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CyTOF Data Normalization

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singh423

Participant

Posts: 12

Joined: Mon Feb 12, 2018 10:42 pm

Location: University of Minnesota

Post Mon Apr 17, 2023 5:12 pm

CyTOF Data Normalization

Hello CyTOFER

This is Sam Menge, a researcher at SDI at the University of Minnesota Medical School. I was running CyTOF samples last week and realized after completing the run for all of the samples that I did not have the 140etc (Beads) channel in my panel on the computer application, even though I did add the bead mix to each of the samples. When I was trying to normalize the data later, the program we use would not let me normalize it as it kept saying beads were missing from the application or something similar of that nature. I was wondering if we could find some way to add the 140etc channel to the data so that we can normalize and still use it. If you have any other ideas about how I could get past this normalization issue, please let me know.

Thanks for your time and help,
Sam Menge
and
Amar Singh
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mleipold

Guru

Posts: 6001

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Apr 17, 2023 5:34 pm

Re: CyTOF Data Normalization

HI Sam and Amar,

I think there are 2 separate issues here:

1. Ce140 channel missing from panel and therefore from FCS file.
- It is possible to use something like the Panel Editor function in Premessa (https://github.com/ParkerICI/premessa#p ... d-renaming) to add a Ce140 channel to your files.

However, this will give you empty/zeroes in the Ce140 channel. Which, won't really help with the normalization process other than allowing the normalizer to execute.....your other bead channels would have non-zero numbers changing over time. But, might get you close enough in your norm?
*note: if you do this, the rewriting of the FCS file by Premessa will probably results in the stripping out of the Fluidigm/SBio-specific FCS section that is required for SBio-normalizing. You can see this by the FCS file size dropping by at least 50% pre vs post. In other words, you'd have to use Premessa or something else to normalize your files.

2. There is at least one way to normalize a file with Custom channels. Rachel Finck's original MATLAB normalizer has defaults for DVS Beads (now called EQ4; these are the regular ones), Beta beads (the original non-EQ4 beads from the Finck et al paper), and then an option for Custom, where you can select your bead channels.

https://github.com/nolanlab/bead-normalization

Please note: as stated on the Github page, this is not being actively maintained, so depending on your OS version, etc, it may take some effort to get up and running. But this would allow you to select the remaining bead channels for a Custom normalization.


It would be really nice if Premessa offered this function..........


Mike
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singh423

Participant

Posts: 12

Joined: Mon Feb 12, 2018 10:42 pm

Location: University of Minnesota

Post Mon Apr 17, 2023 9:08 pm

Re: CyTOF Data Normalization

Mike
Thank you so much
We will try the Panel Editor function in Premessa.
Warm regards
Amar/Sam

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