Sta$s$csforGenomics(140.688)

Instructor: Jeff Leek

Slide Credits: Paul Robinson (Purdue), ABDSerotec, Raphael Gottardo (FHCRC), http://www.usuhs.mil/bic/cytometry/

pdf/tutorial.pdf.

5/14/10 – Rough draft of project due. Please send with subject line: [project rough draft]

BasicProblem

Abunchofcells

(1) Whattypeofcellsarethere?

(2) Whatfrac$onareapar$culartype?

(3)Whatproper$esdothecellshave?

(4)Canwesortthecellsbytype?

ThingsYouCanMeasureWithAFlowCytometer

1.  Size and complexity of cells 2.  Cell pigments 3.  Copy number variation (Flow-FISH) 4.  Protein expression/localization 5.  Nuclear antigens 6.  Cell surface molecules

hNp://en.wikipedia.org/wiki/Flow_cytometry#Applica$ons

Publica$onsusingthekeyword“flowcytometry”from

7:36AM ©1990‐2010J.PaulRobinson,PurdueUniversityBMS631Page4

The field of flow cytometry continually grows

•1975-1976

•1976-1977

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2011

0 1000 2000 3000 4000 5000 6000 7000 8000

9000 10000 11000

105,000references(2009)

CommercialInstruments

7:36AM ©1990‐2010J.PaulRobinson,PurdueUniversityBMS631Page5

Millipore/Guava

Apogee

Luminex

Accuri

Bryte

BC

BD

BD

BC

BC

BC

Amnis

Bay Biosciences

BD Influx

BD Aria

LocalResources

FACSCalibur(BDBiosciences)

Flow‐Cytometer

hNp://www.abdserotec.com/uploads/Flow‐Cytometry.pdf

HydrodynamicFocusing

hNp://www.abdserotec.com/uploads/Flow‐Cytometry.pdf

Flow‐Cytometer

hNp://www.abdserotec.com/uploads/Flow‐Cytometry.pdf

ForwardandSideScaNer

Introduc$ontoFlowCytometry:ALearningGuide;BectonDickinson

CellMorphology

hNp://probes.invitrogen.com/resources/educa$on/tutorials/4Intro_Flow/player.html

FlowCytometry(Data)

hNp://probes.invitrogen.com/resources/educa$on/tutorials/4Intro_Flow/player.html

Flourochromes

Introduc$ontoFlowCytometry:ALearningGuide;BectonDickinson

Monoclonalvs.PolyclonalAn$bodies

GuidetoFlowCytometry;DakoCytoma$on

Polyclonal Monoclonal

FlourochromesHaveOverlappingEmissionWavelengths

BDLSRIIUser’sGuide;BectonDickinson

StokesShih

OverlappingWavelengthsLeadstoSpillover

SpectralViewer

hNp://www.invitrogen.com/site/us/en/home/support/Research‐Tools/Fluorescence‐SpectraViewer.html

Compensa$ngforSpillover

1.  Run each dye separately in a control experiment, get readings like: 8% of A spills into B, 10% of B spills into A

2.  Reading for A = Total for A – 10% of B Reading for B = Total for B – 8% of A

ClusterofDifferen$a$onMarkers

Electrosta$cFlowSor$ng

hNp://www.abdserotec.com/uploads/Flow‐Cytometry.pdf

Applica$onsofFC–Flow‐FISH

hNp://en.wikipedia.org/wiki/File:Flow‐FISH_1.JPG

Applica$onsofFC–CellCycle

hNp://mbpl.bmes.nthu.edu.tw/re$red/chinese/class/ns5249/NS5249‐04/5.%20Biological%20pplica$ons%20of%20FACS_04.19.04.pdf

Applica$onsofFC–Clinical

NormalPa$ent Pa$entw/ALL

Jennings,C.&Foon,K.(1997).Blood,90(8),2863‐2892.

BonemarrowcellsareexaminedonSSCandCD45todiagnoseacutelymphoblas$cleukemia(ALL)

Applica$onsofFACS–GeneExpressionMiceengineeredtoexpressGFPinganglioniceminence(GE)derivedneuralprogenitorcells.

Marshetal.(2008)DevelopmentalNeurobiology68:434‐445

DataAnalysis–PreNyOpen

Bashashan$andBrinkman(2009)AdvancesinBioinforma$csAr$cleID584603

FlowPlots2DContourPlot 2DScaNerPlot

1DHistogram

hNp://facs.scripps.edu/ni0706‐681.pdf

ScaleMaNers

“Logicle”Displays

hNp://facs.scripps.edu/ni0706‐681.pdf

LogicleTransform

Linear near 0 and log (exponential) for big values

Ga$ng

www.users.path.ox.ac.uk

MoreComplicatedGa$ng(Manual)

AutomatedGa$ng

MixtureModels

Loetal.Automatedga$ngofflowcytometrydataviarobustmodel‐basedclustering.CytometryA(2008)

Mul$pleSamples–Normaliza$on(1D!)

Hahneetal.Per‐ChannelBasisNormaliza$onMethodsforFlowCytometryDataCytometryA(2009)

EffectofNormaliza$on

Hahneetal.Per‐ChannelBasisNormaliza$onMethodsforFlowCytometryDataCytometryA(2009)

AutomatedClassifica$on

Step2Algorithm

Bashasha$etal.(2009)IEEEngMedBiolSoc.2009:4945‐8