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Debarcoding concatenated files

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Posts: 19

Joined: Wed Mar 02, 2016 10:25 am

Post Mon Jan 29, 2018 10:46 am

Debarcoding concatenated files

Hi all,

I just finished a run of barcoded samples. In total it took about ~10 hours of Helios running time so I split the sample tube into different tubes when I started. Now I'm wondering what's everyone's preferred sequence for debarcoding and concatenating.

The way I see it there are 4 actions that need to be taken:
1. Normalization of the signal based on the EQ Beads.
2. Sample clean up through gating
2a. excluding portions of time where flow was unstable
2b. (optional) excluding beads and debris
3. Concatenation of different runs
4. Debarcoding to obtain samples fcs files.

These can be done in different orders, in previous experiments I followed the described order. However playing around with this dataset I see differences in absolute cell yield when comparing the sum of debarcoded runs versus debarcoding the concatenated version of the same data. I suspect signal drift over the course of the day to impacts CVs which in turn affects the debarcoding of the concatenated file.




Posts: 44

Joined: Wed Nov 20, 2013 12:50 pm

Location: Zurich, Switzerland

Post Tue Jan 30, 2018 10:06 am

Re: Debarcoding concatenated files

Hi Jan,

I first concatenate, then bead-normalize and then debarcode. The steps you further described I leave for later...

Pre-gating is easier and more precisely done after the debarcoing.


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