Quad gates not working

[sbg30]

Hi,

I am currently facing a problem previously reported but the provided solution did not resolve it (https://github.com/RGLab/openCyto/issues/201), I am trying to add a quad gate to an existing gatingset :

wsfile <- open_flowjo_xml('~/Documents/ICS/240506/fcs_files/07-May-2024.wsp') gatingset <- flowjo_to_gatingset(wsfile)

class(wsfile)

plot(gatingset) autoplot(gatingset[[2]])

gs_add_gating_method_init(gatingset)

gs_add_gating_method( gatingset, alias = "A,B,C,D", pop = "*", parent = "cd3neg", dims = "Comp-BUV563-A,Comp-BV605-A", gating_method = "gate_quad_sequential", gating_args = list(gFunc = 'mindensity') )

But it creates the following error:

Error in (function (fs, pp_res, gFunc, popAlias, channels, gFunc_args) : failed at 181503.fcs_0000000000000123906 Error in UseMethod("filter") : no applicable method for 'filter' applied to an object of class "flowFrame"

I also tried to create all the gates manually from raw data:

fcsfilesloaded <- load_cytoset_from_fcs(fcsfilesraw, transformation = F, compensation = F) [...] But I end up facing the same error

I made sure I had the last version of the different packages:

R version 4.4.1 (2024-06-14) Platform: aarch64-apple-darwin20 Running under: macOS Ventura 13.2.1

Matrix products: default BLAS: /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.12.0

locale: [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: Europe/Paris tzcode source: internal

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

other attached packages: [1] patchwork_1.2.0 paletteer_1.6.0 ggsci_3.2.0 forcats_1.0.0 rstatix_0.7.2 [6] ggpubr_0.6.0 stringr_1.5.1 gt_0.10.1 purrr_1.0.2 dplyr_1.1.4 [11] ggcyto_1.33.0 ncdfFlow_2.51.0 BH_1.84.0-0 ggplot2_3.5.1 flowAI_1.35.0 [16] CytoML_2.17.0 openCyto_2.17.1 flowWorkspace_4.17.0 flowCore_2.17.0

loaded via a namespace (and not attached): [1] changepoint_2.2.4 tidyselect_1.2.1 farver_2.1.2 fastmap_1.2.0 XML_3.99-0.17 [6] digest_0.6.36 lifecycle_1.0.4 magrittr_2.0.3 compiler_4.4.1 rlang_1.1.4 [11] tools_4.4.1 utf8_1.2.4 yaml_2.3.8 data.table_1.15.4 knitr_1.47 [16] ggsignif_0.6.4 labeling_0.4.3 htmlwidgets_1.6.4 plyr_1.8.9 xml2_1.3.6 [21] RColorBrewer_1.1-3 abind_1.4-5 withr_3.0.0 RProtoBufLib_2.17.0 BiocGenerics_0.51.0 [26] grid_4.4.1 stats4_4.4.1 fansi_1.0.6 colorspace_2.1-0 scales_1.3.0 [31] cli_3.6.3 rmarkdown_2.27 generics_0.1.3 rstudioapi_0.16.0 reshape2_1.4.4 [36] zlibbioc_1.51.1 parallel_4.4.1 BiocManager_1.30.23 matrixStats_1.3.0 vctrs_0.6.5 [41] jsonlite_1.8.8 carData_3.0-5 cytolib_2.17.0 car_3.1-2 S4Vectors_0.43.0 [46] RBGL_1.81.0 Rgraphviz_2.49.0 hexbin_1.28.3 tidyr_1.3.1 glue_1.7.0 [51] rematch2_2.1.2 stringi_1.8.4 gtable_0.3.5 munsell_0.5.1 tibble_3.2.1 [56] pillar_1.9.0 htmltools_0.5.8.1 graph_1.83.0 R6_2.5.1 evaluate_0.24.0 [61] lattice_0.22-6 Biobase_2.65.0 backports_1.5.0 broom_1.0.6 Rcpp_1.0.12 [66] flowClust_3.43.0 gridExtra_2.3 xfun_0.45 zoo_1.8-12 pkgconfig_2.0.3

Is there something I am doing wrong ?

Thanks

[sbg30]

EDIT: I have found a conflicting package that had another "filter" function and removed it, allowing for the gate_quad_sequential function to operate.

However, I still face some difficulties while trying to use it in a gatingTemplate csv file:

When I tried to use this:

alias	pop	parent	dims	gating_method	gating_args
A,B,C,D	*	cd3neg	cd16,cd56	gate_quad_sequential	gFunc='mindensity'

I face the same error as was described before (https://github.com/RGLab/openCyto/issues/241) : Error in pops[[2]] : subscript out of bounds

I then assumed that the new version had not been merged yet and that pop=* is not yet available for the quadGate.seq ?

But then when I tried

alias	pop	parent	dims	gating_method	gating_args
'*	+/-+/-	cd3neg	cd16,cd56	gate_quad_sequential	gFunc='mindensity'
nk	+	cd3neg	cd16,cd56	boolGate	!cd16-cd56-

Then I face another error : Error in h(simpleError(msg, call)) : error in evaluating the argument 'from' in selecting a method for function 'addEdge': Not able to to find reference to: cd16-cd56- (same thing if I use the channels BUV563-A and BV605-A instead of the marker names). Even though the quad gates are correctly designed (displayed through ggcyto), it looks like the reference gating names created by the quadgate are not correctly included in the gating tree ?

Thanks !

[feanaros]

hello @sbg30 , which package did you use for filtering?

[djhammill]

OK so the issue is that there is still a check that looks for the default quadrant names here: https://github.com/RGLab/openCyto/blob/323430ebb251e69db7f694751f2a9a2e5cba4d26/R/gating-methods.R#L384-L393

We will probably need to bypass it when pop = "*". Working on a fix now.

[djhammill]

@sbg30 I have added the fix. There are a number of changes coming to openCyto over the coming days so there might a delay in getting the fix onto BioConductor for you. In the meantime, you can install directly from GitHub as follows:

devtools::install_github("RGLab/openCyto", ref = "bugfix/ODC-185-Bypass-Quadrant-Selection")

Your openCyto version won't change but your code should run as expected. Please let us know if you encounter any issues after updating.

[djhammill]

You can track the progress of getting this fix on BioConductor here: https://github.com/RGLab/openCyto/pull/263.

[sbg30]

Thank you so much ! I'll try it as soon as possible !