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Error in COMPASSContainerFromGatingSet due to boolean gate? #83

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pbMCs opened this issue Aug 7, 2024 · 0 comments
Open

Error in COMPASSContainerFromGatingSet due to boolean gate? #83

pbMCs opened this issue Aug 7, 2024 · 0 comments

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@pbMCs
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pbMCs commented Aug 7, 2024

Hello! It's my first time using COMPASS and I have tried to resolve this issue by myself and looking at other people's scripts but I can't figure it out... My cytokine gates for IL-2 and IFNg came from a quadrant gate previously gated on flowjo, so for this analysis I previously created two new gates through a boolean filter taking the events from the corresponding quadrants with the gs_pop_add function from the flowWorkspace package:

bgIL2<- booleanFilter(CD4+/Q2: IL2+ , IFNG+|CD4+/Q3: IL2+ , IFNG-, filterId = "IL-2")
gs_pop_add(gs1, bgIL2, parent= "CD4+")

bgIFNG<- booleanFilter(CD4+/Q1: IL2- , IFNG+|CD4+/Q2: IL2+ , IFNG+, filterId = "IFNg")
gs_pop_add(gs1, bgIFNG, parent= "CD4+")

These gates seem correctly added to the gatingset and appear as "child nodes" of CD4+ when running gs_get_pop_path():

gs_get_pop_paths(gs1)
[1] "root"
[2] "/Time"
[3] "/Time/Single Cells SSC"
[4] "/Time/Single Cells SSC/Single Cells FSC"
[5] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes"
[6] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells"
[7] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+"
[8] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+"
[9] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+/CD40L+"
[10] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+/GRZB"
[11] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+/IL-4"
[12] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+/IL-13"
[13] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+/IL-21"
[14] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+/TNFa"
[15] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+/IL-2"
[16] "/Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+/IFNg"

But then when creating the compass container from the gating set I get this error (which disappears when removing those nodes):

Error in COMPASSContainerFromGatingSet(gs1[[2]], node = parent_node, individual_id = id, :
IL-2|IFNg are not the children node of /Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+

To Reproduce

library(flowWorkspace)
#> Warning: package 'flowWorkspace' was built under R version 4.3.3
#> As part of improvements to flowWorkspace, some behavior of
#> GatingSet objects has changed. For details, please read the section
#> titled "The cytoframe and cytoset classes" in the package vignette:
#> 
#>   vignette("flowWorkspace-Introduction", "flowWorkspace")
library(COMPASS)
library(CytoML)
library(flowCore)
#> Warning: package 'flowCore' was built under R version 4.3.3
setwd("C:/Users/mcanyelles/Documents/Malaria Immunology/Studies/MAL067 T Cell Study/Results/Exploratory ICS MainBoost/COMPASS")

fcs_path<- "C:/Users/mcanyelles/Documents/Malaria Immunology/Studies/MAL067 T Cell Study/Results/Exploratory ICS MainBoost/COMPASS/data/fcs/20220526 TFH FCS"
ws1<- open_flowjo_xml("C:/Users/mcanyelles/Documents/Malaria Immunology/Studies/MAL067 T Cell Study/Results/Exploratory ICS MainBoost/COMPASS/data/20220526 ICS.wsp")
keywords2import=c("TUBE NAME","EXPERIMENT NAME")
SampleGroup<- "Test Samples"

gs1<- flowjo_to_gatingset(ws1, name=SampleGroup, keywords= keywords2import, path= fcs_path, extend_val= -10000)
# 
# pop_lists <- lapply(gs1, gh_get_pop_paths)
# unique(pop_lists)

gs1 <- gs_remove_redundant_channels(gs1) 

# will need to handle the IFNG and IL2 gates being quadrants 
# create a boolean gate to unify the 2 positive quadrants for each marker 

bgIL2<- booleanFilter(`CD4+/Q2: IL2+ , IFNG+|CD4+/Q3: IL2+ , IFNG-`, filterId = "IL-2")
gs_pop_add(gs1, bgIL2, parent= "CD4+")
#> [1] 262
bgIFNG<- booleanFilter(`CD4+/Q1: IL2- , IFNG+|CD4+/Q2: IL2+ , IFNG+`, filterId = "IFNg")
gs_pop_add(gs1, bgIFNG, parent= "CD4+")
#> [1] 263
recompute(gs1, "IL-2")
#> done!
recompute(gs1, "IFNg")
#> done!
# autoplot(gs1, "IL-2")
# autoplot(gs1, "IFNg")
# 
# gs_pop_remove(gs1, "CD4+/Q1: IL2- , IFNG+")
# gs_pop_remove(gs1, "CD4+/Q2: IL2+ , IFNG+")
# gs_pop_remove(gs1, "CD4+/Q3: IL2+ , IFNG-")
# gs_pop_remove(gs1, "CD4+/Q4: IL2- , IFNG-")
# gs_pop_remove(gs1, "CD40L+ or Q2: IL2+ , IFNG+ or Q3: IL2+ , IFNG-+ or TNFa+")

