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• Click the Merged counts data node
• Click Exploratory analysis in the toolbox
• Click PCA
• Click Finish to run the PCA with default settings (Figure ?1)

A 388pxA PCA task node will be added to the pipeline under the Analyses tab and a circular PCA output data node will be produced (Figure ?2).

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 The PCA plot will open in a new data viewer session. A 3D scatterplot will be displayed on the canvas (Figure ?3).

• Click and drag the Scree plot from the Available plots card New plot under Setup on the left onto the canvas
• Drop it over the Replace option (Figure ?4)

• Select PCA as data for the new Scree plot (Figure ?5)

The Scree plot (Figure ?6) shows the eigenvalues on the y-axis for each of the 100 PCs on the x-axis. The higher the eigenvalue, the more variance explained by each PC. Typically, after an initial set of highly informative PCs, the amount of variance explained by analyzing additional components is minimal. By identifying the point where the Scree plot levels off, you can choose an optimal number of PCs to use in downstream analysis steps like graph-based clustering and UMAP.

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• Click and drag over the first set of PCs to zoom in (Figure ?7)

• Mouse over the Scree plot to identify the point where additional PCs offer little additional information (Figure ?8)

In this data set, a reasonable cut-off could be set anywhere between around 10 and 30 PCs. We will use 15 in downstream steps.

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Graph-based clustering

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• Click the project name near the top to go back to the Analyses tab
• Click the circular PCA data node
• Click Exploratory analysis in the toolbox
• Click Graph-based clustering
• Click to Compute biomarkers
• Set the number of principal components to 15 (Figure ?9)
• Click Configure under Advanced options and change the Resolution to 1.0
• Click Finish to run the task

A Graph-based clustering task node will be added to the pipeline under the Analyses tab and a circular Graph-based clusters output data node will be produced (Figure ?10)

UMAP

Once the graph-based clustering task has completed, we can visualize the results with a UMAP plot. You could use the same steps here to generate a t-SNE plot. For this tutorial, we will use UMAP, as it is faster on several thousand cells.

• Click the circular Graph-based clusterscircular PCA data node
• Click Exploratory analysis in the toolbox
• Click UMAP
• Set the number of principal components to 15 (Figure ?11)
• Click Finish to run the task

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A UMAP task node will be added to the pipeline under the Analyses tab and a circular UMAP output data node will be produced (Figure ?12)

Notes on Performing Exploratory Analysis with Protein or Gene Expression Data Only

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It can be interesting to perform exploratory analysis on the two feature types separately. For example, you might be interested to see how the clustering of the same cells differs between protein expression profiles vs. gene expression profiles. To do this

To perform exploratory analysis on the two feature types separately, select the Merged counts data node, click Pre-analysis tools, followed by Split by feature type from the toolbox. A new task, Split by feature type, will be added to the pipeline resulting in two output data nodes: Antibody capture (protein data) and Gene expression (mRNA data). Both contain the same high-quality cells.

Performing exploratory analysis with gene expression data is the same as for the merged counts. Because there are a large number of genes, you will need to reduce the dimensionality with PCA, choose an optimal number of PCs and perform downstream clustering and visualization (e.g. graph-based clustering and UMAP/t-SNE). Performing exploratory analysis with protein data is different. There is no need to reduce the dimensionality as there are only a handful of features (17 proteins in this case), so you can proceed straight to downstream clustering and visualization. Figure ? 13 shows an example of how the pipeline might look if the data is split and analyzed separately.

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You can then use the Data viewer to bring together multiple plots for comparison (Figure ?14).

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