Hello all, I was planning on using plateCore for multiple plates analysis, almost a 100 plates overall and I am having some difficulties as to how it's best to proceed. So I have a few questions: 1. Using xyplot(`FL1-H` ~ `FSC-H` | as.factorWell.Id),transform("FL1-H"=log10) %on% emkFPcg2[1:12], displayFilter=TRUE,smooth=FALSE, col=c("red","blue"), filter="Negative.Control") I get plots with FL1-H scale from 0 t0 3, but all my events are from 2.5 to 3. All the samples have most of the events on the first decade. Why is this happening? I did not have this problem with other samples.... 2. I do not have negative control in each plate, only in one well in one plate, how can I use this for all of the plates? I am running batches of 10+ plates, single color, 5K collected. What would the max number of plates that could be combined? See history below Thanks Jose Sancho Genzyme FCCF wellAnnotation<-read.table(file("C:/Documents and Settings/sanjo05/Desktop/PLATES/table.txt"),header=TRUE, stringsAsFactors=FALSE) emkPlate10<-read.flowSet(path="C:/Documents and Settings/sanjo05/Desktop/PLATES/EMK092110P10",transformation=FALSE) wellAnnotation<-read.table(file("C:/Documents and Settings/sanjo05/Desktop/PLATES/table.txt"),header=TRUE, stringsAsFactors=FALSE) emkFP10 <- flowPlate (emkPlate10, wellAnnotation, plateName="EMK092110.001") pData(phenoData(emkPlate10))[1,] fs <- plateSet(emkFP10) xyplot(`SSC-H` ~ `FSC-H` | as.factorWell.Id), emkFP10[1:4], displayFilter=TRUE, smooth=FALSE, col=c("red", "blue")) normGate <-norm2Filter("FSC-H", "SSC-H", scale.factor=2) xyplot(`SSC-H` ~ `FL1-H` | as.factorWell.Id), emkFP10[1:4], displayFilter=TRUE, smooth=FALSE, col=c("red", "blue"),filter=normGate) emkFP10.gated <- Subset(emkFP10,normGate) emkFPcg10<- setControlGates(emkFP10.gated,gateType="Negative.Control",numMads=5) emkFPcg10 <-applyControlGates(emkFPcg10) wells <-unique(subset(emkFPcg10@wellAnnotation,Negative.Control=="A01", select="name")[,1]) xyplot(`FL1-H` ~ `FSC-H` | as.factorWell.Id),transform("FL1-H"=log10) %on% emkFPcg2[1:12], displayFilter=TRUE,smooth=FALSE, col=c("red","blue"), filter="Negative.Control") [[alternative HTML version deleted]]

Hi Jose, 1. Using xyplot(`FL1-H` ~ `FSC-H` | > as.factorWell.Id),transform("FL1-H"=log10) %on% emkFPcg2[1:12], > displayFilter=TRUE,smooth=FALSE, col=c("red","blue"), > filter="Negative.Control") I get plots with FL1-H scale from 0 t0 3, but > all my events are from 2.5 to 3. All the samples have most of the events > on the first decade. > > Why is this happening? I did not have this problem with other > samples.... > > I don't see this behavior in the sample data set, so I'm not sure what's causing your problem. Maybe the events are piling up on the axis? The plateCore %on% function is a wrapper for the flowCore %on% function. The function attempts to transform the Negative Control Gates (if they exist) and then transform the flowSet/plateSet. If something were wrong with flowCore log10 transforms I think we'd see a lot of people reporting problems. > > 2. I do not have negative control in each plate, only in one well > in one plate, how can I use this for all of the plates? I am running > batches of 10+ plates, single color, 5K collected. What would the max > number of plates that could be combined? > > The easiest way might be to give the negative control file a new name and well id, and then copy the fcs file into each folder. You'd have to update the well annotation table to reflect this change. If you're using 96-well plates, create an fcs file with the well identifier of something like I01 so you don't overwrite existing wells. Alternatively you could read in the plate and the negative control wells as two separate flowSets and then combine them before creating a plateSet. You'd still have to assign the negative control well a new id that doesn't overlap with anything on the plate. Lastly, if you know where you'd like to set your gate, 100 units for example, you could directly assign the value using plateSet@wellAnnotation$Negative.Control.Gate <- 100, which should set the value at 100 for all the wells on the plate. The maximum number of plates depends upon how many events are on each plate and how much memory you have available. I had no trouble combining 9 plates (1 million events per plate) on a 64-bit Red Hat machine with 32GB of RAM. If you filter the plates to remove events that are not of interest, then you should easily be able to combine 10-20 plates if you're only collecting 5K per well. Sincerely, Errol Strain [[alternative HTML version deleted]]