Scanpy is an open-source toolkit for the analysis of single-cell and spatial expression data.

Squidpy is a tool for the analysis and visualization of spatial molecular data.

Stereopy is a package for spatial transcriptomics data analysis of STOmics data.

Matplotlib is a Python 2D plotting library.

Seaborn is a data visualization library based on Matplotlib.

SpatialDE is a method to identify genes which significantly depend on spatial coordinates in non-linear and non-parametric ways.

LeidenAlg is a Python implementation of the Leiden community detection algorithm.

Pandas is a data manipulation and analysis library for Python.

NumPy is a fundamental package for scientific computing with Python.

Scanorama enables batch-correction and integration of heterogeneous scRNA-seq datasets.

To search for a gene (heatmap) using the search bar:

1. Click on the search bar located at the top left of the menu panel.
2. Start typing the name of the desired gene.
3. Select from the auto-completed results by clicking any of the buttons below the search bar.
4. The gene expression values are normalised and can be referenced using the colour gradient legend located at the bottom left of the screen.

To search for a gene (binary) using the search bar:

1. Move the slider below the search bar from Heatmap to Binary
2. Click on the search bar.
3. Start typing the name of the desired gene.
4. Select the desired gene from the autocompleted buttons below the search bar.
5. The gene expression values are normalised and can be referenced using the colour gradient located at the bottom left of the screen.

By default, gene names entered into the search bar will only return results that start with the input.

To also include gene names that contain the input, uncheck the Starts with Input box:

• Click on the Starts with Input checkbox to unselect it.
• The search will now include gene names that contain the input in addition to those that start with the input.

By default, each location in the dataset is coloured according to the normalised gene expression values. If no value is found for a location, the location will be cleared.

The threshold slider is set to 0% by default, which means that all locations with an expression value of 0 or Lower of the normalised values will be visualised. To focus on regions with a Low expression level of the gene, you can adjust the slider to a Lower position:

• Move the threshold slider to the desired position.
• All regions of the current gene expression pattern that are lower than the adjusted percentage of the normalised values will be cleared.

ROIs can be selected by using the selection tool.

To select a group of ROIs:

1. Click on the selection button.
2. An context menu will appear below.
3. Choose a group number from 1 to 4.
4. Click on individual locations in the dataset to add them to the respective group.
5. Selected locations will be colored according to the chosen group number.

Single locations can be removed from the ROI selection.

To remove a single location from the ROIs:

1. In the selection tool context menu use the slider.
2. Changing the slider value from + to -.
3. Click on the location that should be removed from the group 1 - 4 selected the selection.
4. Changing the slider to + again allows adding new locations to the selected group.

All locations can be removed from all groups at once.

To remove all selections:

• In the selection tool context menu use the bin button 🗑.
• This will remove all selections made.

ROI selections can be exported. This will export all current selected locations and the respective groups they are in for later analysis.

To export selections:

• Select ROIs by using the selection tool.
• Click on the export button ↑ (Arrow up).
• A .txt file will be generated to save the current selections.

To import previously saved ROI selections:

• Ensure no locations are currently selected. If unsure use the trash button 🗑.
• Click on the import button ↓ (Arrow down).
• The last saved selection will be applied

In VR-Omics, it is possible to make 3D selections of ROIs if a 3D dataset (e.g. multiple Visium slides) is used. To make a 3D selection:

1. Select the 3D selection tool.
2. Choose a group number from 1 to 4.
3. Click on a location in the dataset.
4. Underlying locations associated with the clicked location will be selected as well.

This allows the user to select 3D regions of interest. These selections can be exported as described above.

To toggle between 3D and 2D selection, use the 3D selection button.

1. From the menu select the dropdown menu spatial
2. Select UMAP

1. From the menu select the dropdown menu spatial
2. Select t-SNE

1. From the menu select the dropdown menu UMAP or t-SNE
2. Select Spatial

If cluster information is available it can be visualised in the VR-Omics Visualiser:

• Expand the top menu in the top center of the screen
• Click on the Cluster button
• Each location will be highlighted according to the cluster information found. Locations that are not found in any cluster will become black.

1. Expand the top menu in the top center of the screen
2. Click on the Cluster button
3. From the cluster legend at the bottom click on the coloured icon of the cluster to visualise.

The dataset can be overlaid with the respective tissue image. Use the tissue button to activate the tissue image overlay. The image is aligned automatically.

The image will be automatically downloaded if the dataset was downloaded using VR-Omics Automated Workflow.

If using a different dataset, ensure that the tissue file is called tissue_hires_image.png and saved within the sample folder.

The tissue image provides additional spatial orientation.

The tissue opacity can be adjusted using the opacity slider in the tissue context menu.

Move the slider to the left decreased the tissue opacity.

Move the slider to the right increases the tissue opacity.

The side-by-side comparison feature allows comparing two different gene expression patterns next to each other.

To enable the feature, click on the "Compare" button in the menu.

The current dataset will be duplicated.

Additionally, a slider with the label Duplicate will appear under the search box.

If the slider is set to left, and a new gene is selected from the search bar, the gene will be applied to the left dataset. If the slider is set to right, the next gene will be applied to the right side of the dataset.

This feature can be combined with the ROI selection tool, where ROIs will be visualized in both datasets.

If the side-by-side comparison feature is disabled while two different genes were selected, a dialog menu will open asking to merge or discard the selection.

If the user decides to merge the selection, a vector based difference analysis is performed.

The difference between the both genes selected in each location will be calculated and visualised in a heatmap like manner. This allows easily to visualise regions of similarity or high differences.

