Question: How can I use the Filter Function in Loupe Browser?
Answer: The Filters function in Loupe Cell Browser 3.1 or Loupe Browser 4.0 or higher allows the selection of a particular set of barcodes by specifying a set of rules using boolean operators (AND/OR).
In this demonstration we are going to use the PBMC Gene Expression + Antibody Barcode .cloupe file and PBMC T-Cell Receptor .vloupe file. This dataset was generated using approximately 8,000 PBMCs labeled with a panel of 17 TotalSeq™-C antibodies. Amplified cDNA was used to prepare TCR enriched and Ig enriched V(D)J libraries. The goal of this demonstration is to use the Filters function to select a subset of T cell clonotypes based on the expression of canonical T cell markers.
First, we are going to import the V(D)J clonotypes into Loupe Browser by selecting the PBCM T-Cell Receptor .vloupe file:
Next, we are going to select all clonotypes and create a new category called “All T Cells” and a cluster called “T Cells”:
Now, we are going open the Filter interface in the right panel and start creating a set of rules allowing us to identify CD4+ helper T cells and CD8+ cytotoxic T cells. We can filter barcodes exclusively in the “All T Cells” cluster by clicking the "Create New Rule" button, then the "in [cluster name]", enter or search for the category and cluster name “All T Cells: T cells”:
This selects all 2987 barcodes in the “T Cells” cluster:
Next, to find CD4+ helper T cells, we want to find barcodes that are included in this cluster and express both CD3E and CD4. We create a new rule by clicking the “Create New Rule” button and add a rule to threshold on count. In this example, we use “CD3E > 0” and “CD4 > 0” in the “Threshold by log2(count)” field :
This rule set identifies 1029 barcodes. We can further refine our selection by excluding cells expressing CD8A or CD8B. Since each ruleset is restricted to a single boolean operator, we need to add a new ruleset. Click the Add new ruleset button, then click the Create new rule button and select the equal to (=) symbol in the “Threshold by log2(count)” field. Create a rule that reads “CD8A = 0”; repeat this step for CD8B. Since we are interested in excluding cells expressing CD8A or CD8B, we accomplish this by clicking the toggle switch in the top right of this rule and selecting “Or”.
This new set of rules will identify 1001 barcodes. We can now create a new cluster called “CD4+ Helper” in the “All T Cells” category by clicking the Assign 1001 Barcodes button:
Now let’s assign barcodes to CD8+ cytotoxic T cells. First, we want to find barcodes that are in cluster “T Cells”, express CD3E but do not express CD4. Then, we need to create a new ruleset allowing us to select barcodes expressing either CD8A or CD8B. This new set of rules will identify 776 barcodes.
We can create a new cluster called “CD8+ Cytotoxic” in the “All T Cells” category. Next, we can split the view to better visualize these clusters and run a locally distinguishing comparison analysis to identify genes significantly expressed in each cluster:
Finally, the expression of these canonical markers in each cluster can be visualized by making use of the Violin Plots feature (here below is the violin plot for CD4).