When working with groups of samples or several panels, gating groups on the Cytobank platform allow you to create different gates and population hierarchies per group. Here, we will give an overview of how to build and manage gating groups per experiment in the Cytobank platform. Please see Overview of Gating in the Cytobank platform to learn more about gating and gating-based visualization.
Video overview
For a complete overview of gating in the Cytobank platform, see our video below!
Using gating groups in an Experiment
When working with different groups of samples or more than one Panel in one experiment, you may want to apply different gates or populations to different groups. With gating groups you can set up multiple gating strategies within one experiment. The organization of groups of files into gating groups is managed using the gating groups sample tag dimension. Assigning files to gating groups provides you with the ability to have distinct sets of gates and their associated populations per individual gating group. Note, however, that per file only one gating strategy can be used. For gates that are present in multiple gating groups, gate locations can be synced either for all files within one group, across all files in all groups where the gate is present, or on a per-file basis. For more details on synchronizing gate locations, see Sync settings: adjust the location of a gate to files, populations and gating groups.
Gating groups can also be used to filter files in other parts of the platform to facilitate setting up Advanced analyses or Illustrations. For more details, see How to configure and run a dimensionality reduction analysis, How to set up a new automatic gating analysis by training a new model, How to Configure and Run a FlowSOM Analysis, How to Configure and Run a SPADE Analysis, How to Configure and Run a CITRUS Analysis, and Overview of figure generation using the Illustration Editor.
Setting up gating groups using the Sample Tag Manager
To setup Gating groups from the Gating Editor, select the Define gating groups link under Gates. This will open the Sample Tag Manager window.
(Gating Editor Define gating groups link.)
Alternatively, you can navigate directly to the Sample Tag Manager from anywhere in an Experiment by selecting Sample tags in the experiment level navigation bar and selecting Sample Tag Manager.
In the Sample Tag Manager window, click on Add dimension and select Gating groups. You can now complete the column with the appropriate gating group name for any files you would like to assign to a gating group. Once you are done, click on Apply changes. You can return to the Sample Tag Manager and update your gating group assignment at any point. For details on working with the Sample Tag Manager, see How to annotate data with sample tags in Cytobank (tutorial).
(Sample Tag Manager with Gating groups dimension and Apply changes button.)
Using gating groups in the Gating Editor
If no gating groups have been defined, all files will be assigned to the Default gating group. When gating groups have been assigned, both gates and populations are organized in gating groups based on the gating groups sample tag dimension in the Gating Editor. To facilitate orientation in experiments with one or several complex gating strategies, groups can be collapsed and expanded as needed.
The gating group assignment of the FCS file visible in the gating plot determines which gates can be visualized. You can only show gates that belong to the same gating groups as the file that is currently shown. All gates in the Gates list belonging to other gating groups are greyed out. When you select one of the greyed-out gates from another gating group, the gating plot is automatically updated to show the first FCS file from the relevant gating group along with the selected gate. If a parent input population can be unambiguously identified, this will also be updated together with the channels used to draw the gate. When you select a gate that belongs to a gating group that does not currently have any FCS files assigned to it, you will see a prompt to assign files using the Sample Tag Manager but you will still be able to perform gate actions such as renaming that gate, making changes to the gate sync settings etc.
Moving gates and populations between gating groups
When a gate is created on a file, the gate and associated population become part of the gating strategy for only the gating group the FCS file belongs to.
It is possible to use the same gate and population in several gating groups. To achieve this, there are two options:
- Copy gate to a population in a new gating group: gates can be copied to a population in a different gating group by dragging and dropping the gate. A new population is generated adding the dragged gate to the target population as parent. Alternatively, you can select the Copy to population function in the Gates section for the gate to be copied and then click on the desired parent population. When using the Copy to population button, a dialogue box appears allowing you to select whether the gate is copied to the selected group only or to all applicable groups. You can temporarily save your selection for future Copy to population actions by checking the Remember this decision for 5 minutes check box. The newly created population is named based on the gate name with the name of the parent population added in parenthesis.
- Copy a population to another gating group: populations can be copied to other gating groups by dragging and dropping the population to the gating group name in the Population tree. The gate position in the destination group will be set to match the position in the originating group. If gates required to define the population are missing from the destination gating group, a dialogue box appears asking you whether you want to copy the missing gates between groups. It is possible to multi-select several populations to copy them to another gating group in one step.
Note that population names must be unique within an experiment. You cannot copy a gate or population to another gating group if the action would result in same-named populations in the destination group. Gates in different gating groups may share the same name when drawn on the same channels and have the same shape. Populations in different gating groups sharing the same gate logic may also share a name.
If a population is copied to a new gating group without the required gates for the definition of that population, the newly created population will have a red error sign. This error can be resolved by using the Copy gate to functionality and choosing an FCS file from the destination gating group as a target.
Synchronizing gate positions
For every gate, you have several options to synchronize gate positions across files and populations. The synchronization option chosen is visible in the Gates section of the Gating Editor. When you click on the syncing icons for any gate, the Gate position dialogue opens, where you can choose to sync the gate position across all files or populations for which the gate is available, all files or populations within each gating group, or none.
If you want to learn more about the different gate sync modes, clicking on the hover tip in the Gate positions window opens the Learn about: Gate position sync modes window. In this box you can explore interactive examples for the different modes without making changes to your actual gating strategy.
Possible gate synchronization combinations are:
When a gate is created on a file in a gating group, by default the gate location is set to be synced across all files and all populations.
For more details, see also Sync settings: adjust the location of a gate to files, populations and gating groups.
Managing gating groups
Once gating groups have been set up using the Sample Tag Manager, you can always go back and change the file assignment. In addition, you can rename a gating group in the Gating Editor, just note that every gating group needs to have a unique name within the experiment.
You can also delete gating groups in the Gating Editor. When deleting a gating group that does not contain any gates and populations, no further warning will be given. When you delete a gating group that contains gates and populations, you are prompted to choose whether you also want to delete the gates and populations within this group. If you choose not to delete the gates and populations, they will be moved to the Default group.
For more details on managing gates and populations, also see The impact of deleting gates or populations.
Further support for gating groups
Gating groups are also part of the extended functionality in the Gating Editor, such as undo/redo and saving/loading gate versions. It is possible to undo actions like deleting and renaming gating groups. For more details, see Undo and Redo options in the Gating Editor .
The gating groups themselves, names of gating groups, and associated gates and populations are saved as part of gate versions and are loaded when loading a previously saved gate version. Note that the FCS file assignment is not part of the saved gate version, i.e., when restoring deleted gating groups by loading a previously saved gate version, you have to re-assign the FCS files to the restored gating group in the Sample Tag Manager. For more details on saving and loading gate versions, see Gate versioning .
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