Week #12 - General Discussion Thread

[UMGCCCFCSR]

This is the Week # 12 - Spectral Signatures - Discussion Thread.

Having too much fun extracting signatures, visualizing them as interactive plots via plotly, and have come up with a grand unified theory of autofluorescence you want to send off to Cytometry Part A? Or is your data from a non-Cytek Aurora, found a bug, and want to report it so that the bug fixes can quickly get made before next week? Either way, welcome!

If it doesn't merit it's own discussion thread, this is the place for it 🥳

Best-
David

[ToniAms]

I try to reinstall flowgate and luciernaga but I get error messages, see below. What should I change?

library(remotes)

• install_github("NKInstinct/flowGate") # Alternatively via BiocManager::install for the "developmental" branch
• install_github("DavidRach/Luciernaga")
  Warning message:
  package ‘remotes’ was built under R version 4.5.3
  Using github PAT from envvar GITHUB_PAT. Use gitcreds::gitcreds_set() and unset GITHUB_PAT in .Renviron (or elsewhere) if you want to use the more secure git credential store instead.
  Downloading GitHub repo NKInstinct/flowGate@HEAD
  These packages have more recent versions available.
  It is recommended to update all of them.
  Which would you like to update?

1: All
2: CRAN packages only
3: None
4: ggcyto (e5ed28665... -> 2484d0d88...) [GitHub]

Enter one or more numbers, or an empty line to skip updates:
1
ggcyto (e5ed28665... -> 2484d0d88...) [GitHub]
Downloading GitHub repo RGLab/ggcyto@HEAD
── R CMD build ──────────────────────────────────────────────────────────────────────────────────
✔ checking for file 'C:\Users\toniv\AppData\Local\Temp\RtmpMzQL1Y\remotes53c445524ec2\RGLab-ggcyto-2484d0d/DESCRIPTION' ...
─ preparing 'ggcyto': (566ms)
✔ checking DESCRIPTION meta-information ...
─ installing the package to process help pages
-----------------------------------
─ installing source package 'ggcyto' ...
** this is package 'ggcyto' version '1.41.1'
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
Warning message:
package 'ggplot2' was built under R version 4.5.3
Error in conformMethod(signature, mnames, fnames, f, fdef, definition) :
in method for 'transform' with signature '_data="filter"': formal arguments (_data = "filter", ... = "filter") omitted in the method definition cannot be in the signature
Error: unable to load R code in package 'ggcyto'
Execution halted
ERROR: lazy loading failed for package 'ggcyto'
─ removing 'C:/Users/toniv/AppData/Local/Temp/RtmpAT54So/Rinst402411fc3efe/ggcyto'
-----------------------------------
ERROR: package installation failed

Error:
! Failed to install 'flowGate' from GitHub:
Failed to install 'ggcyto' from GitHub:
! System command 'Rcmd.exe' failed

Show Traceback

[DavidRach]

Hi @ToniAms , can you check your ggplot2 version that is installed?

packageVersion("ggplot2")

If it is still a version 3.5. and not version 4. go ahead and reinstall it as well.

Best-
David

[ToniAms]

Hi David,,
thanks for your quick answer!
it is 4.0.3

[DavidRach]

May be something new then, I will check it out when I get to work. What are your current ggcyto, flowGate and Luciernaga versions at?

[ToniAms]

packageVersion("ggcyto")
[1] ‘1.39.1’

packageVersion("flowGate")
[1] ‘1.10.0’

Luciernaga, don't know, I removed the package and now I try to reinstall the way we did in week 11 but it fails.

[carloscgomiz]

Hi! I actually had an issue running the script during the stream. When adding the corrected metadata to the gating set, I get the following error:

pData(BeadsSC_GatingSet) <- Updated_bead_pd

Error:
! 1 not found!

I have tried removing one variable at a time, but it does not fix it. Also, it happens with all GatingSets, not only with beads. I have tried updating all packages, including flowWorkspace.

Since I have neither been able to render the .qmd file due to other errors (related to load_gs function), I imagine it has something to do with the flowGate package. Has someone else encountered this?

[DavidRach]

Hi @ToniAms AND @carloscgomiz,

I suspect this merged pull request to ggcyto is the issue for both of you. Relates to this broader change to Bioconductor transform

For now, please install this version immediately pre-change for ggcyto and then retry the install/run the workflow. Let me know if this resolves the issue!

Best-
David

remotes::install_github("RGLab/ggcyto", ref="6c11f2e")

[carloscgomiz]

Hi David! Thank you for your response. I have tried to reinstall ggcyto, but it does not seem to do the trick. I had problems with that before, but now I am able to do the gating, it is adding the metadata and loading the GatingSet I am having problems with. Here's the issue I get when rendering the .qmd file btw:

104/229 [unnamed-chunk-52]
Error:
! Failed to parse GatingSet.
Backtrace:
▆

1. └─flowWorkspace::load_gs(SaveUpdatedCellSCHere)
2. ├─methods::new(...)
3. │ ├─methods::initialize(value, ...)
4. │ └─methods::initialize(value, ...)
5. └─flowWorkspace:::cpp_loadGatingSet(...)

