Problem with CytoNorm Prep

[MBakker06]

Hi, I'm running into an issue when trying to run the following code during batch alignment:

align.model <- prep.cytonorm(dat = ref.dat, cellular.cols = c(cellular.cols, paste0(cellular.cols, crs.append)), cluster.cols = paste0(cluster.cols, crs.append), batch.col = batch.col, dir = fine.dir, xdim = 14, ydim = 14, meta.k = 8)

It gets stuck during step 2/4 and then I receive the following error:

Step 1/4 - Splitting files for use with original FlowSOM function
Step 2/4 - Running FlowSOM
Reading 1.fcs
Error in if (sampleWithReplacement & (nrow(f) < cFile)) { :
argument is of length zero

I followed the Batch alignment code from the general discovery workflow:
https://github.com/ImmuneDynamics/Spectre/blob/master/workflows/Classic%20workflows/Spectre%20general%20discovery%20workflow/2%20-%20batch%20alignment.R

Does anyone have an idea how to resolve this issue, any help is much appreciated! :)

[tomashhurst]

Hi @MBakker06 , thanks for reaching out! If you run the code here on the workflow page (https://wiki.centenary.org.au/display/SPECTRE/Discovery+workflow+with+batch+alignment+using+CytoNorm), do you get a successful result?

[ghar1821]

Can you run sessionInfo() and post the content? This is to see what versions of what packages you are using.

[MBakker06]

sessionInfo()
R version 4.3.0 (2023-04-21 ucrt)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19045)

Matrix products: default

locale:
[1] LC_COLLATE=English_Netherlands.utf8 LC_CTYPE=English_Netherlands.utf8 LC_MONETARY=English_Netherlands.utf8
[4] LC_NUMERIC=C LC_TIME=English_Netherlands.utf8

time zone: Europe/Amsterdam
tzcode source: internal

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

loaded via a namespace (and not attached):
[1] Spectre_1.0.0 compiler_4.3.0 tools_4.3.0 rstudioapi_0.14 data.table_1.14.8

[ghar1821]

Ah I'm hoping to find the version of FlowSOM and Cytonorm you have installed. Can you find that and paste them here? You can use packageVersion("flowsom") function (replace flowsom with cytonorm for cytonorm). Also, did u install Spectre from our master or development branch?

[MBakker06]

I installed Spectre from the master branch.
FlowSOM v2.8.0
CytoNorm v0.0.17
(CytoNorm was installed with install_github('saeyslab/CytoNorm') using devtools

[ghar1821]

Can you try re-installing Spectre using the development branch? You can use install_github(immunedynamics/Spectre, ref="development"). We may have had some issues with the alignment functions in the past which may have been resolved in our development branch but not yet pushed to the master branch.

[mortadelo93]

I have the same issue.
Did anything work?

[ghar1821]

we recently deployed a new version. Can you try re-installing and see if it solves the problem?

[mortadelo93]

Hi
Thanks for the replay.

Im afraid the error still persist.

I uninstalled Spectre and installed again using
devtools::install_github("immunedynamics/spectre")

I run again the workflow and nothing changed.
The error is the same as @MBakker06

Step 1/4 - Splitting files for use with original FlowSOM function
Step 2/4 - Running FlowSOM
Reading batch1.fcs
Error in if (sampleWithReplacement & (nrow(f) < cFile)) { :
argument is of length zero

package.version('Spectre')
[1] "1.1.0"
package.version('FlowSOM')
[1] "2.10.0"
package.version('CytoNorm')
[1] "2.0.0"
session_info()
─ Session info ─────────────────────────────────────────────────────────────────────────────────────────────────────────────────────
setting value
version R version 4.3.2 (2023-10-31)
os macOS Sonoma 14.1
system aarch64, darwin20
ui RStudio

[SamGG]

Error in if (sampleWithReplacement & (nrow(f) < cFile)) { :
argument is of length zero

& should be replaced by &&

[mortadelo93]

Should I modify original code using trace() or something like that?

[SamGG]

This line of code belongs to the FlowSOM package... I referenced the issue in saeyslab/FlowSOM#18.
The question still why either sampleWithReplacement or nrow(f) or cFile is of length zero...
I don't know how to modify code using trace().
What I would do is to fork the github repo, modify the code changing & by && and adding a browser() command, and install the modified version FlowSOM. But only if you feel confident.
Best.

[mortadelo93]

I guess I could have a try.
Hoping developers fix it too.
Thanks for the info @SamGG

[mortadelo93]

Just an update if someone has the same issue.
The error as @SamGG said is in FlowSOM, specifically in FlowSOM::AggregateFlowFrames() function.

What I do to fix it is to run trace(FlowSOM::AggregateFlowFrames, edit = T) and replace the & by &&.
I have to do this every new session but I could not modify the original version of the package.

Thanks again

[tomashhurst]

The prep.cytonorm issues should be fixed in the beta of v1.2 which you can test out with the following install code.

if(!require('remotes')) {install.packages('remotes')} # Installs the package 'remotes'
remotes::install_github(repo = "immunedynamics/spectre", ref = 'v1.2.0-beta') # Install the Spectre package

Don't forget the ref = 'v1.2.0-beta'

We are hoping to have v1.2 out as the main release next week.