Error message

[mleipold]

Hi,

I'm not sure this is exactly an error, per se.

I have a time series of samples using the same barcode. In certain donors, a specific timepoint might be missing. In that case, I have edited the CSV key to remove the "missing" sample.

I noticed that when I leave the blank line, the CSV uploads properly, and only after I select the FCS file and it starts working, do I then get the following error message:

"Error: Barcoding schems with a variable number of positive channels per sample are not currently supported"

If I take out the blank line, then it works normally.

So, a couple things:

1. If I have a CSV key line that doesn't exist in the sample, is that OK? Like, if my BC5 sample isn't present in the sample, can I still leave in the BC5 line without problems? Obviously, I won't get a file, but will it interfere with the debarcoding?

2. If there's a problem with the CSV key, I would suggest that it should be flagged when the CSV file is selected, rather than waiting until later.

Mike

[mleipold]

I also got the same error when I had no blank lines, but had different numbers of pos channels: for instance, having both 00101 and 00100 (pipetting error messed up the BC sample in channel 5).

If I remove all other barcodes and have just 00100, then it will execute on the FCS file.

So, I guess this raises the question: is Premessa unable to handle N! exhaustive (all possible combinations) barcoding like in the original Bodenmiller paper? For example, would Premessa be unable to handle 10000, 11000, 11100, 11110, and 11111 all in the same debarcoding CSV?

[pfgherardini]

Hi Mike,

Can you send me a copy of the barcoding key that fails? WIth respect to your questions:

1. Yes you can have a barcode key that includes barcode combinations that are not really in your experiment.
2. No at the moment the software does not handle barcoding scenarios where the number of positive channels is different across barcodes combinations. For instance in the standard Fluidigm kit, each barcode sample is positive for 3 channels. The program will not handle cases where one BC combination is positive for 2, and another one is positive for 3. I could implement that, but it's been low on the priority list, since practically everybody uses the Fluidigm barcoding scheme. Do you have a dataset where that's a limitation? If you can share such a dataset with me, I can work on it (time permitting).

[mleipold]

Hi Federico,

I've uploaded two CSV Keys: one that works, and one that doesn't. They differ only by the presence or absence of a blank line.

You can find the files and the FCS file at the following link (let me know if you have trouble accessing): https://drive.google.com/drive/folders/1JARwEgIoy_3DiE6Fi06U_lcrhyYf2gUk?usp=sharing

1. If I can have a key with a BC that doesn't exist in my sample, then that fixes the problem for me.

2. I don't use the Fluidigm BC reagents: I use CD45. I therefore often use a scheme other than 3-of-6 (in the above case, it's a 2-of-5). I don't normally plan to mix, say, 1-of-5 with 2-of-5: this recent case was an experimental problem where a pipetting error left out one of the 2 reagents in a particular BC cocktail (eg, added A but left out B). I therefore modified the CSV key and tried to debarcode again: this would force the cells to "compete" against each other so that the 1-of-5 would hopefully not "steal" cells from the other 2-of-5 barcodes that contained A.

Instead, I had to do it as a separate run on the parent FCS, using a separate CSV key which contained only one barcode....though I guess I could have used the separate CSV on the Unassigned from the first (2-of-5) run, and effectively had the same "competition".

[angel611]

Hello, i need some help,
I am getting this error when i run the debarcoder-GUI.

Listening on http://127.0.0.1:3706
Warning: Error in [: subscript out of bounds
200: calculate_bcs_separation
199: debarcode_data_matrix
198: premessa:::debarcode_data
197: reactive:debarcoderui_get_bc_results [/Library/Frameworks/R.framework/Versions/3.5/Resources/library/premessa/debarcoder_shinyGUI/server.R#102]
195: .func
192: contextFunc
191: env$runWith
184: ctx$run
183: self$.updateValue
181: debarcoderui_get_bc_results
168: renderPlot [/Library/Frameworks/R.framework/Versions/3.5/Resources/library/premessa/debarcoder_shinyGUI/server.R#127]
166: func
126: drawPlot
112: reactive:plotObj
96: drawReactive
83: origRenderFunc
82: output$debarcoderui_plot1
2: shiny::runApp
1: premessa::debarcoder_GUI
Warning: Error in [: subscript out of bounds
183:
Warning: Error in [: subscript out of bounds
89: calculate_bcs_separation
88: debarcode_data_matrix
87: debarcode_data
86: premessa:::debarcode_fcs
73: observeEventHandler [/Library/Frameworks/R.framework/Versions/3.5/Resources/library/premessa/debarcoder_shinyGUI/server.R#179]
2: shiny::runApp
1: premessa::debarcoder_GUI