dput(unname(pData(parameters(gh_pop_get_data(gs1[[1]])))[,2]))
#> structure(c(NA, NA, NA, NA, "CD45RA", "IL21", "PD1", "CXCR5", 
#> "IL2", "IL4", "TNFA", "CCR7", "IL13", "CD8", "CD40L", "CD3", 
#> "CD14_AVID", "IFNG", "CD56", "CD4", "GRZMB", NA), class = "AsIs")
# structure(c(NA, NA, NA, NA, "CD45RA", "IL21", "PD1", "CXCR5", 
#             "IL2", "IL4", "TNFA", "CCR7", "IL13", "CD8", "CD40L", "CD3", 
#             "CD14_AVID", "IFNG", "CD56", "CD4", "GRZMB", NA), class = "AsIs")

markernames <- c("FSC-A", "FSC-H", "SSC-A", "SSC-H", "CD45RA", "IL21", "PD1", "CXCR5", 
                 "IL2", "IL4", "TNFA", "CCR7", "IL13", "CD8", "CD40L", "CD3", 
                 "CD14_AVID", "IFNG", "CD56", "CD4", "GRZMB", "Time" )
names(markernames) <- pData(parameters(gh_pop_get_data(gs1[[1]])))[,1]
markernames(gs1) <- markernames
pData(parameters(gh_pop_get_data(gs1[[1]])))[,c(1,2)]
#>                         name      desc
#> $P1                    FSC-A     FSC-A
#> $P2                    FSC-H     FSC-H
#> $P3                    SSC-A     SSC-A
#> $P4                    SSC-H     SSC-H
#> $P5  Comp-640-780_60-750LP-A    CD45RA
#> $P6        Comp-640-660_20-A      IL21
#> $P7  Comp-561-780_60-750LP-A       PD1
#> $P8  Comp-561-610_20-600LP-A     CXCR5
#> $P9  Comp-561-582_15-570LP-A       IL2
#> $P10 Comp-488-710_50-685LP-A       IL4
#> $P11 Comp-488-525_50-505LP-A      TNFA
#> $P12 Comp-403-780_60-750LP-A      CCR7
#> $P13 Comp-403-710_50-685LP-A      IL13
#> $P14 Comp-403-670_50-635LP-A       CD8
#> $P15 Comp-403-620_40-595LP-A     CD40L
#> $P16 Comp-403-585_42-570LP-A       CD3
#> $P17 Comp-403-525_50-505LP-A CD14_AVID
#> $P18       Comp-403-450_50-A      IFNG
#> $P19 Comp-350-730_45-685LP-A      CD56
#> $P20       Comp-350-379_28-A       CD4
#> $P21 Comp-640-710_50-690LP-A     GRZMB
#> $P22                    Time      Time
nodes_to_remove<- c("CD8+", "CD3-, CD56dim","CD3-, CD56hi",  "NKT-like","CD8-, CD4 -", "Q5: CCR7- , CD45RA+", "Q6: CCR7+ , CD45RA+ (Naïve)", 
                    "Q7: CCR7+ , CD45RA- (TCM)", "Q8: CCR7- , CD45RA- (TEM)", "CXCR5")

lapply(nodes_to_remove, function(node){
  gs1<- gs_pop_remove(gs1, node)
})

# plot(gs1, fontsize=70, bool=T)

get_stim <- function(x) {
  if (grepl("DMSO", x)) {
    "NC"
  } else {
    "CSP"
  }
}

pData(gs1)$stim<- sapply(pData(gs1)$name, get_stim)

get_id <- function(tube_name) {
  sub("_.*", "", tube_name)}


pData(gs1)$ID<- sapply(pData(gs1)$`TUBE NAME`, get_id)

# save_gs(gs1, file.path(getwd(),"out/GatingSet"))

# save_gslist()

set.seed(123)

parent_node<- "CD4+"

id<- "ID"

markernames(gs1)
#> Comp-640-780_60-750LP-A       Comp-640-660_20-A Comp-561-780_60-750LP-A 
#>                "CD45RA"                  "IL21"                   "PD1" 
#> Comp-561-610_20-600LP-A Comp-561-582_15-570LP-A Comp-488-710_50-685LP-A 
#>                 "CXCR5"                   "IL2"                   "IL4" 
#> Comp-488-525_50-505LP-A Comp-403-780_60-750LP-A Comp-403-710_50-685LP-A 
#>                  "TNFA"                  "CCR7"                  "IL13" 
#> Comp-403-670_50-635LP-A Comp-403-620_40-595LP-A Comp-403-585_42-570LP-A 
#>                   "CD8"                 "CD40L"                   "CD3" 
#> Comp-403-525_50-505LP-A       Comp-403-450_50-A Comp-350-730_45-685LP-A 
#>             "CD14_AVID"                  "IFNG"                  "CD56" 
#>       Comp-350-379_28-A Comp-640-710_50-690LP-A 
#>                   "CD4"                 "GRZMB"
markermap<- list("IL2", 
                 "IL4", "IL21", "TNFA", "IL13", "CD40L", "IFNG", "GRZMB")

