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Concatenating barcoded samples from different runs

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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: 2

Joined: Thu Nov 23, 2017 11:09 am

Post Sat Mar 21, 2020 10:15 am

Concatenating barcoded samples from different runs

Dear all,

I have a set of files from multiple acquisitions of the same pool of barcoded samples (14 clinical samples and 1 reference). We split the pool of samples to multiple vials, froze them, and then thaw on the day of acquisition. Here's how the CD3 expression looked like across the acquisitions. I do not think there's a prominent shift of marker expression distributions, but I can see that the lines (which indicate quantiles) are not aligned. What would be the best way to proceed?


Would it be wise to concatenate all files and then debarcode them?

I appreciate all of your insights.

Best regards,



Posts: 16

Joined: Wed Mar 02, 2016 10:25 am

Post Tue Mar 24, 2020 3:27 pm

Re: Concatenating barcoded samples from different runs

Hi Mikhael,

This would be a good application of CytoNorm (https://github.com/saeyslab/CytoNorm) or CytofBatchAdjust (https://github.com/CUHIMSR/CytofBatchAdjust) I would say. The barcoding would probably be separate from these issues, so it would not matter for the debarcoding if you debarcoded before or after the batch correction.



Posts: 79

Joined: Wed Dec 21, 2016 9:22 pm

Location: Marseille, France

Post Tue Mar 24, 2020 4:57 pm

Re: Concatenating barcoded samples from different runs


I am not an expert, but I would say "move foward".
From the image, I don't see see any effect, not knowing which samples belong to a batch or another.
After debarcoding, you could get as in Fig 6 of cytoNorm
https://onlinelibrary.wiley.com/doi/ful ... to.a.23904
Or get an overview of any batch effect by coloring samples using their batch id on a tSNE (downsampling OK).


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