Visualisation and Analysis of Spatially Resolved Transcriptomics Data.

VR-Omics

 

Abstract

Spatially resolved transcriptomics (SRT) technologies produce complex, multi-dimensional data sets of gene expression information at subcellular levels that enable biologist to systematically investigate spatiotemporal gene expression patterns. However, analysis of SRT data remains broadly underpowered by complexity and lack of adequate software suited to non-computational users. We introduce VR-Omics, a freely available programme for comprehensive analysis, visualisation, and exploration of single or multi-section SRT data generated from a variety of platforms as desktop application or immersive virtual environment. VR-Omics accommodates various SRT technologies, including 10x Genomics' Visium and Xenium, Vizgen's MERFISH, open-source Tomo-seq, and custom SRT data. The system can be implemented as a local 3D desktop application or as an immersive environment in virtual reality using head-mounted display. This allows biologists to interact with the data in novel ways that were previously inaccessible, which includes visually exploring the gene expression patterns of an organ and comparing genes based on their 3D expression profiles.

Download VR-Omics

Documentation

Automated Workflow

Quick Access Visualisation Features

VR-Omics Setup
1 Download VR-Omics

Click the Download VR-Omics button to obtain VR-Omics from our repository.

Download VR-Omics
2 Download VR-Omics

Click the Download button

3 Unzip the folder

To unzip the folder, right-click on the zip file and select Extract here.

4 Start VR-Omics

Once the folder is unzipped, open it and double-click on the VR-Omics.exe.

VR-Omics Automated Workflow
Visium
Download Data
1 Choose the Download menu
  1. From the main menu select Visium
  2. Select Download
VR-Omics main menu.

2 Selecting datasets and filtering
  1. Choose a dataset from the dropdown menu.
  2. Optional: Filter dataset by entering the respective parameters into the input fields, or leave them blank to skip filtering.

3 Identify spatially variable genes

Tick the Spatially Variable Genes box to perform spatial analysis.

Spatially variable genes are genes that exhibit differences in expression levels across different regions or cell types within a tissue or organism.

4 Download the dataset

Select Download to download and process the dataset.

The data will be downloaded from the 10X Genomics Resources Website and processed to be read in VR-Omics

The data will be stored in the VR-Omics folder on your local machine. ../VR-Omics/Assets/PythonFiles/sample/ To Visualise the data select the Visium Load function and select the whole folder from the Browse menu.

Load data from local machine
Loading a single Visium slide
1 Load a single Visium dataset
  1. From the main menu select Visium
  2. Select Load
2 Select sample folder
  1. Select the main folder of the dataset
  2. Click Load to confirm
Select the main folder of the processed Visium sample.
Samples processed using the VR-Omics workflow can be found in the VR-Omics folder as shown above.
3 Confirm selection
  1. The selected sample's tissue image will be shown.
  2. Expand the tissue image using the expand button.
  3. Delete the sample by clicking the 🗑 button.
  4. Select the sample by clicking Align

If the tissue image wasn't found a default VR-Omics placeholder will be shown. To fix this, please ensure that the respective file within the sample folder is named tissue_hires_image.png

The dataset can be visualised and explored without using the tissue image with some limited functionalities.

4 Rotate the samples in the 3D mapping tool
  1. Use the slider to set the transparency of the tissue image.
  2. Use the + or - Button to rotate the slide.
  3. Leave the distance field blank.
  4. Click the Go! button if all adjustments are made to visualize the combined dataset in the VR-Omics Visualizer.
Concatenate multiple Visium slides in 2D space

1 Select the Concat menu
  1. From the main menu select Visium
  2. Select Concat
2 Select the Visium samples to merge
  1. Click Browse
  2. From the Explorer find the main folders of the datasets to merge.
  3. Click Load to load the samples.
3 Check the selected samples
  1. The selected samples will be shown
  2. Expand the tissue image or delete a sample using the 🗑 button.
  3. Click Concat to confirm the selection.
The order of the samples is not important for the following steps.
3 Arrange the slides
  1. Arrange the slides by using the mouse.
  2. Drag a sample by using left click to move the sample into position
  3. Hold down the left mouse button and click the right mouse button once to rotate a sample by 90 degrees.
  4. Click Concat to confirm.
Ensure that no samples overlap with each other.
4 Processing the joint dataset
  1. (Optional) Enter filter parameters to filter the joint dataset or leave fields blank.
  2. (Optional) Select T-SNE and UMAP analysis
  3. (Optional) Select SVG analysis to run spatial analysis.
  4. Click Process to confirm.
Click here for more information on filter parameters. Selecting T-SNE and UMAP as well as SVG analysis increases processing time of the sample.

Loading multiple Visium slides in 3D
1 Load multiple Visium datasets in 3D
  1. From the main menu select Visium
  2. Select Load
2 Select sample folders
  1. Click Browse
  2. Using the explorer select the main folders of the samples to load.
  3. Click Load to confirm selection.
The samples processed using the VR-Omics workflow are stored in the VR-Omics application folder on your local machine. ../VR-Omics/Assets/PythonFiles/sample/
3 Confirm selection
  1. Tissue images can be expanded.
  2. Change the order of the samples using ˄ or ˅.
  3. Remove samples by clicking 🗑
  4. Confirm selection and order by clicking Align

If the tissue image wasn't found a default VR-Omics placeholder will appear. To fix this, please ensure that the respective file is named tissue_hires_image.pngin the directory.

