatelythawedandprofiled
Expansionofmyeloidprogenitors(CD34+)(44%)
#ofcells:6500,#ofrawreads/cell:38k
CD34+ Myeloid Progenitors
CD14+ Monocytes
CD14+ Monocytes
CD34+ Myeloid Progenitors
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Singlecellprofilingenablesmoresensitivecomparativeanalysis
TSNE1
TSN
E2
Normal PBMCs AML PBMCs
Myeloid population
Myeloid population
TSNE1
TSN
E2Significant gene sets
Vs.
"Bulk"cells(54)
Myeloidcellscomparison
(114)14
Upregulation of HPGDS, CD34, KIT Upregulation of SOCB2 & LAPTM4B
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ExpansionofBcellsinCLLBMMCs
Healthyindividual CLLpatient(untreated)
CLLpatient(treatedbutrelapsed)
CD19+ B cell
~82% abnormal B cells (CD19+/CD5+) cells detected by
immunophenotyping of cell surface markers
79%86%
82% abnormal B cells (CD19+, CD20+, CD5+, CD10-, CD11c-,CD38-, Lambda+) detected by
immunophenotyping of cell surface markers
CD19+ B cellCD19+ B cell
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DistinctExpansionsinCLLvsAMLBMMCs
Healthyindividual CLLpatient(untreated)
AMLpatient(untreated)
CD34+ Progenitors
CD19+ B cell
Expansion of B cells (86%) Expansion of myeloid progenitors (69%)
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AdditionalEarlyAccessData:Hanlee JiatStanford
–Follicularlymphoma:expansionofBcells–AZD4547treatedKATOIIIcells
CD19+ B
control
AZD treated
42Confidential— Donotdistribute
AdditionalEarlyAccessData:CalvinKuo atStanford