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Verity Cen-se?

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dtelad11

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Post Mon Sep 11, 2017 11:59 pm

Verity Cen-se?

Are there any resources online that describe the method? Is it available anywhere?

I know that it was presented in Cyto2017 but I cannot find any additional material.
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mleipold

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Post Fri Sep 15, 2017 4:25 am

Re: Verity Cen-se?

Hi El-ad,

The Cen-se' algorithm was described in Bruce Bagwell's CYTO 2017 talk. It happened to be something that was recorded, and has been posted to CYTO U (http://cytou.org).

It's located under "Education", then "CYTO Meeting Recordings". The title of the talk: "Stochastic Neighbor Embedding Methods: T-Sne and Verity Cen-se"

All CYTO U recordings are free for ISAC members (many of us are, since it's usually included in CYTO registration), otherwise it's $35.


In the talk, he goes through all the background of SNE/tSNE algorithms, and the extension to Cen-se'. So, if you have a chance to watch it, you might be able to make the adaptations; I believe he explicitly stated that there's no R package.


Mike
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dtelad11

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Post Fri Sep 15, 2017 11:40 am

Re: Verity Cen-se?

Thank you! I will look into it.
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mleipold

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Post Thu Oct 05, 2017 8:20 pm

Re: Verity Cen-se?

Hi El-ad,

You probably received the Fluidigm email today, with the links to the 2017 Fluidigm UGM talks that are now webinars: https://www.fluidigm.com/articles/mass- ... mit-videos

While all the talks are good and relevant, Bruce Bagwell's "A New Analytic Approach for Live Singlet Identification" is probably of particular interest to you. He mentions some of the Cen-se stuff (this was a few days before his CYTO 2017 talk in the previous cytou.org link), as well as getting into Singlet identification (including finally finding a use for Center/Offset/Width/Residual!).


Mike
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dtelad11

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Post Thu Oct 05, 2017 10:42 pm

Re: Verity Cen-se?

Thank you! Unfortunately the talk doesn't describe the method itself. Since Bagwell has first discussed Verity Cen-se(TM) several months ago, I am sure that he is in the process of preparing a manuscript that explains the details, I'll wait until that is on bioRxiv or published.
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kunicki

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Post Wed Oct 25, 2017 10:29 pm

Re: Verity Cen-se?

Hello,

I can see the value behind Bagwell's alternative singlet gating procedure, utilizing the parameters offset, center, residual, and width that are automatically derived from an event's total ion current. In his talk at the 6th Annual Mass Cytometry Summit, Bagwell highlights differences in DNA-Ir signal between singlet populations that would otherwise be disproportionately lost by manual gating of DNA signal alone. My question, in waiting for a publication on this subject, is whether there is a general consensus in the community, for- or against-, this filtering technique. I think it would be worthwhile reporting acquisition yield , and intra- and inter-assay variability between standard and ion current based singlet gating methods. Has anyone tested this extensively yet?

The idea that there are physical parameters from total ion current that we could utilize to filter cell singlets, is spectacular!
However, couldn't known background contaminants (e.g., Pb/Ba background) and variation in instrument performance (e.g., detector voltage) have a larger impact on filtering singlets by this method, being the values from the 4 aforementioned parameters are more susceptible to these technical issues? I think in implementing this practice, it would be crucial to understand how severe batching affects Bagwell's gating process, and what level of CyTOF QC/performance is necessary to still use this technique for biological samples. Perhaps this isn't as much of a concern as I imagine?


I couldn't find another thread related to the whole ion current singlet gating process, so my apologies if responding within the Cen-se(TM) thread was a bad idea. (I'd be happy to create a new thread)
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dtelad11

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Post Wed Oct 25, 2017 10:44 pm

Re: Verity Cen-se?

I doubt there is a consensus given that 1) none of this has been published and 2) many (most?) labs can't even extract these parameters from the instrument. There is no way to test this. As far as I know only Bagwell has ever utilized this method or presented any data using it. And from what I see he is focusing on the method, with no benchmarks, comparisons, or biological results.
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GregBehbehani

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Post Wed Oct 25, 2017 10:55 pm

Re: Verity Cen-se?

