Screen Mining with KNIME - idisk-srv1.mpi-cbg.de€¦ · Outline Martin Stöter, MPI-CBG, Dresden, Germany 2 - IntroducAon into High-Content Screening (HCS) data and the HCS Tools

Screen Mining with KNIME

A user-friendly framework for high throughput / content data analysis

Mar$nStöterHT-TechnologyDevelopmentStudio(TDS),[email protected]

KNIMEworkshopFebruary27th2016,Berlin

Outline

Martin Stöter, MPI-CBG, Dresden, Germany 2

-  IntroducAonintoHigh-ContentScreening(HCS)dataandtheHCSToolsnodes

-  Hands-onsessionHCSTools-  IntroducAonintoScripAngIntegraAonnodes-  Hands-onsessionScripAonIntegraAon

Technology Development Studio (TDS)


MPI-CBG,Dresden,Germany

Screeningfacilityforacademiclaboratories

ProvidefullserviceforautomaAonandcell-basedscreens,RNAiand

chemicalscreens

Equipment:liquidhandlingrobots,dropdispensers,platewashers,platereaders,

HighContentScreeningplaTorms

Data Analysis is a Bottleneck in HCS!

4

Dataanalyst

ComplexExperimentsLotsofdata(toomuchforExcel)Fancydataanalysis/miningManyscienAsts,butfewdataanalystsSomeAmesdifferentlanguagesDataanalysisisoYenaboZleneck!

Scien$sts

HCSTools+ +

+…

High-Content Screening (HCS) data


DatageneraAon-Cells(RNAi,compounds)-Microscopy->images-Imageanalysis-Cellfeatures/parameters->welldata

Tasks/problems-ReaddatafromvarioussourcesSQLdatabase,XML,Excel,various.csv…-ScreeningspecificstaAsAcs-ScreeningspecificuAliAes-Datamining,visualizaAon

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24AB DMSO DMSO DMSOC 0.001 DMSO DMSO 0.001D 10 DMSO DMSO 10E 10 DMSO DMSO 10F 3 DMSO DMSO 3G 3 DMSO DMSO 3H 1 DMSO DMSO 1I 1 DMSO DMSO 1J 0.3 DMSO DMSO 0.3K 0.3 DMSO DMSO 0.3L 0.1 DMSO DMSO 0.1M no AB no AB 0.1 DMSO DMSO 0.1N no AB no AB 0.1 DMSO DMSO 0.1O DMSO DMSOP

HCS Tools for KNIME

DataImportImageAnalysisReaders(Opera,OpereQa,Mo$onTracking)PlateReaders(Envision,GeniusPro,MSDSectorImager)Other(ExampleData,GenericXML)

NormalizaAonPercent-of-control(POC),Normalizedpercentinhibi$on(NPI)Z-score,B-scoreVectorLengthNormaliza$on(clustering)Op$onal:robuststa$s$cs(Median+MAD)Selectwellstonormalize(controls,samples)

QualityControlZ-primefactor(Z‘),Mul$variateZ‘,SSMDCV(coefficientofvariance)Op$onal:robuststa$s$cs(Median+MAD)Selectwellstonormalize(controls,samples)

HCSTools

HCS Tools for KNIME

UAliAesHandlebarcodes,wellsandrowleQersJoinLayoutfromExcel(wellannota$on,metadata)CreateWellPosi$on(NEW)

VisualizaAonPlateHeatmapViewerDoseResponse(dependentonR!)

AdvancedStaAsAcsBinningAnalysisDataManitupaAon/Pre-ProcessingSplit/CombineColums(byheader)NumberFormaQer(NEW)RangeFilter,SpliQerOutlierRemoval

HCSTools

HCS Tools: Standardized Data Format

-EnforcestandardizaAonofdataformat

-Differentreadernodestoshapeacommondatastructure

-Lowertheknowledgeentrybarrierfornewusers

“barcode”,“plateRow”,“plateColumn”,param1,param2,…

->EasesuptheusageofotherHCSToolsnodes

HCS Tools: Expand well

StandardizaAonofthewellcoordinates:-“plateRow”and“plateColumn”asintegervaluesresemblewellposi$onmatrix(insteadofwell)

