spillover::flowCore problem: Error: Unable to match stains with controls based on intensity: a single stain matches to several multiple controls.
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@samuel-ahuno-14031
Last seen 3.1 years ago
Ghana/KNUST

My spillover matrix fails to generate. Details of problem below. Any help in that regard will be gladly appreciated. Thanks

myflowset3 = read.flowSet(files=list.files(path=".",pattern="fcs", full.names=TRUE))

summary(myflowset3)

spx=spillover(myflowset3 , unstained = "Sample 2_Unstained.fcs", patt = NULL, fsc = "FSC-A",  ssc = "SSC-A", method = "median", stain_match = c("intensity", "ordered", "regexpr"), useNormFilt=FALSE, pregate = FALSE, plot = FALSE)

Error: Unable to match stains with controls based on intensity: a single stain matches to several multiple controls. 

 

I have the following experimental files in my flowSet named "myflowset3"

  1. Compensation Controls_APC-H7 Stained Control_018.fcs
  2. Compensation Controls_APC Stained Control_017.fcs           
  3. Compensation Controls_Aqua Amine Stained Control_020.fcs
  4. Compensation Controls_BV421 Stained Control_019.fcs          
  5. Compensation Controls_FITC Stained Control_013.fcs           
  6. Compensation Controls_PE Stained Control_014.fcs                
  7. Compensation Controls_PerCP-Cy55 Stained Control_015.fcs 
  8. Sample 1_CEF.fcs                                                                              
  9. Sample 1_CMV.fcs                                                                               
  10. Sample 1_Unstim.fcs                                                                      
  11. Sample 2_CEF.fcs                                                                               
  12. Sample 2_Unstained.fcs                                                                  
  13. Sample 2_Unstim.fcs                                                                          
  14. Sample 3_CEF.fcs                                                                               
  15. Sample 3_CMV.fcs                                                                               
  16. Sample 3_Unstim.fcs                                                                           

 

flowCore spillover compensation matrix bioconductor flow cytometry • 518 views
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Entering edit mode

The flowset should consists in the compensation controls plus one unstained sample. Moreover you should set only one value for the stain_match option. I would recommend "ordered" and organize the controls in the order of the channels in the flowset you give to the spillover function. Doing so you are sure of the matching.

For more information look at the code

https://github.com/RGLab/flowCore/blob/37c4546341f1475fda49d7a1d63c82bdce5489aa/R/flowSet-accessors.R#L610
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