Week # 10 - General Discussion Thread

[UMGCCCFCSR]

This is the Week # 10 - Down-sampling and Concatenation - Discussion Thread.

Focused on the Down-sampling portion, and wondering how to modify it to your particular workflow? Ventured into the Concatenation bonus-content and perplexed by the sausage-making process that is creating new .fcs files? 🗡️

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

Best-
David

[osterbar]

Hi, I was wondering if you could offer any suggestions. I am getting this error when running flowGate::gs_apply_gating_strategy(SFC_GatingSet, gating_strategy=GatingTable)

Error in purrr::pmap():
ℹ In index: 1.
Caused by error in .f():
! unused argument (filterDetails = .l[[1]][[i]])

part of this, is also an error from flowGate, which is
Warning: Error in as: no method or default for coercing “gg” to “ggcyto_flowSet”

flowGate is v0.99.2.
purr is 1.2.2
ggplot2 4.0.2
R 4.5.2

The flowGate has been installed from your github modified version. The gatingset has 3 files in it. If you have any suggestions, that would appreciated.

Thanks,
Andrew

[osterbar]

I managed to fix one of the problems. The Warning: Error in as: no method or default for coercing “gg” to “ggcyto_flowSet”

can be fixed by (claude vibe coding bug fixes):

Fix 1 — preparePlot.R

1. Open preparePlot.R in RStudio (File → Open File…)
2. Press ⌘ + F to open Find, then click the Replace toggle to expand it
3. Enter:
   • Find: sample.gs <- gs[[sample]]
   • Replace: sample.gs <- gs[sample]
4. Click All to replace, then ⌘ + S to save

---

Fix 2 — gs_gate_transform_interactive.R

1. Open gs_gate_transform_interactive.R
2. Press ⌘ + F → expand Replace
3. Enter:
   • Find: sample.gs <- gs[[sample]]
   • Replace: sample.gs <- gs[sample]
4. Click All, then ⌘ + S to save

Important: This file also contains lines like gs[[1]] and gh_pop_get_gate(gs[[1]], node) — leave those alone. The find/replace above is specific enough that it won't touch them.

---

Verify in the RStudio Console

grep("gs\\[sample\\]", readLines("~/path/to/preparePlot.R"), value = TRUE)
# Should print:  sample.gs <- gs[sample]

Swap in your actual file path, then re-run your gating workflow — the error should be resolved!

[UMGCCCFCSR]

Hi Andrew, what is your ggcyto version at? The ggplot2 version and flowGate version look good, but that is the other piece of the puzzle. Bioconductor's semi-yearly release happened last night, so any new problems may be due to the update. Best- David

[osterbar]

Okay, i found the other problem and it was a typo i didnt see until now. With the typo fixed, and the modifications above everything works now.

Thanks.

[DavidRach]

Hi @osterbar, can we try the code without the modifications above? That section of code was working on our end and for the in-class participants without any edits required, so that they were needed on your end to avoid errors is concerning.

Going from [[]] to [] generally means you are working with different input type objects than what the function anticipated using.

The Claude suggestions appear to be modifying the actual flowGate R package files themselves, which is not something users normally do, so you are basically creating a parallel package version and risking having it behave in odd ways later on in the course, so you might encounter additional errors going forward.

Given flowGate is not actively maintained, it is also possible you found an actual bug in the package. Since its a forked version, I can fix it, so if we can pinpoint the cause that led to the error, we can change the code at the package level, roll out an update, so that it behaves accordingly for everyone.

Can I get your package version for ggplot2, ggcyto and flowGate?

packageVersion("ggcyto")

And also a screenshot for the output when running via the terminal:

quarto render

And the code right before the error was returned. When pasting into GitHub, place four before the code, and then four after.

Sorry for the hassle, but debugging early pays off in the long run 🥰

Best-
David

[osterbar]

Hi, I tried it again this morning and everything worked. But to make sure it wasnt a version of flowGate I had modified, I deleted the flowGate folder in the R library path, quit positron and reinstalled flowWorkspace and the modified flowGate from your github, and now everything works. My apologies for the extra work this caused. Best, Andrew

…

________________________________ From: David Rach ***@***.***> Sent: Thursday, April 30, 2026 6:56 PM To: UMGCCCFCSR/CytometryInR ***@***.***> Cc: Osterburg, Andrew (osterbar) ***@***.***>; Mention ***@***.***> Subject: Re: [UMGCCCFCSR/CytometryInR] Week # 10 - General Discussion Thread (Discussion #180) External Email: Use Caution Hi @osterbar<https://github.com/osterbar>, can we try the code without the modifications above? That section of code was working on our end and for the in-class participants without any edits required, so that they were needed on your end to avoid errors is concerning. Going from [[]] to [] generally means you are working with different input type objects than what the function anticipated using. The Claude suggestions appear to be modifying the actual flowGate R package files themselves, which is not something users normally do, so you are basically creating a parallel package version and risking having it behave in odd ways later on in the course, so you might encounter additional errors going forward. Given flowGate is not actively maintained, it is also possible you found an actual bug in the package. Since its a forked version, I can fix it, so if we can pinpoint the cause that led to the error, we can change the code at the package level, roll out an update, so that it behaves accordingly for everyone. Can I get your package version for ggplot2, ggcyto and flowGate? packageVersion("ggcyto") And also a screenshot for the output when running via the terminal: quarto render And the code right before the error was returned. When pasting into GitHub, place four before the code, and then four after. Sorry for the hassle, but debugging early pays off in the long run 🥰 Best- David — Reply to this email directly, view it on GitHub<#180 (reply in thread)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/BB3AMFEY3VILOSWXLOYVFAD4YPKZXAVCNFSM6AAAAACYJ3GT7SVHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTMNZXGQ3TEOI>. Triage notifications on the go with GitHub Mobile for iOS<https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android<https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>. You are receiving this because you were mentioned.Message ID: ***@***.***>

