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$onsusingthekeyword“flowcytometry”from
7:36AM ©1990‐2010J.PaulRobinson,PurdueUniversityBMS631Page4
The field of flow cytometry continually grows
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105,000references(2009)
CommercialInstruments
7:36AM ©1990‐2010J.PaulRobinson,PurdueUniversityBMS631Page5
Millipore/Guava
Apogee
Luminex
Accuri
Bryte
BC
BD
BD
BC
BC
BC
Amnis
Bay Biosciences
BD Influx
BD Aria
LocalResources
FACSCalibur(BDBiosciences)
Flow‐Cytometer
hNp://www.abdserotec.com/uploads/Flow‐Cytometry.pdf
HydrodynamicFocusing
hNp://www.abdserotec.com/uploads/Flow‐Cytometry.pdf
Flow‐Cytometer
hNp://www.abdserotec.com/uploads/Flow‐Cytometry.pdf
ForwardandSideScaNer
Introduc$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