Hi Matthew,

I think this is a great point to bring up. I would certainly agree with you that this would need validation (preferably using barcoded samples), but I'm also sure that the parameters that are used in the analysis could also be tweaked to exclude things like lead contaimination, etc.

Two add my two cents, I have for a long time believed that parameters such as offset, center, residual, and width would be very useful for doublet discrimination, if you watch the raw data from the machine (the rain), one can commonly visually discriminate doublet events that are almost certainly being counted as single events by the CyTOF sofware. I'm sure there is a software approach that could identify these. However, in just playing with the currently available metrics ( offset, center, residual, and width) with manual gating, I've never been able to convince myself of a way to really use them currently.

If the approach could be validated, I think it would be great for excluding doublets, but I'm not sure if one could ever accurately recreate what the data would have looked like had the doublet not formed. Thus, I think non-redundant barcoding and running slowly will remain the most important tools for dealing with this issue (unless a fluidics method can be developed to get the cells to evenly space themselves).

best,

Greg
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mleipold

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Post Wed Oct 25, 2017 11:08 pm

Re: Verity Cen-se?

El-ad: those parameters are present in Helios data. Considering that the Helios instruments have been out for more than 2 years (and a number of CyTOF2 instruments have undergone the Helios upgrade), a large number of people should have data that could be looked at.

I agree, I wish the paper would come out so we could read it and evaluate more than what two talks have stated.


If you could give Bruce a wishlist for benchmarking, what would it be? And what sort of biological results are you meaning? Probably the main point from the Fluidigm UGM talk was basically being able to gate your LiveIntact Singlets away from Debris, Doublets, Dead Cells, and Beads in one go.
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dtelad11

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Posts: 113

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Post Wed Oct 25, 2017 11:32 pm

Re: Verity Cen-se?

mleipold wrote:El-ad: those parameters are present in Helios data. Considering that the Helios instruments have been out for more than 2 years (and a number of CyTOF2 instruments have undergone the Helios upgrade), a large number of people should have data that could be looked at.

I've been working with Mount Sinai and as far as I know they don't have access to this data. Also, judging from public data sets, I couldn't find these channels. I might be doing something wrong though, please correct me if there are any public data sets that include these.

mleipold wrote:If you could give Bruce a wishlist for benchmarking, what would it be? ... Probably the main point from the Fluidigm UGM talk was basically being able to gate your LiveIntact Singlets away from Debris, Doublets, Dead Cells, and Beads in one go.

I would want to see an experiment where doublets can be easily identified through an orthogonal mean. That orthogonal mean would be the "gold standard" we are trying to identify. Two possible examples:

- Take one sample, split it to ten subsamples, barcode each, combine them, and acquire them using a mass cytometer. Cells which have two or more barcodes are doublets.
- Take one sample, split it to ten subsamples. In each subsample, measure CD45 using a different mass. Cells which have two or more CD45 channels are doublets.

Then, we could take several doublet identification methods, such as Bagwell's method, the standard event length versus DNA, backgating, clustering, what-have-you. Compare methods using precision/recall, F-score, AUC, or other metric of choice.

mleipold wrote:And what sort of biological results are you meaning?

Let's say I currently gate doublets using DNA versus event length. I'm really good at it, or I have a trained technician who does it, or my lab's expertise revolves around it. It removes most doublets and probably a small number of legit cells.

Now a magical method comes out that removes *all* doublets and *only* doublets from a sample (I'm exaggerating here for the argument's sake).

Why should I switch to the new magical method? Why should I invest time and effort in integrating it into my pipeline?

In my opinion, one way to show the utility of the new method is to provide an experiment where utilizing it allows the identification of biological insights that the current method failed to identify. A lower bar (which I would also accept) is to show some statistical relevance of magical method over existing method -- even something as simple as lower p values on a set of statistical tests.

Another option is to show that magical method is cheaper, faster, easier, or otherwise superior to the existing methods. If there was an easy way to run this doublet detection, great!

There are a lot of mass cytometry methods coming out right now. From my point of view, Bagwell's method warrants no special attention until there is some evidence that it provides a benefit over existing methods. I admit that the current methods are not very good, but still, there should be *something* coming from Bagwell to show that this warrants further attention.
Last edited by dtelad11 on Wed Oct 25, 2017 11:34 pm, edited 1 time in total.
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