Calculates the single color control matrix for a given quantile cutoffs and a given statistic

Calculates the single color control matrix for a given quantile cutoffs and a given statistic

Usage

Luciernaga_SingleColors(
  x,
  sample.name,
  removestrings,
  subset,
  PanelCuts,
  stats,
  SignatureView,
  Verbose = FALSE,
  returntype = "data"
)

Arguments

x

A Gating Set Object

sample.name

The Keyword for which the Fluorophore Name is stored

removestrings

Values to remove from the name

subset

A desired Gating Hierarchy level of cells to filter in

PanelCuts

A .csv or dataframe containing columns Fluorophore, From and To Fluorophore name should match sample.name style

stats

Whether to use "mean" or "median"

SignatureView

Whether to also return a normalized signature plot.

Verbose

Provides debugging for removestrings

returntype

Allows to modify default "data" to instead return the "plots"

Value

A tibble row for each flow object containing the summarized data.

Examples

library(flowCore)
library(flowWorkspace)
library(openCyto)
library(data.table)
library(dplyr)
library(purrr)
library(stringr)

StorageLocation <- file.path(tempdir(), "LuciernagaTreeExample")
if (!dir.exists(StorageLocation)) {dir.create(StorageLocation)}

File_Location <- system.file("extdata", package = "Luciernaga")
FCS_Files <- list.files(path = File_Location, pattern = ".fcs",
  full.names = TRUE)
CellSingleColorFiles <- FCS_Files[grep("Cells", FCS_Files)]
CellSingleColors <- CellSingleColorFiles[!str_detect("Unstained", CellSingleColorFiles)]
MyCytoSet <- load_cytoset_from_fcs(CellSingleColors,
  truncate_max_range = FALSE,transformation = FALSE)
MyGatingSet <- GatingSet(MyCytoSet)
MyGates <- fread(file.path(path = File_Location, pattern = 'Gates.csv'))
MyGatingTemplate <- gatingTemplate(MyGates)
#> Adding population:singletsFSC
#> Adding population:singletsSSC
#> Adding population:singletsSSCB
#> Adding population:nonDebris
#> Adding population:lymphocytes
gt_gating(MyGatingTemplate, MyGatingSet)
#> Gating for 'singletsFSC'
#> done!
#> done.
#> Gating for 'singletsSSC'
#> done!
#> done.
#> Gating for 'singletsSSCB'
#> done!
#> done.
#> Gating for 'nonDebris'
#> done!
#> done.
#> Gating for 'lymphocytes'
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> done!
#> done.
#> finished.

UnstainedFCSFiles <- FCS_Files[grep("Unstained", FCS_Files)]
UnstainedCells <- UnstainedFCSFiles[-grep("Beads", UnstainedFCSFiles)]
MyUnstainedCytoSet <- load_cytoset_from_fcs(UnstainedCells[1],
  truncate_max_range = FALSE,transformation = FALSE)
MyUnstainedGatingSet <- GatingSet(MyUnstainedCytoSet)
gt_gating(MyGatingTemplate, MyUnstainedGatingSet)
#> Gating for 'singletsFSC'
#> done!
#> done.
#> Gating for 'singletsSSC'
#> done!
#> done.
#> Gating for 'singletsSSCB'
#> done!
#> done.
#> Gating for 'nonDebris'
#> done!
#> done.
#> Gating for 'lymphocytes'
#> The prior specification has no effect when usePrior=no
#> Using the serial version of flowClust
#> done!
#> done.
#> finished.

removestrings <-  c(".fcs", "(", ")", "Cells")
FileLocation <- system.file("extdata", package = "Luciernaga")
pattern = "AutofluorescentOverlaps.csv"
AFOverlap <- list.files(path=FileLocation, pattern=pattern, full.names = TRUE)

