1. The use of R statistical software to analyze flow cytometry data M. Ramón 1 , A. Maroto-Morales 1 , O. García-Álvarez 2 , P. Jimenez-Rabadán 2 , MD. Pérez-Guzmán 2 , F. Martínez-Pastor 3 , AJ. Soler 1 , JJ. Garde 1 1 IREC, (CSIC-UCLM-JCCM), Albacete; 2 CERSYRA, Valdepeñas; 3 ITRA-ULE-INDEGSAL, León 10º Congreso de la Asociación Española de Reproducción Animal Cáceres, 2-5 Junio 2010

2. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 R Statistical Software Integrated suite of software facilities for data manipulation, calculation and graphical display. WinMDI Version 2.9 Windows Multiple Document Interface for Flow Cytometry. The Scripps Research Institute (TSRI) WEASEL Version 2.7.4 Walter & Eliza Analysis Software: Eclectic & Lucid. The Walter and Eliza Hall Institute (WEHI)

3. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 Main Goal: To provide widespread access to a broad range of powerful statistical and graphical methods for the analysis of genomic data. Bioconductor provides a unified framework to develop methods to analyze and interpret Flow Cytometry data. Most common Packages α FlowCore α FlowUtils α FlowViz α FlowQ α FlowStats α FlowClust Bioconductor Open Source Software for Bioinformatics

5. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 1. READ DATA FILES > flowData <- read.flowSet( path =“.”, alter.names=TRUE, phenoData= “annotation.txt&quot;) > wf <- workFlow(flowData, name=&quot;Workflow #1&quot;)

8. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2. STANDARD FLOW OPERATIONS Scatter plot matrix

9. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2. STANDARD FLOW OPERATIONS Scatter plot matrix Some transformation is needed for better visualization of data

10. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2a. DATA TRANSFORMATION > tf <- transformList(colnames(flowData), asinh, transformationId=&quot;asinh&quot;) > add(wf,tf) > flowDataT <- transform(flowData, tf)

12. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2a. DATA TRANSFORMATION Scatter plot matrix

13. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2b. IDENTIFICATION OF SPERM POPULATION FSC vs. SSC scatter plot > xyplot(`SSC.H` ~ `FSC.H` | sampleId, data=wf [[&quot;asinh&quot;]])

14. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2b. IDENTIFICATION OF SPERM POPULATION FSC vs. SSC scatter plot > xyplot(`SSC.H` ~ `FSC.H` | sampleId, data=wf [[&quot;asinh&quot;]]) Debris

15. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2b. IDENTIFICATION OF SPERM POPULATION FSC vs. SSC scatter plot > sp.gate <- polygonGate(.gate=r01) > add(wf, sp.gate, parent='asinh')

16. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2b. IDENTIFICATION OF SPERM POPULATION FSC vs. SSC scatter plot WinMDI WEASEL

17. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 3. MEMBRANE INTEGRITY STUDY YO-PRO®-1 (FL1.H) vs. PI (FL3.H) scatter plot > xyplot(`FL3.H` ~ `FL1.H` | sampleId, data=wf[[&quot;asinh&quot;]]) RAW DATA

18. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 3. MEMBRANE INTEGRITY STUDY YO-PRO®-1 (FL1.H) vs. PI (FL3.H) scatter plot > xyplot(`FL3.H` ~ `FL1.H` | sampleId, data=wf[[&quot;asinh&quot;]]) Debris must be removed !!! RAW DATA

19. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 3. MEMBRANE INTEGRITY STUDY YO-PRO®-1 (FL1.H) vs. PI (FL3.H) scatter plot > xyplot(`FL3.H` ~ `FL1.H` | sampleId, data=wf[[&quot;asinh&quot;]]) RAW DATA SUBSET SPERM POPULATION

20. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 3. MEMBRANE INTEGRITY STUDY YO-PRO®-1 (FL1.H) vs. PI (FL3.H) scatter plot > xyplot(`FL3.H` ~ `FL1.H` | sampleId, data=wf[[&quot;asinh&quot;]]) Is a compensation of data needed? Must be applied before data transformation !! SUBSET SPERM POPULATION

23. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2c. DATA COMPENSATION COMPENSATION MATRIX

24. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2c. DATA COMPENSATION COMPENSATION MATRIX WEASEL

26. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 3. MEMBRANE INTEGRITY STUDY IDENTIFICATION OF DIFFERENT SUBPOPULATIONS RECTANGLE REGION POLYGON REGIONS QUADRANT REGIONS

27. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 3. MEMBRANE INTEGRITY STUDY IDENTIFICATION OF DIFFERENT SUBPOPULATIONS QUADRANT REGIONS

28. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 3. MEMBRANE INTEGRITY STUDY IDENTIFICATION OF DIFFERENT SUBPOPULATIONS Definition of Regions/Quadrants Regions are different for each semen sample Data normalization will allow the use of a single region or quadrant set

29. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2d. DATA NORMALIZATION > norm <- normalization(parameters=param, normalizationId=&quot;norm&quot;, + normFunction=function(x, parameters, ...) warpSet(x,parameters)) > add(wf, norm, parent=&quot;asinh&quot;) BEFORE NORMALIZATION

30. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 2d. DATA NORMALIZATION > norm <- normalization(parameters=param, normalizationId=&quot;norm&quot;, + normFunction=function(x, parameters, ...) warpSet(x,parameters)) > add(wf, norm, parent=&quot;asinh&quot;) AFTER NORMALIZATION

31. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 3. MEMBRANE INTEGRITY STUDY IDENTIFICATION OF DIFFERENT SUBPOPULATIONS Summarize statistics FL1.H-FL3.H- FL1.H+FL3.H- FL1.H+FL3.H+ Y.PI.MT.0 47.77% 33.68% 18.37% Y.PI.MT.3 53.65% 30.28% 15.15%

34. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 7. iFLOW

35. The use of R statistical software to analyze flow cytometry data 10º Congreso de la Asociación Española de Reproducción Animal (AERA) Cáceres, 2-5 Junio 2010 THANK YOU FOR YOUR ATTENTION