If the SVG analysis was performed using the VR-Omics Autoamted Workflow, the Visualiser is able to highlight the SVGs in the search results.

If a term is entered into the search bar, the autocomplete feature will show the search results for the available genes. The buttons for SVGs will be highlighted in cyan.

If the SVG analysis was performed using the VR-Omics Autoamted Workflow, the SVG results can be browsed from the Visualiser:

• From the Top menu select the SVG button.
• Scroll through the results. The SVG names together with their p- and q-values are shown.
• Close the window by clicking on the SVG button again.

Your current session can be saved and continued to a later time point.

This will include:

• The current SRT method and dataset.
• The current camera position.
• Current selected gene.
• Selected ROIs

To save the session select the top menu and click on the Save button.

To continue a saved session, from the main menu select the Continue Session button

Keep in mind that saving the current session will override the last session saved. The sessions are saved as json files within the data structure.

Screenshots can be made from the visualiser. For the screenshot only the dataset will be shown.

To make a screenshot:

1. Expand the Top Menu.
2. Click the screenshot button
3. The screenshot will be automatically saved locally.

ROI selections can be exported. This will export all current selected locations and the respective groups they are in for later analysis.

To export selections:

• Select ROIs by using the selection tool.
• Click on the export button ↑ (Arrow up).
• A .txt file will be generated to save the current selections.

To import previously saved ROI selections:

• Ensure no locations are currently selected. If unsure use the trash button 🗑.
• Click on the import button ↓ (Arrow down).
• The last saved selection will be applied

If the VR-Omics Automated Workflow was used to download the Visium data optionally the output plots can be generated.

The plots will be saved within the sample folder in the VR-Omics folder ../VR-Omics/Assets/PythonFiles/sample/figures.

The plots can also be quick accessed from the top menu through the Visualiser

VR-Omics allows to easily switch between the desktop application and the VR environment:

• Before starting VR-Omics ensure that your VR headset is connected.
• From the Automated Workflow click the VR button (Icon will change) and then put the headset on.
• From the Visualiser click the VR button (Icon will change) and then put the headset on.
• To leave the VR environment click the VR button again and take off the headset.
• The keyboard can be used in VR to enter text into the search bars

We used the VR toolkit plugin (XR Interaction Toolkit version 2.0.3) supported are all devices that are supported by the toolkit.

The following headsets have been confirmed to work with the plugin but not all are tested with VR-Omics: Windows Mixed Reality (eg: HP Reverb, Samsung Odyssey), Oculus Quest & Quest 2, Pimax 5K & 8KX, Varjo Aero & VR-3, Valve Index, HTC Vive, HTC Vive Cosmos, Pico Neo 3 & 4.

We recommend using the Oculus Quest 2 or Quest Pro

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If spatial plots are available they can be visualised in the VR-Omics Visualiser:

More information on which plots are available with the VR-Omics Automated Workflow can be found here

To show the plots in the Visualiser:

• Expand the top menu in the top center of the screen
• Click on the Plots button
• A new window will open showing the available plots.
• Navigate through the plots using the top menu
• Close the window by pressing the X button.

The opacity of the 3D object can be adjusted:

• Use the slider on the left side in the menu panel.
• Adjusting the slider to the left will decrease the opacity of the 3D Object until fully invisible.
• Adjusting the slider to the right will increase the opacity of the 3D Object until not transparent at all.

The 3D object can be moved independently from the dataset to align the object:

• Expand the top menu in the top center of the screen.
• Click on the 3D Object button.
• A selection of three additional buttons appears.
• Click on the move button to unlock movement.
• Once the move button was pressed the Object can be moved.
• Up: T
• Down: G
• Right: H
• Left: F
• Forward: Shift + G
• Back: Shift + T
• Lock movement by clicking the move button again.

The 3D object can be rotated independently from the dataset to align the object:

• Expand the top menu in the top center of the screen.
• Click on the 3D Object button.
• A selection of three additional buttons appears.
• Click on the rotate button to unlock rotation.
• Once the rotate button was pressed the Object can be rotated.
• Front Up: T
• Front Down: G
• Right: H
• Left: F
• Side up: Shift + G
• Side down: Shift + T
• Lock rotation by clicking the rotate button again.

The 3D object can be resized independently from the dataset to align the object:

• Expand the top menu in the top center of the screen.
• Click on the 3D Object button.
• A selection of three additional buttons appears.
• Click on the resize button to unlock the resize feature.
• Once the resize button was pressed the Object can be resized
• Increase width: T
• Decrease width: G
• Increase height: H
• Decrease height: F
• Increase depth: Shift + G
• Decrease depth: Shift + T
• Lock the resize feature by clicking the resize button again.

Per default the Symbol to visualise a location is a sphere (except of Tomo-Seq). This can be changed to a Cube:

• Extend the top menu from the top center of the screen.
• Click on the change symbol button.
• The locations will be changed to the selected symbol and the button changes accordingly.
• Click the new button again to toggle back to the original symbol.

The symbol size can be changed:

• From the menu panel on the left use the Size slider to adjust the size of the symbols.
• Move the slider to the left to make the symbols smaller.
• Move the slider to the right to increase the symbol size.

To reset the view to the origin click the reset view button in the bottom left of the screen.

The background colour can be changed from dark to bright mode.

• From the menu panel on the left click on the bright mode button if the background is currently set to black.
• The background will change to white and the bright button will be replaced with a dark button.
• To change the colour back to black click the dark button.