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Cytofkit

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Also, please note that this is a mixed bag of math-gurus and mathematically challenged, so choose your words wisely :-)
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Sep 04, 2018 9:44 pm

Re: Cytofkit

Hi Conor,

Are you finding it with *all* your files, or just merged ones like Tax was seeing?

Out of curiosity: have you tried opening an old (pre-Cytofkit) and a new (post-Cytofkit) file together in Premessa's Panel Editor? https://github.com/ParkerICI/premessa#p ... d-renaming

Just to see whether both *can* be read by another software program in R....


Also: if you do the results GUI in Cytofkit using the *.RData file, can you get the visualizations there properly? Just to try to see whether it's a global export corruption of some sort.....


Mike
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taxkourel

Participant

Posts: 14

Joined: Sun Sep 03, 2017 3:46 pm

Post Tue Sep 04, 2018 9:54 pm

Re: Cytofkit

Conor,
When I tried uploading shorter file names it worked. I have no idea if I had "Weird" characters in the file name or if I hit a limit or what but if you have very long file names as it can happen after cloning experiments etc try shortening them. I asked on github and they weren't sure but here is the long version of this conversation if you want to reply or take a look.

https://github.com/JinmiaoChenLab/cytofkit/issues/47

Good luck!

Tax
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sgranjeaud

Master

Posts: 123

Joined: Wed Dec 21, 2016 9:22 pm

Location: Marseille, France

Post Tue Sep 04, 2018 10:15 pm

Re: Cytofkit

Hi,

@Conor, could you share one of your "incorrect" FCS and tell us the version of flowCore using packageVersion("flowCore")?

@Tax, it would be nice if you reply to the last answer in the issue you opened on github.

Best.
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conorisco

Participant

Posts: 2

Joined: Tue Sep 04, 2018 4:40 pm

Post Wed Sep 05, 2018 8:43 am

Re: Cytofkit

Update:

So after you mentioned checking the version of FLowCore. I simply updated FlowCore with

  Code:
install.packages("devtools")
devtools::install_github("RGLab/flowCore", ref = "trunk")


and reloaded the required packages and bingo it worked. FCS files are exporting correctly now. Thank you

The problem pervious to this was that not only was all the data corrupt for each parameter, but also the FCS files were missing the new parameters such as Phenographcluster_ID and TSNE1 etc. Interestingly I often used strange characters. Since the issue is fixed I suppose there is no need to test if it was just to weird character use.


Thanks for the reply, I also have another bug that I want to get advice on. It regards renaming Markers.

When I rename the marker names in the shiny App the markers get renamed but now become jumbled up. For example, if I rename Y89<CD45> to CD45, and so on, this works as there is now a list of simple names, including "CD45". However, when looking at the expression plots I see that the marker called CD45 is obviously not CD45 but has been mixed up with another marker. This happens for every marker, thus making the analysis unusable. Perhaps there is another way to rename the channels outside of R, but it would be great if this bug could be reported as it could catch out a lot of people and lead to erroneous conclusions.

I could use the great little permessa panel but while I can simplify the parameter name from e.g. "Di209<CD11c>" to "CD11c<CD11c>" in permessa it still appears as "CD11c<CD11c>" in cytofkit rather than the publication-ready "CD11c".
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Tue Oct 02, 2018 2:57 pm

Re: Cytofkit

I am also having problems reported by others - the new fcs files generated after cytofkit runs do not have the tsne or phenograph cluster id channels added as parameters.This is a pain as I would like to look at the clusters in flowjo and generate some figures. Any news on this bug getting fixed ?
Naeem
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sgranjeaud

Master

Posts: 123

Joined: Wed Dec 21, 2016 9:22 pm

Location: Marseille, France

Post Tue Oct 02, 2018 3:25 pm

Re: Cytofkit

I think you should update the flowCore package as stated previously. This solves most of the problems encountered in cytofkit, Catalyst...
As Bioconductor 3.8 will be released on October the 31th, I think there will be no new release of flowCore 1.46.
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Fri Oct 05, 2018 2:39 pm

Re: Cytofkit

I have tried updating flowcore but am still not getting the tsne1 or clusterID channels in the new FCS files when using FlowJo. When I upload them into Cytobank it actually says there are errors in the FCS files and they cannot be read. I have gone back and tried cytofkit with older fcs files where things had worked properly, but no luck with them either.

Is it possible for some-one to forward the series of code lines they are using to compare please?

I have been using the following wwhich worked until recently. I had to install additional packages due to error messages - these went away once I installed the packages

source("https://bioconductor.org/biocLite.R")
biocLite("cytofkit")
biocLite("flowCore")
install.packages("XML")
install.packages("data.table")
library("cytofkit")
cytofkit_GUI()

The GUI loads up okay and everything seems to work during the running of the algorithm. The visne plots and clustering is all good when viewed in SHINY too. Problem remains with the new FCS file.,
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sgranjeaud

Master

Posts: 123

Joined: Wed Dec 21, 2016 9:22 pm

Location: Marseille, France

Post Fri Oct 05, 2018 7:07 pm

Re: Cytofkit

Please, read carefully the post. You didn't update flowCore to the latest development version. You simply reload the current version available from Bioconductor, and this version is incorrect concerning your problem. In order to get a version that should be Ok, you have to run the following commands :
  Code:
install.packages("devtools")
devtools::install_github("RGLab/flowCore", ref = "trunk")

Best.
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Mon Oct 08, 2018 10:43 am

Re: Cytofkit

Thanks SG. I have tried the code you kindly posted and still have the same issue. Is it possible I am doing things in the wrong order? Can I get the whole sequence of code that some-one is using where output files contain the new channels please?
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sgranjeaud

Master

Posts: 123

Joined: Wed Dec 21, 2016 9:22 pm

Location: Marseille, France

Post Mon Oct 08, 2018 10:58 am

Re: Cytofkit

Hi Naeem,
What is the current version of flowCore after having installed the update?
  Code:
> packageVersion("flowCore")
[1] ‘1.47.8’
> packageVersion("cytofkit")
[1] ‘1.11.0’

When updating a package, the package must not be active in any R session. You should close all RStudio sessions, R terminals... Then you could open RStudio, start a new empty project and try the update.
HTH
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