Future support for new Fluidigm 6-element beads?

[mleipold]

Fluidigm has launched new normalizer beads with 6 elements:
"Description: EQ™ Six Element Calibration Beads (EQ6 beads) is a 1X stock solution (used at 0.1X for sample acquisition) of polystyrene bead standards containing known concentrations of the natural abundance metal isotopes yttrium (89Y), indium (115In), cerium (140Ce), terbium (159Tb), lutetium (175Lu), and bismuth (209Bi)."

https://www.fluidigm.com/binaries/content/documents/fluidigm/search/hippo%3Aresultset/tds-00691---201245---eq-six-element-calibration-beads/fluidigm%3Afile

Note that these beads contain Y, In, Tb, and Bi, but do not contain the Eu and Ho from the 4-element beads.

Do you have plans to add the Six-Element beads to the Premessa normalizer, like the current 4-element beads or the original Nolan-Synthesis beads?

[nvall]

Hi, I just changed a bit of the scripts so EQ6 beads will be compatible. Now waiting for the pull request to be evaluated. In the mean time, you could checkout my fork and try it with your data. I've checked it with my first data in which there were few batch effects thus I'm not sure that it is fully working.

[mleipold]

Hi Nicolas,

I'm finally starting to generate some test data with the 6-element beads, and wanted to try testing the effects of 4-element vs 6-element normalization.

As far as I can tell, the current Premessa v0.3.2 still doesn't have the 6-element included. Just to confirm, when you say "checkout my fork and try it with your data", you mean running the command line R using the code here:

https://github.com/ParkerICI/premessa/blob/90d8beee25a1e807d5bd8da3f5bf20445687369b/R/shiny_helpers.R

Thanks,
Mike

[nvall]

Hi Mike,
I think you should pull my git repo https://github.com/nvall/premessa on your local computer. Then load it with devtools. Should be something like this:

library(devtools) 
load_all("<path to your local folder>/premessa") 

Then you can use the shiny interface and this should be working.
If you don't want to pull locally you can install_github("nvall/premessa") but this will replace the package version of @ParkerICI

[mleipold]

hH Nicolas,

Thanks for those details...I'm not a coder, so that really helped!

I decided to do the full installation using "install_github("nvall/premessa")", which appeared to work: I had installed v0.3.2, but after installing your fork, RStudio said I was running v0.3.1; also, when I ran Premessa Normalizer, I saw EQ6 now as a Bead option.

However, I don't think it's pulling the correct channels for EQ6: it still plots the 5 EQ4 channels (including 151Eu, 153Eu, and 165Ho which are not present in EQ6), and do not include 89Y, 159Tb, or 209Bi which are unique to EQ6. Note: this sample had both EQ4 and EQ6 beads in it, which is why you still see Ir-neg Bead-pos events.

See attached screenshots.

Mike

[nvall]

Hi Mike,
Thanks for your comment and sorry for the late answer. Have you tried selecting EQ6 then visualize bead without visualizing the EQ4 before? I think that this is relative to the number of channels to display but I am not sure. I will check this as soon as possible.
Best,
Nicolas

[mleipold]

Hi Nicolas,

OK, I tried that: I opened Premessa, pointed to the folder containing the files. That loaded the GUI. I selected Normalize data. EQ4 was the default; I switched it to EQ6, then selected an FCS file, then hit Visualize Beads.

Unfortunately, that did not fix the problem. The bivariate plots at the bottom were still showing the channels for EQ4 (eg, the Eu channels which are part of EQ4 but not EQ6).

So, I don't think it's pulling the right channels for EQ6.

Mike

[nvall]

Hi Mike, that's odd, it was ok on my config. I'll try to check what is wrong asap. Thanks trying it though!
Best,
Nicolas

[SansMorel]

@mleipold
Feel free to try out my fork at https://github.com/sansmorel/premessa

There is one known bug where plots/channels accumulate if you first plot one type of beads then change bead type and visualize beads a second time.

[mleipold]

@SansMorel @nvall

Thanks for the fork! I installed it using "install_github("sansmorel/premessa")" and the correct EQ6 channels did appear (89Y, ... 209Bi) when I selected Fluidigm2 beads (I was careful and didn't select the normal EQ4 first).

I was able to generate new EQ6-normed files as output.

I will let you know if I have further issues, but at least the package executed!

Mike