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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 :-)
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mdmanurung

Participant

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?

https://ibb.co/X2NbjWc

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

I appreciate all of your insights.

Best regards,
Mikhael
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Verhoeff

Participant

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.
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sgranjeaud

Master

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

Hi,

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).

Best,
Samuel

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