-Somenodesselectthesecolumnsasdefault(JoinLayout,PlateHeatmapViewer)

-Compa$blewith96,384and1536wellformat

-PlateRowConverter(leQer↔integer)

-CreateWellPosi$on(sortablewellstring)

NEWNODE

HCS Tools: Barcode Standard

RegularexpressionforinterpretaAonofbarcode:-Standardizedtablestructure->connec$ontoourTDScompounddatabase

-(?<libplatenumber>[0-9]{3})(?<projectcode>[A-z]{2})(?<date>[0-9]{6})(?<replicate>[A-z]{1})

-ConfigurableinPreferences->KNIME->HCATools

-Mul$plebarcodes/regularexpressionspossible

-Finalreleaserecently













HCS Tools: Annotate Experiment

ExcelisthetoolforexperimentdocumentaAonandassaydevelopment

JoinLayoutnodeisExcelReaderfordefinedspreadsheet

PlateformatwithmulAplewellaZributes(1platelayout->1columninKNIME)

-TitleoflayoutstartsincellC5

-Twoemptyrowsbetweenthelayout

HCS Tools: Normalization

Tocomparedatafromdifferentplates,daysorrunsdatamustbenormalizedperplate

SelectablereferencewellpopulaAonperplatePercent-of-control(POC),Normalizes-percent-of-

inhibiAon(NPI),Z-ScoreRobuststaAsAcs(median&madinsteadofmean&sd)

withstaAsAcstableassecondoutput











HCS Tools: Quality Control (QC)

QualitycontrolstaAsAcmeasuretheassayperformanceSelectable(mulAple)referencewellpopulaAonperplateZ-Primefactor(Z’),mulAvariateZ’,strictlystandardized

meandifference(SSMD),coefficientofvariance(CV)RobuststaAsAcs(median&madinsteadofmean&sd)







HCS Tools: Binning Analysis

BinninganalysisdescribeschangesindistribuAonsGreattoolformovingfromcelltowelldata(insteadof

justtakingmeanperwell)

"CellProfiler and KNIME: open source tools for high content screening.". Methods in molecular biology (Clifton, N.J.) 2013 986, S. 105-22









HCS Tools: Plate Viewer (discontinued)


179platesx384wells=~70.000datapointsAmesxparameters

HCS Tools: Plate Heatmap Viewer

VisualizaAonofscreeningcampaignswithmetadataEasytofindvisuallypaZers,driYs,errors…Newfeatures:

-KNIMEColors-HiLitesupport-representaAonofimages-manydifferentconfiguraAons,e.g.colorscale…







-10x384wellplate-3replicates-~10,000datapoints-Rawdata-Metadatafrombarcode-Normalizeddata-Differentreadout-Metadatafromlayout-Browsingsingleplate-Viewingthewelldata-Displayofimages-…more





























HCS Tools: what was / is cooking?

Newnodes-CreateWellPosiAon-NumberFormaZer

Enhancements-DoseResponse(R)

PlateViewerwasdisconAnuedBinningAnalysisworkinprogress

-BinningCalculate

-BinningApply

-BinningQC&ModelModifier

Transformsnumberstodefinedstring

-imageoutput(insteadofview)-moresta$s$csintableoutput(e.g.Hillcoefficent)-moreplotop$ons(SEM)-newmodelport

?

HCS Tools: the demo

Ok…nowlet’sgototheworkflowandseethenodes…

Thedataset:CellProfilerImagedata(pre-cleanedupasa.tabledueto

technicalreasons)

-10x384wellplatesin3replicateswith3imagesperwell

Acknowledgements

36

SoYwareDevelopmentAntjeJanoschTimNicolaisenMagdalenaRucinskFelixMeyerhofer(past)HolgerBrandl(past)

HCSTools

TDSteam(MPI-CBG)

KNIMEMichaelBertholdandtheKNIMEteam

Screen Mining with KNIME - idisk-srv1.mpi-cbg.de€¦ · Outline Martin Stöter, MPI-CBG, Dresden, Germany 2 - IntroducAon into High-Content Screening (HCS) data and the HCS Tools

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