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.
Cheers,
Jan
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.
Cheers,
Jan