Normalization FAQ - nolanlab/bead-normalization GitHub Wiki

Check that the folder does actually contain files with the FCS extensions, or that those files include the Ir191 and/or Ir 193 channels.

Yes, you can. This requires 4 steps.

1. Acquire an FCS file that contains BOTH types of beads. This file need not contain cells.
2. Create two FCS files from the file of both beads: one that contains only the 'old' beads and one that contains only the 'new' beads. You can do this by either running the Normalizer twice on the file created in Step 1, once selecting the 'old' bead masses and once selecting the 'new' bead masses, or by manually gating out the two bead types.
3. Normalize the subset of your files that contain the 'old' beads to the 'old' beads file you've created in Step 2. Do this by normalizing to a 'saved FCS file' and selecting the file of 'old' beads.
4. Normalize the subset of your files that contain the 'new' beads to the 'new' beads file you've created in Step 2. Do this by normalizing to a 'saved FCS file' and selecting the file of 'new' beads.

The Normalizer works by extrapolating the correction factor to each cell from the beads acquired before and after that cell. This will be inaccurate if the events are not in order, which can happen when multiple files are concatenated without attention to the time. If you get this message, check that Time column of your FCS is steadily increasing throughout the file. This error has also been seen when an FCS file was corrupted, and in that case was solved by re-converting the original IMD file into an FCS file.