Single Cell Sales Deck V2 - biomedsupport.utexas.edu · •High quality UMI and Cell Barcode reads ... , Python, R BAM HDF5 MEX LOUPE BCL. 15 •Flexible demultiplexingfeatures •Built-in

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

3

SingleCellAnalysis:BiologyinHD

“Tumor”

“Blood”

• Cancerstemcells

• Stromalcells

• Tumor-infiltratinglymphocytes

• Plateletsandredbloodcells

• Immunecells

• Circulatingtumorcells

TumorimageattributedtoWikimediaCommons

Bulk SingleCellResolution

4

SingleCellPartitioninginGEMs

Cell

GelBeadwithBarcodedRTPrimers

RTReagentsinSolution

PartitioningOil

5

High-diversityLibrary

HighDiversityBarcodeLibrary

FunctionalOligowithBarcodeGelBeadScaffold

• ~750,000DiscreteReagentsinOneTube• Definedbarcodesequences• Highlyuniformsizeandbarcoderepresentation• Built-insequencingadapter,barcodeandprimer

6

SingleCellPartitioning,LysisandBarcoding

7

SingleCell3’DigitalGeneExpression

• Rapidpartitioningandlysisofcellsin<7minutes

• Lowcellloss• Nolowerlimitoncellsize

• Output:Digitalgeneexpressionprofilesfromeverypartitionedcell

8

•Partitions100- 10,000+cellsperchannelin<7minutes

•Recovers~65%ofallloadedcells

•Lowdoubletrate:0.9%per1,000cells

RapidandEfficientMicrofluidics

9

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

10

•Standardsequencingconfigurations

•Easiertomultiplexwithnon-SClibraries

•HighqualityUMIandCellBarcodereads

•Highperformanceonpatternedflowcells

3’AssayScheme- GelBeadRTPrimersforInlineBarcoding

11

•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

12

293THumanEmbryonicKidneyCells NIH/3T3MouseFibroblasts

v2ReagentsandWorkflowBoostsSensitivity

v2

v1

v2

v1

13

•Choosetheoptimalsequencerforthescaleofyourexperiments

•HiSeq 4000enablessequencingof10,000s-100,000+cellsperflowcell

CompatibilitywithAllIlluminaSequencers

14

• CompleteLinux-basedsoftwarepackageforsinglecellanalysis

• BundledwithSTARforefficienttranscriptomealignment

• OutputsarestandardformatsplusLoupevisualization

• Runsanywhere:LocalModeandClusterMode

CellRanger™– InformaticsWorkflow

CellRanger™AnalysisPipelines

Loupe™CellBrowser

StandardInformaticssamtools,Python,R

BAM

HDF5

MEX

LOUPE

BCL

15

•Flexibledemultiplexing features

•Built-indifferentialgeneexpresson,PCA,t-SNEandclusteringanalysis

•Newfunctionalityforaggregatingdatafrommultiplesequencingruns,librariesand/orsamples

•Highperformanceandscalability:Enablesanalysisof1,000,000+celldatasets

CellRanger™1.2Updates

16

Million-scaleSingleCellAnalysis

136~10,000celllibraries

17ChromiumSCv2chips

4HiSeq 4000flowcells

E18MouseCortex,HippocampusAndVentricularZone

CellRanger1.2

1,308,421SingleCellExpressionProfiles

17

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

19

•ASingleCellSolutionforeverylab

•SupportsChromiumSingleCell3’v1andv2

•SamethroughputanddynamicrangeasthegeneralChromiumController

•$75,000

Chromium™SingleCellController

Confidential— Donotdistribute©10xGenomics,Inc.2016 FORRESEARCHUSEONLY.Notforuseindiagnosticprocedures.

21

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

26Confidential— Donotdistribute

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

27Confidential— Donotdistribute

•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

28

Example5:PrimaryCellPopulations

PlateletsandPlasmaComponents

Erythrocytes(RBCs),EosinophilsandNeutrophils

MyeloidCells LymphoidCells

Monocytes

MyeloidDendriticCells

WholeBlood

Macrophages

PlasmacytoidDendriticCells

BCells NKCells TCells

MononuclearCells(PBMCs)• Acomplexmixtureofdifferentcell

types• Well-studiedandreadilyavailableprimarycells

PeripheralBloodMononuclearCells(PBMC)

29

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

31

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

32

Referencepopulationsclassifycelltypes

bead purifiedPurified cells

Single cell RNA-SeqPBMCS

33

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

34Confidential— Donotdistribute

MajorpopulationsofPBMCsaredetected

35

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

37

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

38Confidential— Donotdistribute

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

39

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

40Confidential— Donotdistribute

DistinctExpansionsinCLLvsAMLBMMCs

Healthyindividual CLLpatient(untreated)

AMLpatient(untreated)

CD34+ Progenitors

CD19+ B cell

Expansion of B cells (86%) Expansion of myeloid progenitors (69%)

41

AdditionalEarlyAccessData:Hanlee JiatStanford

–Follicularlymphoma:expansionofBcells–AZD4547treatedKATOIIIcells

CD19+ B

control

AZD treated

42Confidential— Donotdistribute

AdditionalEarlyAccessData:CalvinKuo atStanford