Hi,

I'm trying to use the gate_mindensity2 from the openCyto package to gate out debris from my .fcs files. I'm using https://flowrepository.org/id/FR-FCM-ZZ36 as an example. I can gate out the debris at the lower end (i.e. left hand side of the gate) but if I want to apply a gate to remove the events on the right hand side, I am unable to do so, even if I set the positive argument to TRUE. I've probably just missed something, but has the positive=TRUE argument to gate_mindensity2 been removed from the recent version of openCyto.

Reprex and link to reprex output below. The issue I have is from applying FCSgate2fun to the gating set and expecting the filter to take the real line to the left of the cutpoint

Many thanks, Miha

https://github.com/Biomiha/FACS/blob/master/FACS_reprex.html

library(tidyverse)
library(openCyto)
library(ggcyto)

PBMC_luca <- list.files(path = "FR-FCM-ZZ36/", pattern = ".fcs", full.names = TRUE) %>% 
  grep(pattern = "luca", value = TRUE) %>% 
  read.ncdfFlowSet(.)

PBMC_luca[[1]] %>% 
  ggcyto(aes(x = `FSC-A`)) +
  geom_density()

PBMC_luca_gatingSet <- GatingSet(PBMC_luca)
FCS_gate1_fun <- function(fr, channel = "FSC-A"){
  peaks_found <- openCyto:::.find_peaks(x = as.numeric(unname(unlist(exprs(fr[, channel])))), num_peaks = 3)
  openCyto::gate_mindensity2(fr, channel = channel, gate_range = sort(peaks_found$x)[1:2])
}
FCS_gate1_fun(getData(PBMC_luca_gatingSet[[1]]))

if(require(openCyto)){
  thisData <- getData(PBMC_luca_gatingSet)
  nonDebris_gate <- fsApply(thisData, FCS_gate1_fun)
  add(PBMC_luca_gatingSet, nonDebris_gate, parent = "root", name = "nonDebris")
  recompute(PBMC_luca_gatingSet)
}

getData(PBMC_luca_gatingSet[[1]]) %>% 
  ggcyto(aes(x = `FSC-A`)) +
  geom_density() +
  geom_gate(FCS_gate1_fun(getData(PBMC_luca_gatingSet[[1]])))

getData(PBMC_luca_gatingSet[[1]], y = "nonDebris") %>% 
  ggcyto(aes(x = `FSC-A`)) +
  geom_density()

FCS_gate2_fun <- function(fr, channel = "FSC-A"){
  openCyto::gate_mindensity2(fr, channel = channel, positive = TRUE) #not sure if this argument exists anymore
}

getData(PBMC_luca_gatingSet[[1]], y = "nonDebris") %>% 
  ggcyto(aes(x = `FSC-A`)) +
  geom_density() +
  geom_gate(FCS_gate2_fun(getData(PBMC_luca_gatingSet[[1]], y = "nonDebris")))

if(require(openCyto)){
  thisData <- getData(PBMC_luca_gatingSet, y = "nonDebris")
  nonDebris_gate2 <- fsApply(thisData, FCS_gate2_fun)
  add(PBMC_luca_gatingSet, nonDebris_gate2, parent = "nonDebris", name = "nonDebris2")
  recompute(PBMC_luca_gatingSet)
}

getData(PBMC_luca_gatingSet[[1]], y = "nonDebris") %>% 
  ggcyto(aes(x = `FSC-A`)) +
  geom_density()

getData(PBMC_luca_gatingSet[[1]], y = "nonDebris2") %>% 
  ggcyto(aes(x = `FSC-A`)) +
  geom_density()

sessionInfo()

I'd recommend openCyto gating functions to be used with its template-based gating framework. Instead of iterating through each sample (i.e. fsApply) of each gated population (i.e. getData) and invoking mindensity and adding gates manually, you can simply submit the gating strategy to the framework and let openCyto do these boilerplate jobs for you. It can be done either through a csv template or incrementally by add_pop API. To select lower(left) end of 1d gate, simply set pop column(or argument) to "-" in csv (or add_pop() call)

Here is the example http://rpubs.com/wjiang2/464414, which is more succinct and easy to read/understand.

P.S. With more recent development version of openCyto (trunk branch of github repo), you will able to quickly undo add_pop through newly added 'remove_pop' API.(instead of manually remove gates through 'Rm' function)