Hello, I am trying to code in a gating strategy in flowCore and I cannot figure out why my last gates are not showing up. I have followed the flowcore guide to a T, and yet my displayed plots/gates are not applying to the transformed fluorescent channels. Has anyone seen this before?

Here is my workflow from start to finish.

`require(flowAI) require(flowCore) require(ggcyto) require(flowStats) require(flowWorkspace) require(openCyto) require(flowViz)

Incorporation of FCS files

fcs.dir <- file.path("C:", "Users", "very.confused", "FCSfiles") frames <- lapply(dir(fcs.dir, full.names=TRUE), read.FCS, transformation=FALSE, emptyValue = FALSE, min.limit=0) fs <-as(frames, "flowSet")

Check FSC SSC plot

autoplot(fs[[1]], "FSC-H", "SSC-H")

Setting up for gating

fs <- as(fs, "flowSet") gs <- GatingSet(fs)

Data transformation

gh <- gs[[1]] fr <- ghpopget_data(gh) chnls <- markernames(gh) trans <- estimateLogicle(gh, chnls) gs <- transform(gs, trans)

HEK gating

gsgetpoppaths(gs) HEKgate <- rectangleGate("FSC-A"=c(80000, Inf),"SSC-A"=c(30000,Inf)) gspop_add(gs, HEKgate, parent = "root", name = "HEKgate") recompute(gs) autoplot(gs[[1]], "HEKgate")

Setting singlet gate

mat <-matrix(c(70000,70894,252144,252144,5037,61224,196501,155613), nrow = 4) colnames(mat) <-c("FSC-A", "FSC-H") singgate <- polygonGate(mat) gspopadd(gs, singgate, parent = "HEKgate", name = "singlet") recompute(gs,"HEKgate") autoplot(gs[[1]], "singlet")

Setting PE-A gate

rg2 <-rectangleGate("FL4-A"=c(2000, Inf)) gspopadd(gs, rg2, parent = "singlet", name = "PEGate") recompute(gs,"singlet") plot(gs)

autoplot(gs[[2]])

Setting APC gate

rg3 <-rectangleGate("FL7-A"=c(2000, Inf)) gspopadd(gs, rg3, parent = "singlet", name = "APCGate") recompute(gs,"PEGate")

autoplot(gs[[2]])`

Whenever I run the autoplot or the stats on either of the final two gates, I am not able to see any gate on the plot, or on the data itself. Does this have to do with the transformation performed? Or maybe how the data is incorporated into the flowset? Any help would be greatly appreciated.

check the data scale for FL channels before setting your gates, i.e.

autoplot(gs_pop_get_data(gs, "singlet"), "FL4-A")

2000 is probably way out of your transformed data range