After cytoqc, error in CytoML::flowjo_to_gatingset (missing compensation parameter)

[PedroMilanezAlmeida]

@gfinak and @mikejiang, great package, thanks for making it publicly available!

Our wet-lab collaborators acquired some samples with and others without an open autofluorescence channel.

I was able to clean up the dataset with cytoqc (amazing btw!) but now when I try to open their FlowJo workspace, I get the following error:

gs <- CytoML::flowjo_to_gatingset(ws,
                                   name = 1,
                                   subset = basename(sampleFCS_path),
                                   extend_val = -Inf,
                                   cytoset = cs,
                                   additional.sampleID = TRUE)

Error in (function (ws, group_id, subset, execute, path, cytoset, backend_dir,  : 
  compensation parameter 'AF-A' not found in cytoframe parameters!

How would you guys go about editing the compensation matrices now?

Any help would be much appreciated, thanks in advance!

[mikejiang]

You can pass a custom compensation through compensation argument to skip flowjo's compensation.
see details from ?flowjo_to_gatingset

[PedroMilanezAlmeida]

Sounds good, thank you!

[Biomiha]

Hi @mikejiang,

Can I hijack this issue please since I am getting the same error? I think the cause of my issue is that I have got a discrepancy between the channel names in my cytoframe and my workspace. After running cytoqc to match the channels, I have the following:

cs <- cytoset(cqc_data)
cytoqc::cf_get_panel(cs[[16]])
# A tibble: 36 × 2
   channel    marker      
   <chr>      <chr>       
 1 FSC-A      NA          
 2 FSC-H      NA          
 3 FSC-W      NA          
 4 SSC-A      NA          
 5 SSC-H      NA          
 6 SSC-W      NA          
 7 V 431/28-A CD1 BV421
 8 V 525/50-A CD2 BV480  
 9 V 586/15-A CD3 V575    
10 V 605/40-A CD4 BV605 
# … with 26 more rows

Note that the channel names contain a / (slash).
If I try to parse the flowjo workspace to a gatingSet I still get the following error:

flowjo_to_gatingset(ws, 7, cytoset = cs, compensation = comp) # *see below for details on how I obtained `comp`

Error in (function (ws, group_id, subset, execute, path, cytoset, backend_dir,  : 
  compensation parameter 'B 530_30-A' not found in cytoframe parameters!

Note that here the parameter contains an _ (underscore). I presume flowjo automatically corrected the illegal character / to a _ in the worskpace creating the discrepancy.

If I change the execute argument to FALSE I can parse it just fine but obviously this doesn't transform and compensate properly.

gs <- flowjo_to_gatingset(ws, 7, cytoset = cs, execute = FALSE)
gs
A GatingSet with 6 samples

* I used this gs to extract the compensation matrix comp

comp <- gs_get_compensations(gs)

What would be the easiest / quickest way to correct the illegal characters in the channel names?

Many thanks in advance and thanks again for both the really useful packages and the prompt responses to the issues.
Best wishes,
Miha

[mikejiang]

how about colnames(cs) <- gsub("/", "_", colnames(cs))

[Biomiha]

Thanks @mikejiang. That now works!