This article walks through an example of how to annotate data with Sample tags.
If you haven't already, please read the Overview of Sample tags for background on the subject.
If you want to follow along, look for the public PBMC (fluorescence) experiment on the Cytobank server on which you have an account. This experiment is a PBMC sample stimulated with four conditions: unstimulated, IL10, IL6, and LPS.
To make a copy of the experiment for your own use, open the experiment, and Selective Clone it without Sample tags (annotations), such that you can add them yourself.
Why do we need to annotate files with sample tags?
Sample annotation is an essential step for generating the desired reports for your data analysis. In the Cytobank platform, we create illustrations based on the sample tags. It is important to tag your files before creating analysis reports for Dots plots, Heatmaps, Stain index charts, and Summary plots(statistical inference). During the statistical test for Summary plots, pairing and non-pairing tests are dependent on the sample tags. Please see this article on How to run a paired statistical test to learn more. Below is an example showing how we use sample tags to generate statistical plots to compare the significance between two populations.
Fig 1. FCS files of donors suffering from rheumatoid arthritis are annotated by two groups, that is HC and AE groups. Each group has eight samples. The CD8+ population of lymphocytes is compared between two annotated groups by Student’s t-test, and the statistical analysis shows the difference is significant.
How to annotate data with Sample tags
After the experiment has been cloned, or when opening an experiment you already own, you will be directed to the Set up new experiment page. Please refer to How to set up a new experiment? to select appropriate data scaling and compensation settings for the newly cloned experiment. Click Sample tags on the top navigation bar to annotate files with Sample tags.
We are using the "Conditions" dimension because it fits with the variable of this experiment, which is stimulations with different compounds. For more discussion on the theory of Sample tags, please see the Overview article for Sample tags.
The resulting page can be used for one experiment variable dimension in this experiment. Now that we're inside the dimension, we can add experimental attributes (Sample tags) to it. These tags can be entered as a comma-separated list:
After clicking to add these Sample tags to the experiment, the file names and tube/sample ID metadata will be scanned for character strings that match what has been entered. Where matches are found, files are automatically tagged. This highlights the value of carefully labeling your files during acquisition.
(Files have been automatically tagged from character string matching)
If you have files that aren’t assigned correctly, simply drag them to the correct Sample Tag. In this case, we will remove the extra "Unstim" file because only one is needed for analysis. Another tool at your disposal on the Sample tags page is the file filter. Enter characters into the filter to find matching files and hide others. When matching files are found, they can be sent to the desired Sample tag:
(Click to enlarge animation - Reassigning files to different Sample tags)
To change the name of any Sample Tag after creating it, simply click the name of the Sample Tag and edit it.
(rename an existing Sample Tag)
This experiment only has one variable with four conditions. Because of this, we only need to visit this single page in order to accomplish annotating this experiment. If there were other variables such as patients or timepoints, we would need to visit more pages to apply Sample tags for those classes of variables. See the Sample tags Overview for more information on multi-dimensional tagging.
Now that the files have been tagged, we're done! The Sample tags will be available in their respective dimensions boxes within the Illustration, ready to be used for generating layouts.
Also read about annotating files with Sample tag using spreadsheets.
Have more questions? Submit a request