names(markermap)<- c("IL-2", "IL-4", "IL-21", "TNFa", "IL-13", "CD40L+", "IFNg", "GRZB")

cc<- COMPASSContainerFromGatingSet(gs1[[2]],
                                   node= parent_node,
                                   individual_id= id,
                                   mp= markermap,
                                   countFilterThreshold = 5000,
                                   filter.fun= NULL)
#> Extracting cell counts
#> Fetching CD4+
#> Fetching child nodes
#> common markers are:
#> FSC-A FSC-H SSC-A SSC-H CD45RA IL21 PD1 CXCR5 IL2 IL4 TNFA CCR7 IL13 CD8 CD40L CD3 CD14_AVID IFNG CD56 CD4 GRZMB Time
#> Extracting single cell data for IL-2|IL-4|IL-21|TNFa|IL-13|CD40L+|IFNg|GRZB
#> Error in COMPASSContainerFromGatingSet(gs1[[2]], node = parent_node, individual_id = id, : IL-2|IFNg are not the children node of /Time/Single Cells SSC/Single Cells FSC/Lymphocytes/Live Cells/CD3+/CD4+

Created on 2024-08-07 with reprex v2.1.1

sessionInfo():
R version 4.3.1 (2023-06-16 ucrt)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19045)

Matrix products: default

locale:
[1] LC_COLLATE=Spanish_Spain.utf8 LC_CTYPE=Spanish_Spain.utf8 LC_MONETARY=Spanish_Spain.utf8 LC_NUMERIC=C
[5] LC_TIME=Spanish_Spain.utf8

time zone: Europe/Madrid
tzcode source: internal

attached base packages:
[1] stats graphics grDevices utils datasets methods base

other attached packages:
[1] ggcyto_1.30.2 ncdfFlow_2.48.0 BH_1.84.0-0 ggplot2_3.5.1 flowViz_1.66.0 lattice_0.22-6
[7] openCyto_2.14.0 COMPASS_1.40.0 flowWorkspace_4.14.3 flowCore_2.14.2 CytoML_2.14.0

loaded via a namespace (and not attached):
[1] gtable_0.3.5 xfun_0.45 latticeExtra_0.6-30 Biobase_2.62.0 vctrs_0.6.5 tools_4.3.1
[7] generics_0.1.3 stats4_4.3.1 parallel_4.3.1 tibble_3.2.1 fansi_1.0.6 cluster_2.1.6
[13] pkgconfig_2.0.3 KernSmooth_2.23-24 data.table_1.15.4 RColorBrewer_1.1-3 S4Vectors_0.40.2 graph_1.80.0
[19] lifecycle_1.0.4 farver_2.1.2 compiler_4.3.1 deldir_2.0-4 pdist_1.2.1 munsell_0.5.1
[25] clue_0.3-65 yaml_2.3.8 hexbin_1.28.3 pillar_1.9.0 tidyr_1.3.1 MASS_7.3-60.0.1
[31] abind_1.4-5 RProtoBufLib_2.14.1 tidyselect_1.2.1 dplyr_1.1.4 purrr_1.0.2 labeling_0.4.3
[37] flowClust_3.40.0 grid_4.3.1 colorspace_2.1-0 cli_3.6.2 magrittr_2.0.3 RBGL_1.78.0
[43] XML_3.99-0.16.1 utf8_1.2.4 withr_3.0.0 scales_1.3.0 matrixStats_1.3.0 jpeg_0.1-10
[49] interp_1.1-6 gridExtra_2.3 cytolib_2.14.1 png_0.1-8 knitr_1.47 rlang_1.1.3
[55] Rcpp_1.0.12 glue_1.7.0 Rgraphviz_2.46.0 BiocGenerics_0.48.1 rstudioapi_0.16.0 jsonlite_1.8.8
[61] R6_2.5.1 plyr_1.8.9 IDPmisc_1.1.21 zlibbioc_1.48.2

Here is the link to the workspace and fcs files to reproduce the error (I only included 2 samples): https://www.dropbox.com/scl/fo/hi5rj6kf7a3gqbjzjiwi2/AKjr-kB96d_V67eLR9PhDIc?rlkey=ujqo7jgbrjhmj6b1mjyrgwuh3&st=ab5jl4yg&dl=0

Thank you very much, any comment or suggestion will be very useful!
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