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Problem with Helios normalizer

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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Tue Aug 02, 2016 5:17 am

Problem with Helios normalizer

Hi everyone,

We recently discovered a weird issue with the Helios normalizer that I thought everyone should be aware of: when normalizing and concatenating multiple files together, the Pd102 channel does not get normalized. This is illustrated in the example below, in which a large barcoded sample was collected as 3 separate acquisitions (on a CyTOF2). The signal distribution of each of the barcode channels is shown in the first three rows. The next 3 rows show the signal distribution when the files were normalized individually, resulting in a characteristic signal boost that we see with our CyTOF data post normalization. All this looks fine. The the 5th row shows the result of concatenation of the files without normalization, and the 6th row shows simultaneous concatenation and normalization (i.e., both boxes checked in the software).

The 6th scenarios is where the issue manifests (highlighted with a red arrow); the post normalization distribution of Pd102 is identical to the pre-normalization, whereas all the other channels are normalized to the level seen when the files are normalized individually.

Slide1.PNG


This causes issues when attempting to debarcode the files. As you can see, when samples are barcoded well, the barcode separation threshold is fairly even for all the files. However, after normalization/concatenation you end up with this bimodal distribution, where samples 1-10 (which all use the Pd102 label) have a much lower separation than 11-20. We've found that to effectively debarcode the normalized data to maximize recovery and minimize doublet contamination you have to run the software twice with two different separation thresholds and select one set of files from one and another set of files from the other (which is a pain).

Slide2.PNG


Slide3.PNG


Turns out that doing a 2 step process where you normalizing the files first and then concatenating with the fluidigm software doesn't work (doing this in fact removes the normalization from the data, which I knew was an issue with version 1 of the CyTOF2 software and I had assumed would have been fixed by now but apparently not). Concatenating first and then normalizing results in the same issue of Pd102 not being normalized.

Slide4.PNG


The best solution I've found so far is to normalize the individual files and then run the Cytobank concatenator tool, which preserves the signal like it's supposed to (doing the reciprocal doesn't work and brings up an error message when you try to normalize).

Slide5.PNG


Unfortunately, this largely eliminates one of the main reasons I use the Fluidigm normalizer in favor of Rachel Fink's one, which is that it's a bit faster and more convenient to be able to simultaneously normalize and concatenate a big batch of files.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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RSchackmann

Participant

Posts: 9

Joined: Fri May 27, 2016 5:26 pm

Post Tue Aug 02, 2016 2:09 pm

Re: Problem with Helios normalizer

Hi Adeeb,
I've been trying to reproduce what you are seeing, but for me it doesn't seem to matter if I normalize first and then concatenate or let the Fluidigm software do it all in the same batch or concatenate first and then normalize

The only effect I see on the barcode signal is the (normal) slight increase in signal intensity after normalization, but my pd102 does not show the lack of normalization you are seeing. Comparing pd102 vs 110 show identical effects of normalizing/concatenating etc.

I've run this on 2 files and also tried it on 3 files (run concatenate and normalize in fluidigm at the same time; concatenate only or concatenate first then normalize), nothing I do gives me the the pd102 problem you are showing

(files collected on a cytof2-Helios upgrade)


I have only used 5 barcodes in this sample as I haven't used a full panel yet (barcodes no. 6-10 which all contain pd 102, so I can not comment on your debarcoding figure), but even then I should see the issue you show in your first figure

which version of the fluidigm software are you using? I'm on 6.5.358

here are some examples of what I see. the red and the brown are the only one's that aren't normalized and hence show a slightly lower signal. pd110 top , pd102 bottom
Untitled.jpg



,
Ron
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Tue Aug 02, 2016 2:52 pm

Re: Problem with Helios normalizer

Thanks, Ron. I'm using version 6.5.273 so maybe that's the issue. I'll download 6.5.358 and see if that hopefully fixes the issue.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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RSchackmann

Participant

Posts: 9

Joined: Fri May 27, 2016 5:26 pm

Post Tue Aug 02, 2016 3:02 pm

Re: Problem with Helios normalizer

I just got an email from our operator Nicole, see below

"
Hi Ron,

Re: your forum post - I can’t log into it but there was a known normalization issue with the older helios software in which the first channel was not normalized in any file. On most of our panels it was only impacting users that had 89Y since I keep it open own all of our panels. If Adeeb did not have 89Y open then most likely 102Pd was the first open channel and why he sees a problem with this. 6.5.358 was released with a fix for that issue.



Nicole
"

Cheers,
Ron
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mleipold

Guru

Posts: 2256

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Aug 02, 2016 3:14 pm

Re: Problem with Helios normalizer

Hi Adeeb and Ron,

Which version of the Fluidigm normalizer are you using? I don't just mean the software (6.5.358), but also which Passport you're using. I know the HIMC Helios instruments have the regular P13... Passport, but also the P13...-ver2. We know that the ver2 is the better of the Passports (ie, after normalization, the beads in each file are almost superimposable).


Mike
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anitamkant

Contributor

Posts: 46

Joined: Mon Nov 18, 2013 6:30 am

Post Tue Aug 02, 2016 3:15 pm

Re: Problem with Helios normalizer

Hello Adeeb,
Thank you for raising the topic on the CytoForum. Fluidigm is aware of the bug in the older version of the software and has corrected the same in version 6.5.358
We have notified all the Helios customers that they should upgrade the software to the latest version.
We strongly encourage you to do the same.
Please find enclosed the release notes for SW 6.5.358.
Please contact your Fluidigm Field Application Scientist for any further questions.
Thanks once again.
Attachments
Helios v6 5 358_data processing_rn_400255v1.pdf
(412.16 KiB) Downloaded 201 times
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Tue Aug 02, 2016 3:49 pm

Re: Problem with Helios normalizer

Thanks all. Just updated the software to version 6.5.358 and can confirm that this resolves the problem.

Mike, I'm using passport ver 2.0

Also, thanks Nicole and Michelle Poulin for further claridying out that this was specifically an issue with the lowest mass channel in a given panel, which would explain why I hadn't noticed this before (this particular experiment did not have the 89Y channel selected).

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC

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