Quitting from index.qmd:622-628 [unnamed-chunk-52]
Execution halted

On the other hand, the error I get adding the metadata seems to trace back to flowWorkspace:

pData(BeadsSC_GatingSet) <- Updated_bead_pd
Error:
! 1 not found!
▆

1. ├─Biobase::pData<-(*tmp*, value = <df[,4]>)
2. └─flowWorkspace::pData<-(*tmp*, value = <df[,4]>)
3. ├─Biobase::pData<-(*tmp*, value = value)
4. └─flowWorkspace::pData<-(*tmp*, value = value)

5. └─flowWorkspace:::set_pheno_data(object@pointer, value)
   

This is the sessionInfo (just for the record, I guess there's not much to do but to see if anyone else gets the same issue):

sessionInfo()
R version 4.6.1 (2026-06-24 ucrt)
Platform: x86_64-w64-mingw32/x64
Running under: Windows 11 x64 (build 26200)

Matrix products: default
LAPACK version 3.12.1

locale:
[1] LC_COLLATE=Spanish_Spain.utf8 LC_CTYPE=Spanish_Spain.utf8
[3] 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] stringr_1.6.0 dplyr_1.2.1 Luciernaga_0.99.10
[4] flowGate_1.13.1 ggcyto_1.41.0 ncdfFlow_2.58.0
[7] BH_1.90.0-1 flowCore_2.24.0 ggplot2_4.0.3
[10] flowWorkspace_4.24.0 remotes_2.5.0

Thanks again!

Carlos

[ToniAms]

Thanks David! This worked for me and I now have the right versions of both Flowgate and Luciernaga.

[DavidRach]

@carloscgomiz, can you send to the flowcore email the .qmd or .R file you are working from, I want to make sure it is not something odd on the file pathing. Also in your console, run getwd() to verify your working environment current location

Are you able to see the metadata after the data tidying, but are not able to append it back in place?

I will also try cross-matching vs. those installed package versions in case its a change Herve made leading to metadata loss.

[carloscgomiz]

Hi David, I'm just trying to run the index.qmd file from the course. The working directory and the output location should be fine both for rendering and for running the code, because I can import the data, do the gating and even export the metadata .csv file. There seems to be something off with the pData() <- value function. Also something weird is that rendering breaks at the load_gs() function, and not at the save_gs() function that comes several times before. Could it be due to some bug with the flowWorkspace package? (I tried reinstalling already)

Regards,
Carlos.

[asalboix]

Hello everyone,

When running the "LLM code" on beads single colors to get fluorophore, antigen and type (see below), I am not getting the expected result. Instead, I get "name", "fluorophore", "antigen", "type". Do you have any idea of why it might be that?

Thank you. Best,

Anna

bead_pd <- pData(BeadsSC_GatingSet)

Updated_bead_pd <- bead_pd |> mutate(
raw = str_extract(name, "(?<=DR_)[^(]+(?=\()"),
Antigen = raw |>
str_trim() |>
str_extract("^\S+"),
Fluorophore = raw |>
str_trim() |>
str_remove("^\S+\s+"),
Type = name |>
str_extract("(?<=\()([^)]+)(?=\))"),
raw = NULL
)

Updated_bead_pd <- Updated_bead_pd |> relocate(Fluorophore, Antigen, Type, .after="name")

[DavidRach]

Hi Anna, can run the following:

colnames(bead_pd)
colnames(Updated_bead_pd)

Double check if it name is present, or was already previously renamed?

[asalboix]

Hello David,

colnames(bead_pd)
[1] "name"
colnames(Updated_bead_pd)
[1] "name" "Fluorophore" "Antigen" "Type"

Name is present, I guess that is the issue why I am seeing it, right?

[asalboix]

Hello everyone,

I did not do yet the Retrieving Signature Matrices, and I am doing now Spectral Signatures.

May it be the reason why I am getting this error after doing:

StoreHere <- file.path(OutputLocation, "CellInitialSignatureMatrix.csv")
CellFluorophoreSignatures <- read.csv(StoreHere, check.names=FALSE)
head(CellFluorophoreSignatures, 3)

Warning message:
In file(file, "rt") :
cannot open file 'outputs/CellInitialSignatureMatrix.csv': No such file or directory
Error in file():
! cannot open the connection

Hide Traceback
▆

1. └─utils::read.csv(StoreHere, check.names = FALSE)
2. └─utils::read.table(...)

3. └─base::file(file, "rt")
   

(So that I have not generated that csv file)

THANK YOU!

[DavidRach]

Hi @asalboix, checking your fork of the class repository, you do have the files present:

As far as why the file.path() argument is breaking, a couple possibilities. If you are working within a new "Week12" project repository, its possible that the above .csv files were not copied over from the CytometryInR folder fork. Alternatively, you might have slightly different folder names between your working environment and the folder where the files are being stored.

Try troubleshooting via

IAmHere <- getwd() #Identify where you are
list.files(IAmHere) # See subfolder names
NowCheckHere <- file.path(IamHere, "outputs_or_however_folder_called)
list.files(NowCheckHere, full.names=TRUE) # repeat the process until you have set found the .csv file, then pass to read.csv

Also remember to sync your fork and pull changes locally (in case it is a bug that I end up catching after the fact)

Best Wishes-
David

[asalboix]

Thank you so much David, have a great weekend!