TheCellAF  <- Luciernaga_QC(x=MyUnstainedGatingSet[[1]], subsets="nonDebris",
 removestrings=removestrings, sample.name="GUID", unmixingcontroltype = "cells",
 Unstained = TRUE, ratiopopcutoff = 0.001, Verbose = FALSE,
 AFOverlap = AFOverlap, stats = "median", ExportType = "fcs",
 Brightness=TRUE, SignatureReturnNow = TRUE, outpath = StorageLocation,
 Increments=0.1, SecondaryPeaks=2, experiment = "FirstExperiment",
 condition = "ILTPanel", Subtraction = "Internal", CellAF=TheCellAF,
 SCData="subtracted",NegativeType="default", minimalfcscutoff=0.01)
#> Normalizing Data for Signature Comparison

 MyGatingSet <- subset(MyGatingSet, !str_detect(name, "CD14"))

SingleColor_Data <- map(.x=MyGatingSet, .f=Luciernaga_QC,
 subsets="lymphocytes", removestrings=removestrings, sample.name="GUID",
 unmixingcontroltype = "cells", Unstained = FALSE, ratiopopcutoff = 0.001,
 Verbose = FALSE, AFOverlap = AFOverlap, stats = "median", ExportType = "fcs",
 Brightness=TRUE, SignatureReturnNow = FALSE,outpath = StorageLocation,
 Increments=0.1, SecondaryPeaks=2, experiment = "FirstExperiment",
 condition = "ILTPanel", Subtraction = "Internal", CellAF=TheCellAF,
  SCData="subtracted",NegativeType="default", minimalfcscutoff=0.02)
#> No second peak
#> No second peak
#> No second peak
#> Only a single detector present. If this was not an autofluorescence overlap
#>               fluourophore, it would suggest there was no antibody staining, or everything
#>               was overstained. Please investigate further.
#> No second peak
#> No second peak
#> No second peak

Unstained_Data  <- map(.x=MyUnstainedGatingSet, .f=Luciernaga_QC,
 subsets="nonDebris", removestrings=removestrings, sample.name="GUID",
 unmixingcontroltype = "cells", Unstained = TRUE, ratiopopcutoff = 0.001,
 Verbose = FALSE, AFOverlap = AFOverlap, stats = "median", ExportType = "fcs",
 Brightness=TRUE, SignatureReturnNow = FALSE, outpath = StorageLocation,
  Increments=0.1, SecondaryPeaks=2, experiment = "FirstExperiment",
  condition = "ILTPanel", Subtraction = "Internal", CellAF=TheCellAF,
   SCData="subtracted",NegativeType="default", minimalfcscutoff=0.01)

PanelPath <- file.path(File_Location, "UnmixingPanel.csv")
MoveThese <- Luciernaga_Tree(BrightnessFilePath = StorageLocation,
 PanelPath = PanelPath)

SortedStorageLocation <- file.path(tempdir(), "LuciernagaSelected")
if (!dir.exists(SortedStorageLocation)) {dir.create(SortedStorageLocation)}

UnmixingPanel <- read.csv(PanelPath, check.names=FALSE)
TheseFluorophores <- UnmixingPanel %>% pull(Fluorophore)

walk(.x=TheseFluorophores, .f=Luciernaga_Move, data=MoveThese,
 input=StorageLocation, output=SortedStorageLocation)
MovedFiles <- list.files(SortedStorageLocation, pattern="fcs", full.names=TRUE)

UnmixingPanel <- read.csv(PanelPath, check.names=FALSE)
ThePanelCuts <- UnmixingPanel %>% select(-Detector) %>%
 mutate(From=0.3) %>% mutate(To=1)

MovedFiles <- list.files(SortedStorageLocation,
  pattern="fcs", full.names=TRUE)
Selected_CS <- load_cytoset_from_fcs(MovedFiles[1],
  truncate_max_range = FALSE, transform = FALSE)
Selected_GS <- GatingSet(Selected_CS)

removestrings=c("DR_", ".fcs")

SCs <- map(.x=Selected_GS[1], .f=Luciernaga_SingleColors,
 sample.name="TUBENAME", removestrings=removestrings, subset="root",
  PanelCuts=ThePanelCuts, stats="median", Verbose=TRUE, SignatureView=TRUE,
   returntype = "plots")
#> After removestrings cleanup the name is CCR4 BUV615
#> The Fluorophore is BUV615 and the ligand is CCR4