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Normalization without all bead channels?

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Please be as geeky as possible. Reference, reference, reference.
Also, please note that this is a mixed bag of math-gurus and mathematically challenged, so choose your words wisely :-)



Posts: 11

Joined: Wed Apr 19, 2017 7:35 pm

Post Sat Mar 10, 2018 3:05 pm

Re: Normalization without all bead channels?

mleipold wrote:Hi Santhosh,

Unfortunately, none of the current methods (Fluidigm, MATLAB/Premessa) will work without all the five major EQ bead channels. And there's no way to "recover" data that was never collected in the first place (since it wasn't in your template).

The best chance you would have would be to talk to someone who could modify the normalization script(s) to leave out the missing channel.


An alternative could be the CATALYST package: https://catalyst-project.github.io/.

There is an implementation of the Finck normalization where you specify the channels you want to use.

There are also at least 3 entry points: 1. The R package itself, if you write your own scripts; 2. Local web interface,
where you install all the packages locally to your R installation and run a web-based app from R; 3. The web-based app http://imlspenticton.uzh.ch:3838/CATALYSTLite/ (can be slow, has some file size limits, more for exploring than production use).

Best, Mark



Posts: 31

Joined: Mon Aug 01, 2016 2:31 pm

Post Mon Mar 12, 2018 2:27 pm

Re: Normalization without all bead channels?

Hi Mark,

Thank you for that suggestion, that definitely seems like what I was looking for. I will try out the web application and see if it works for my samples.




Posts: 5285

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Mar 12, 2018 2:54 pm

Re: Normalization without all bead channels?

Hi Xiao,

There will always be batch effects. They are most often due to day-specific experimental (bench) stuff: pipetting errors, washing efficiency difference, etc. The normalization beads can't completely fix those....they weren't designed to.

Regarding spillovers: that's something that you as a researcher have to determine yourself. That's because it mainly depends on your particular panel design: specific marker+metal combinations, and marker vs marker combinations, etc. Fluidigm doesn't design your panels for you.

Spillover (eg, M+1, M+16) is as a percentage of signal at mass M, so if you have something screaming bright like CD45 or CD57 and you don't titer down, you're going to get spills, particularly if you put them on low mass lanthanides that have higher M+16 spill. The usual tricks of putting, say, a B-cell-specific marker in a channel receiving spill from a T-cell-specific marker, can minimize this sort of thing.


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