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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"
- Compensation Controls_APC-H7 Stained Control_018.fcs
- Compensation Controls_APC Stained Control_017.fcs
- Compensation Controls_Aqua Amine Stained Control_020.fcs
- Compensation Controls_BV421 Stained Control_019.fcs
- Compensation Controls_FITC Stained Control_013.fcs
- Compensation Controls_PE Stained Control_014.fcs
- Compensation Controls_PerCP-Cy55 Stained Control_015.fcs
- Sample 1_CEF.fcs
- Sample 1_CMV.fcs
- Sample 1_Unstim.fcs
- Sample 2_CEF.fcs
- Sample 2_Unstained.fcs
- Sample 2_Unstim.fcs
- Sample 3_CEF.fcs
- Sample 3_CMV.fcs
- Sample 3_Unstim.fcs
>
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