Tips for boolean analysis using Funky Cells

Author: Louis HENRY

 


Combined with Funky Cells, Boolean data analysis lets you explore a wide range of populations based on your markers, and also take a closer look at polyfunctionality. Below, you’ll find a few tips to help you save time and make the analysis smoother.

 

1. Anticipation

When setting up your gating strategy, it’s a good idea to think ahead about whether you want to perform a boolean analysis of your samples. This will help you design a gating strategy that works both for a classical analysis and for the boolean analysis later on. You’ll need to make sure your gates make biological sense, while still being suitable for boolean analysis. Keep in mind that a boolean analysis requires each gate to include all positive events for a given marker, whether or not they form a clearly defined cell population. If you manage to build a strategy that works for both classical and boolean analyses, you can simply duplicate your FlowJo workspace (.wsp), keep one version for the classical analysis, and run the boolean analysis on a separate file.

 

2. Segment your files

For boolean analysis, it is recommended to keep the number of samples per FlowJo file as low as possible. The more markers you plan to include in your boolean analysis, the more important it is to split your work across multiple FlowJo files. As a general rule, try to avoid having more than 60 samples per FlowJo file.

  

3. Apply gates to all sample in one go

You’ll quickly notice that FlowJo becomes very slow because of the large number of gates that get created during a boolean analysis (2^number of variables). To apply the analysis efficiently across all your groups, you can follow the steps below:

 

  • Select all the gates you want to include in the boolean analysis.
  • Go to “Create Combination Gates” in the Tools menu.
  • In the window that opens, rename the acronyms if needed, select all the files you want to include, and validate. FlowJo will then calculate all the gates, which can take quite some time. Do not interact with the software until this step is finished.
  • Once completed, select all the boolean gates and copy them (Ctrl / Cmd + C).
  • Go to the “All Samples” group (this step is important).
  • On a single sample (any one), select the parent populations where you want to apply the boolean analysis. Note that this will only work if the gates below the selected parent have the same gate names as those used to originally create the boolean gates.
  • Once the parent populations are selected, right-click and choose “Select Equivalent Nodes.”
  • Carefully check that the correct parent gates are selected. FlowJo can make mistakes at this stage. If the selection is incorrect, click on a random gate to deselect everything and try again (keeping the parent including the original boolean gates open can help here).
  • When you are sure the correct parent gates are selected, paste the boolean gates (Ctrl / Cmd + V).
  • FlowJo will then start applying all the gates, which can take a long time (up to ~45 minutes depending on the number of gates). Do not interact with FlowJo during this period, not even in another window. If possible, it’s recommended to use another computer in parallel.
  • Once the boolean gates have been applied to all samples, save your work, completely close FlowJo, and reopen it before moving on to the next file. FlowJo keeps data cached, and leaving it open for too long can cause crashes or freeze your computer.
  • This approach has the advantage of letting you do something else while FlowJo applies all the gates to your samples in one go.
  • Good luck…

 

Video tutorial for FlowJO boolean gating implementation.

  

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