Visium data analysis pipeline

A basic example of a spatial data analysis is outlined below and is similar to a single cell RNA-Seq analysis pipeline with the addition of the Spatial report. Note that Single cell QA/QC has not been performed in this example and would be performed from the Single cell counts note with the filtered cells applied to the Single cell counts before the Filter features task. Click here for more information on Single cell QA/QC. 

Performing tasks in the Analyses tab

A context-sensitive menu will appear on the right side of the pipeline. Use the drop-downs in the toolbox to open available tasks for the selected data node. 

An important step in analyzing single cell RNA-Seq data is to filter out low-quality cells. A few examples of low-quality cells are doublets, cells damaged during cell isolation, or cells with too few counts to be analyzed. Click here for more information on Single cell QA/QC. We will not perform Single cell QA/QC in this tutorial. 

Filter Features

• Click the Filtering drop-down in the toolbox
• Click the Filter Features task 
• Choose Noise reduction
• Exclude features where value <= 0.0 in at least 99.0% of the cells 
• Click Finish

A task node, Filtered counts, is produced. Initially, the node will be semi-transparent to indicate that it has been queued, but not completed. A progress bar will appear on the Filter features task node to indicate that the task is running.

Normalization

• Select the Filtered Counts result node
• Choose the Normalization task from the toolbox 

• Click Use recommended  
• Click Finish

Exploratory analysis

• Click the Normalized counts result node
• Select the PCA task under Exploratory analysis in the toolbox
• Unselect Split by Sample
• Click Finish

• Click the PCA result node
• Select the Graph-based clustering task 
• Click Finish

• Click the Graph-based clustering result node
• Select the UMAP task
• Click Finish

• Double-click the UMAP result node

The UMAP is automatically colored by the graph-based clustering result in the previous node. To change the color, click Style. 

Automatic classification 

• Click the Filtered counts node
• From the Classification drop-down in the toolbox, select Classify cell type 
• Using the Managed classifiers, select the human Intestine Garnett classifier
• Click Finish

Double-click the Classify result node to view the cell count for each cell type and the top marker features for each cell type.

• Click the Classify result node
• Select Publish cell attributes to project under Annotation/Metadata 
• Select cell_type from the drop-down and click the green Add button
• Name the cell attribute
• Click Finish

The name of the Cell attribute can be changed in the Metadata tab. 

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