Hello everybody,

I have recently started using BioConductor and its flow packages in particular and now would like to learn openCyto. I have a very simple four-color panel for testing purposes. For one of my gating steps, I would like to use the quadGate.tmix method, since the populations are bleeding into each other a little. During my odyssey through the farthest reaches of the internet, I stumpled upon this issue on github: https://github.com/RGLab/flowWorkspace/issues/175

The image on there shows pretty much exactly what I want to achieve, but unfortunately, I have a hard time figuring out how to use the command to actually produce a filter object I can use. Running the quadGate.tmix function with usePrior="no" returns "A list of 4 filters applied to a flowFrame.", which, as far as I understand, contains the four polygon gates defining the quadrants. How do I succesfully apply this to a flowFrame, and how can I visualize it? I can kind of get it to work if I use as filter the first element of the resulting list:

xyplot(`Pacific Blue-A` ~ `PE-A`, testframe, filter=testfilter[[1]])

This results in the following:

How do I get that to display the full quadrants to me? Also, the thresholding is obviously off, presumably because I ran the filter without any prior. I could not really find any info on that. Maybe I did not look hard enough, but I would much appreciate it if you could point me towards useful resources.

On a different note, I alternatively tried an approach using flowClust. In the openCyto gating template, I set collapseDataForGating to TRUE for this step and was under the impression that this would result in the same gate for all samples. The actual output, however, looks like this:

Is there a way to generate a common gate?

I thank you in advance for any help you can provide. I am aware that these are not very specialised questions and that I am probably missing some fundamental concepts here, still I would very much appreciate some useful pointer.

Best,

VT

I've done some testing, I believe the tmixfilter is broken.. I dont' have more specific details yet. A call to flowClust on the flowFrame works without issue.

Here is from the doc '?quadGate.tmix':

Value

a filters object that contains four polygonGates following the order of (-+,++,+-,–)

That said, the results you've got should be ready to plot as it is. i.e.

library(openCyto)
library(flowClust)
testfilter <- quadGate.tmix(fr, c("PE-A", "Pacific Blue-A"), K = 3, usePrior = "no", prior = list())

xyplot(`Pacific Blue-A` ~ `PE-A`, testframe, filter=testfilter)

Regarding to 'collapseDataForGating', it should be used in conjunction with 'groupBy'.  See package vignette for more details https://www.bioconductor.org/packages/release/bioc/vignettes/openCyto/inst/doc/openCytoVignette.html.

Could you please email me one of the example files that I can reproduce what you've seen?