[DavidRach]

Hi Andrew, no worries! Glad that you got it working 🙂 ☕ Best- David

[okolupaev]

Hi,
I don't know if this is technically a bug but if you try to use concatenate function with the gating set not named SFC_GatingSet, it will throw an error at you. You may want to change this line in the body of the concatenate function
dataFrameList <- purrr::map(.x=SFC_GatingSet, subset=subset, .f=Downsampling, DownsampleCount=DownsampleCount, addon=addon, returnType="data.frame", inverse.transform=inverse.transform, StorageLocation=StorageLocation)

I changed this line:
dataFrameList <- purrr::map(.x=gs, subset=subset, .f=Downsampling, DownsampleCount=DownsampleCount, addon=addon, returnType="data.frame", inverse.transform=inverse.transform, StorageLocation=StorageLocation)

I like to use different name for my variables, helps me to pay attention :-)

[DavidRach]

Hi @okolupaev, nice catch, I had caught that one after the initial release and fixed in the 4b9fadb6 commit. So everyone syncing their forks after Wednesday evening should hopefully avoid the issue 😉 Best- David

[okolupaev]

The price one pays for getting early and not refreshing the page for 3 days.. :-)

[DavidRach]

Naaah, more what happens when David is building the lesson while flying it (way less than ideal) 😄

[alexandragabrielaferreira]

Hi!

1st of all, congrats on the PhD! Awesome presentation and I learned a lot as well both about cytometry and your research! Thank you for sharing!

Back to the lessons and trying to catch up after a long break (life kinda got in the way again ):

I'm trying to run concatenate as you have in the walk-through and I get this error:

I'm not versed enough in functions to understand where the argument is unused. I checked the concatenate code and inverse.transform is set to TRUE so it should default to that if I don't pass this argument on right?

EDIT: checked that the concatenate functions uses the downsampling function and I changed argument terms like subset to gate, and inverse.transform to inverse while writing my downsampling, which meant that when concatenate calls downsampling it has nowhere to use subset and inverse.transform ::facepalm::

[DavidRach]

Hi @alexandragabrielaferreira,

Welcome to the world of making sure that your nested function arguments/names match the inputs that the main function inputs provide or generate 😸 It still happens to me a couple times a month, fortunately its error out message is fairly unique so once encountered once, it sticks as far as the next trouble shoot step.

Thanks!
David

[alexandragabrielaferreira]

Can confirm this happened again while doing problem 1 from the take home problems, and fixed it immediately :)

For the second problem I have some questions:
I actually don't understand what other keywords could we modify with downsampling that can be given through arguments. Do you have a specific example?

Personally, I was thinking of maybe changing the keyword $DATE or create a new keyword so that reflects the date the new file was created, is this even possible? But also thinking about it, it shouldn't require an argument passed to the function in the beginning...

thank you in advance :)
Best
Gabriela

[DavidRach]

So we modified GUID for that example, which often is the equivalent of GROUPNAME and TUBENAME combined. If you were trying to ensure there was no likelihood of confusing your downsampled file with the original, you could modify additional keywords beyond a single location to make it obvious. Modifying $DATE would occur in a similar manner as modifying GUID.

You can also add keywords to the description/keyword list (since it's a list which makes it editable 😄 ) . Since its a named list, you just need to provide the names. An example would be along the lines of:

AddTheseKeywords <- list(FavoriteImmuneCell = "Tcell")
UpdatedList <- c(OriginalKeywordList, AddTheseKeywords)

UpdatedFCS@description <- UpdatedList # Direct Overwrite

In a pass-through from a function argument, you would just swap the argument itself in for "Tcell"

AddTheseKeywords <- list(FavoriteImmuneCell = FunctionArgumentName)

[asalboix]

Hello everyone,

1st of all, congratulations David! Impressive PhD defence. I hope you had time to celebrate the achievement.
Thank you as well for your collaborative mindset and for teaching us on how to analyse flow cytometry data in R. I am learning a lot!

When I want to draw the gates, I am facing however a small issue. Please find attached the screenshot.

I copied the data folder of week 10 to my documents, and I am working from there. I also confimed that my computer identified the 3 .fcs files.

What might I be doing wrong, or how I could overcome this issue?

Thank you!

Anna

[DavidRach]

Thanks Anna, I think this is likely you have the Bioconductor flowGate version installed (still no reply from the maintainer about adding the bug fixes for the ggplot2/ggcyto version changes). For the course, we are currently using my fork with the changes added (should show up as either 0.99.2 or 0.99.3)

To switch out the versions, it would be

remove.packages("flowGate")
remotes::install_github("DavidRach/flowGate")

Going to reach out to the maintainer again later this week. If I don't hear back this time, might hard-fork the package since the course version now includes additional features than the original and can't have every Bioconductor update result in automated install of the non-functional version.

Best-
David

[asalboix]

Thank you David. The issue disappeared when I followed your advice. Have a nice day!