Trouble with IMC spillover R script`

[asasama12]

I've collected IMC images of dotted metal conjugated antibodies and am attempting to use the imc_generatespillmat_long.Rmd script to calculate spillover between channels but am having trouble getting it to run. I am able to load all the packages and data and run the script up to line 125, but when calculating the CATALYST based compensation I get the following error.

Error in CATALYST::assignPrelim(x = ff, y = ss_ms) : unused argument (y = ss_ms) 4. re_from_dat(x, ss_ms = x[!is.na(mass), unique(mass)], minevents = 40, correct_bc = T) 3. FUN(X[[i]], ...) 2. lapply(dats_agg, function(x) re_from_dat(x, ss_ms = x[!is.na(mass), unique(mass)], minevents = 40, correct_bc = T)) 1. lapply(dats_agg, function(x) re_from_dat(x, ss_ms = x[!is.na(mass), unique(mass)], minevents = 40, correct_bc = T))

[votti]

Hi!

The issue underlying this is that CATALYST changed quite a bit since this repository was created. This broke a lot of functionality and is causing the error you are seeing.

I now added a branch with instructions how to install an R environment with an old CATALYST version:
https://github.com/BodenmillerGroup/cyTOFcompensation/tree/environment

Pull request: #2

Installing the Conda environment should allow you to use this scripts - could you give this a try?

Best, Vito

[asasama12]

Ah! That helped progress past line 125, but I am now getting a different error at line 148:
`Error in check_sm(SM, isotope_list) : The supplied spillover matrix seems to be invalid. All isotopes should appear in isotope_list.

5. stop("\nThe supplied spillover matrix seems to be invalid.\n", "All isotopes should appear in ", deparse(substitute(l)), ".")
6. check_sm(SM, isotope_list)
7. .local(bc_ms, SM, ...)
8. CATALYST::plotSpillmat(ss_ms, sms[[i]])
9. CATALYST::plotSpillmat(ss_ms, sms[[i]])`

[votti]

Hi!
I think the problem is that you likely have a non isotope channel (e.g. BCKG153Di or ArAr80Di).

Unfortunately these need to be manually added to the CATALYST isotope list as they are otherwise not supported :/
This is discussed here:
HelenaLC/CATALYST#35

I guess this could be also fixed via running this at the Beginning of the script

CATALYST::isotope_list <-  c(CATALYST::isotope_list, list(BCKG=153, ArAr=80))

(Not entirely sure, let me know if that worked).

[asasama12]

That does not seem to be the issue, I am using standard isotopes and I went through and checked in isotope_list and they are all present there:

I do get an output spreadsheet with what I assume is the spillover matrix

[votti]

Yes, the .csv files are the spillover matrix.
plotSpillmat is indeed just a nicer visualization.

The question is usually rather what channels were selected during acquisitions and are in the files.
This would be the output of:
colnames(sms[[i]])
and
rownames(sms[[i]])

Could well be that instead of Eu153 you acquired BCKG=153 or something like this :/

[asasama12]

Thank you for all your help in this, I really appreciate it.
I checked colnames(sms[[i]]) and rownames(sms[[i]]) and they cambe back
colnames:
[1] "Y89Di" "Ru100Di" "Ru101Di" "Ru102Di" "Ru104Di" "In113Di" "In115Di" "I127Di" "Xe131Di" "Ba138Di" "La139Di" "Pr141Di" "Nd142Di" "Nd143Di"
[15] "Nd144Di" "Nd145Di" "Nd146Di" "Sm147Di" "Nd148Di" "Sm149Di" "Nd150Di" "Eu151Di" "Sm152Di" "Eu153Di" "Sm154Di" "Gd155Di" "Gd156Di" "Gd158Di"
[29] "Tb159Di" "Gd160Di" "Dy161Di" "Dy162Di" "Dy163Di" "Dy164Di" "Ho165Di" "Er166Di" "Er167Di" "Er168Di" "Tm169Di" "Er170Di" "Yb171Di" "Yb172Di"
[43] "Yb173Di" "Yb174Di" "Lu175Di" "Yb176Di" "BCKG190Di" "Ir191Di" "Ir193Di" "Pb208Di" "Bi209Di"
rownames:
[1] "Er170Di" "Eu151Di" "Eu153Di" "Gd158Di" "La139Di" "Lu175Di" "Nd142Di" "Nd144Di" "Nd148Di" "Nd150Di" "Pr141Di" "Sm147Di" "Sm152Di" "Sm154Di" "Yb171Di" "Yb176Di"

