Background
When SPADE is run, events are pulled from a selected population across all compatible files in the Cytobank experiment. The events are pooled together, and the algorithm operates on all of them. The end result is a single minimum spanning tree that represents all of the data. For analysis, however, the files are split back out and visualized individually in context of the global tree. Each file will fill the tree in a unique way.
Looking at individual SPADE trees is useful but presents problems when a quantitative comparison between two trees is desired. Consider the situation depicted below. Two annotated SPADE trees are shown with pSTAT5 intensity as the coloring channel. Differences can be observed between the trees, but it requires looking back and forth on a node-by-node basis. This method doesn't scale and comparisons can be hard to make.
(Comparing basal to IL-7 is scientifically interesting, but hard to do well with separate trees Bendall, Simonds, et al, Science, May 2011.)
This two-tree problem is solved easily in SPADE by overlaying the trees. Each node, instead of displaying a raw value for intensity on a certain channel, will now display a fold intensity. The fold is calculated on a node-by-node basis.
(The two separate SPADE trees can be visualized as one, with fold change intensity indicated as the color. Bendall, Simonds, et al, Science, May 2011.)
(two separate SPADE trees are overlaid to view as a single SPADE tree. Coloring is fold intensity on pSTAT5 Bendall, Simonds, et al, Science, May 2011.)
How to setup SPADE with fold change
SPADE requires configuration for fold change before the run is executed, and samples cannot be compared dynamically after the fact. To use different baselines, a different SPADE will have to be run. (Note: if you are invested in a particular SPADE tree, with effort, any SPADE tree can be manually changed to show whatever values you like).
To get started with configuring fold change, first create a new SPADE run. On the SPADE setup page, click inside the Fold-change groups box or on the Choose option to open the Select fold-change groups dialogue.
(Click the Fold-change groups box to open the Select fold-change groups dialogue.)
The SPADE Select fold-change groups dialogue is visualized below with annotations:
(The SPADE Select fold-change groups dialogue.)
Two important concepts to know when setting up SPADE with fold change are fold-change groups and baselines.
A fold-change group is a collection of files that will be compared amongst each other for the fold-change calculation. In order to compare files with each other, there needs to be a baseline file. Each file will be compared to this baseline in order to calculate fold change. Multiple baseline files can be selected within one group. In this case the statistics for the files will be averaged before serving as the baseline. Again, fold is calculated on a node-by-node basis.
When using multiple fold-change groups, the calculations are encapsulated only within those groups. No calculation is done between groups. Each group will have to have its own baseline to compare between files in that group. Fold change is simply a calculation and visualization and doesn't affect the structure of the SPADE tree.
To set up multiple fold-change groups, click Add group(s) in the Select fold-change groups dialogue, type comma-separated group names and click OK. You can reset the groups by clicking on Reset to match panels button. Once you have added all fold-change groups, assign the files to the groups. Then, check the Baseline box to indicate which samples will act as baseline(s). Use the search box to filter the displayed files. Click on a fold-change group name to access further functionalities: assign all displayed files to that group, view the channels in the file, and rename or delete the group. When all files are correctly assigned to the fold-change groups and the baseline(s) sample defined, click Close to return to the complete SPADE setup.
You can use this dialogue to exclude samples from a SPADE run. Just set the samples that you would like to exclude from the analysis to the Unassigned group.
How to analyze SPADE with fold change
The principal way to characterize a SPADE tree during any analysis is by coloring the tree by a channel. When this is done, each node will report a colorized version of a statistic taken from the events within that node. Besides choosing a channel to display, a metric needs to be chosen for the data in that channel.
The selected metric determines how the SPADE tree is colored. There are five options in SPADE, or three if fold change is not enabled. To view the tree in context of fold change, the metric must be changed to Fold or Raw Fold. At this point the nodes will be colored in overlay mode.
(Change the SPADE metric to Fold or Raw Fold to view the tree in context of fold change to baseline.)
To learn more about SPADE metrics of Medians, Raw Medians, Fold, Raw Fold, and CVs, read the article on SPADE metrics.
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