I crossreferenced these with CATALYST::isotope_list and the only one that was missing was BCKG190Di so I added that to isotope_list as you suggested:

`isotope_list <- c(CATALYST::isotope_list, list(BCKG=190))

for (i in seq_along(sms)){
print(names(dats_agg)[i])
ss_ms = dats_agg[[i]][!is.na(mass), unique(mass)]
p = CATALYST::plotSpillmat(ss_ms,sms[[i]])
print(p)
}`

However, I still get the same error message as above - supplied spillover matrix seems to be invalid. all isotopes should appear in isotope_list

[asasama12]

Solved this with the line p = CATALYST::plotSpillmat(ss_ms,sms[[i]], isotope_list = isotope_list)

I'm now attempting to apply this spillover matrix to my images using the imc_comp_txt_example script, and I'm having the same issue at
img_comp = comp_datimg(img, sm=sm)

Error in check_sm(input_sm, isotope_list) : The supplied spillover matrix seems to be invalid. All isotopes should appear in isotope_list.
19.
stop("\nThe supplied spillover matrix seems to be invalid.\n", "All isotopes should appear in ", deparse(substitute(l)), ".")
18.
check_sm(input_sm, isotope_list)
17.
.local(input_sm, out_chs, ...)
16.
adaptSpillmat(y, flowCore::colnames(x), isotope_list)
15.
adaptSpillmat(y, flowCore::colnames(x), isotope_list)
14.
.local(x, y, ...)
13.
compCytof(x, as.matrix(y), out_path, method)
12.
compCytof(x, as.matrix(y), out_path, method)
11.
.local(x, y, ...)
10.
CATALYST::compCytof(., sm, method = method, ...)
9.
CATALYST::compCytof(., sm, method = method, ...)
8.
function_list[i]
7.
freduce(value, _function_list)
6.
_fseq(_lhs)
5.
eval(quote(_fseq(_lhs)), env, env)
4.
eval(quote(_fseq(_lhs)), env, env)
3.
withVisible(eval(quote(_fseq(_lhs)), env, env))
2.
img_mat %>% flowCore::flowFrame() %>% CATALYST::compCytof(sm, method = method, ...) %>% flowCore::exprs() %>% as.data.table() at spillover_imc_helpers.R#220
1.
comp_datimg(img, sm = sm)

[asasama12]

Trying to track down this issue,
I updated the isotope_list to include BCKG190 as you suggested which allowed me to run plotSpillmat in the other script,

when I run compCytof(img_comp, sm, isotope_list2) I get the error Error in check_sm(input_sm, isotope_list) : The supplied spillover matrix seems to be invalid. All isotopes should appear in 'isotope_list'

If I run compCytof(img_comp, sm, isotope_list = isotope_list2) I get the error Error in .local(x, y, ... ): unused argument (isotope_list = isotope_list2)

I confirmed that all the metal_names from my colnames(img_mat) are included in isotope_list2 including BCKG190

[votti]

Hi,
So the solution to this is to use img_comp = comp_datimg(img, sm=as.matrix(sm), isotope_list=isotope_list)

Somehow the 'isotope_list` argument is only accepted in the compCytof function if sm is a matrix (as opposed to e.g. a data.frame).

I updated the example code in the environment branch to allways coerce the sm to matrix in the comp_datimg function.

[asasama12]

That worked for me! Thank you!

[saisomesh2594]

Hey,

I, too, ran into the same issue due to differences in CATALYST version: Error in CATALYST::assignPrelim(x = ff, y = ss_ms) : unused argument (y = ss_ms)

I tried to install the conda environment from the environment branch of this repo and I am running into issues with installation.

Collecting package metadata (repodata.json): done
Solving environment: failed

ResolvePackageNotFound:
  - bioconductor-ebimage
  - bioconductor-flowcore
  - libgfortran
  

Any idea what could be causing this?

Thanks,
Somesh

[laufre]

Hi,

I'm also experiencing the same problem Error in CATALYST::assignPrelim(x = ff, y = ss_ms) : unused argument (y = ss_ms).
While installing the environment I have the same issue as @saisomesh2594:

`ResolvePackageNotFound:

• bioconductor-ebimage
• bioconductor-flowcore
• libgfortran`

I would really appreciate your help on this, as I would love to try out your IMC spillover script!

[nilseling]

Hi @laufre and @saisomesh2594
not sure if that is still an issue but here are scripts that use the newest version of CATALYST to generate the spillover matrix:

https://bodenmillergroup.github.io/IMCDataAnalysis/spillover-correction.html

@HelenaLC and @votti are you fine with me porting the scripts to the new repo where they are continuously checked?