4 Rotate the samples in the 3D mapping tool
  1. Select a sample from the dropdown menu.
  2. Adjust transparency using the slider for the selected tissue image.
  3. Rotate the sample with the + or - buttons.
  4. Enter slide distances separated by commas.
  5. Click Go! to visualize the combined dataset in the VR-Omics Visualizer.
Process
1

Process Visium dataset from local machine

Combinable with: None

  1. From the main menu select Visium
  2. Select Process
2

Process Visium dataset from local machine

Combinable with: None

  1. Click Browse and select the visium folder from your local machine.
  2. (Optional) Select the Skip Filter Step toggle only if you wish to skip the filter and SVG step.
  3. Click Next to continue.
The folder must contain a spatial folder and a h5 file named filtered_feature_bc_matrix.h5
The samples processed using the VR-Omics workflow are stored in the VR-Omics application folder on your local machine. ../VR-Omics/Assets/PythonFiles/sample/
3

Process and Filter Visium dataset from local machine

Combinable with: Spatially Variable Genes

  1. Select the filter parameters.
  2. Optionally, perform spatial analysis by selecting the SVG Analysis toggle.

The output will be saved in the VR-Omics folder

After clicking Process, the Python executable will be started. Don't close the pop up windows and wait until the pipeline has finished. This might take a couple of minutes.

Demo
1 Explore the Demo dataset
  1. From the main menu select Visium
  2. Select Demo
2 Demo in Visualiser

This will automatically load the demo dataset in the VR-Omics Visualiser ready to be explored.

Filter parameters

Min.count - Filter spots by gene count

Min.count is a filter used to exclude spots with gene expression lower than a specified value from further analysis. It is used to ensure more accurate and robust results in gene expression experiments.

Max.count - Filter spots by gene count

Max.count is a filter used to exclude spots with gene expression Lower than a specified value from further analysis. It is used to ensure more accurate and robust results in gene expression experiments.
Filter spots by percentage of MT count (minimum) MT.count (minimum) is the filter parameter used to exclude spots that have a lower Mitochondrial count than the specified value.
Filter spots by percentage of MT count (maximum) MT.count (maximum) is the filter parameter used to exclude spots that have a Lower Mitochondrial count than the specified value. A Low mitochondria count can be a sign of cell death or cell damage or stress.
Filter out genes that are detected in less spots than this value This minium threshold filter parameter can be used to exclude all genes from the dataset that are detected in less spots than the value provided. Keep in mind the total number of genes of the dataset. VR-Omics will skip this step if this value was selected to Low so that no genes remain or if the value exceeds the total genes number of the dataset.
Filter out spots that have less genes expressed than this value. This minium threshold filter parameter can be used to exclude all spots from the dataset that have less genes expressed than this value. Please keep the maximum number of spots in the dataset in mind. VR-Omics will skip this step if the value was selected so Low, that no spots would remain after analysis.

Further information can be found in the Scanpy spatial analysis documentation.

Required Folder Format for samples not processed with VR-Omics

For samples not downloaded or processed using the VR-Omics Automated Workflow, ensure this folder structure. Further descriptions of the files can be found below. Alternatively, they can be uploaded using the custom data option with limited features.

Required file name ending Full name (VR-Omics) Description Required
tissue_hires_image.png tissue_hires_image.png The high-resolution tissue image (2000 x 2000 pixels) as provided by 10x genomics. false but impacts features
Tissue_positions_list.csv Tissue_positions_list.csv CSV file as provided by 10x genomics. File does not contain headers. 1. Spot IDs; 2. spot on tissue; 3. + 4. row and column of spot; 5. + 6. X and Y coordinates of spot; true
transposed.csv outs_transposed.csv CSV file containing gene counts per spot. Header contains spot IDs. First column contains gene names. Each row contains gene count information per spots (columns). true
metadata.csv outs_metadata.csv Header: in_tissue, array_row, array_col, n_genes_by_counts, log1p_n_genes_by_counts, total_counts, log1p_total_counts, pct_counts_in_top_50_genes, pct_counts_in_top_100_genes, pct_counts_in_top_200_genes, pct_counts_in_top_500_genes, total_counts_mt, log1p_total_counts_mt, pct_counts_mt, n_counts, n_genes, clusters true
gene_panel_IDs.csv outs_gene_panel_IDs.csv CSV file with one column header name gene listing all gene names in the dataset. true
svgtoggle.csv outs_svgtoggle.csv g, FSV, M, g, l, max_delta, max_ll, max_mu_hat, max_s2_t_hat, model, n, s2_FSV, s2_logdelta, time, BIC, max_ll_null, LLR, pval, qval false
obsm.csv outs_obsm.csv 1. column spot IDS. 2. + 3. Spot coordinates in pixels. Followed by a variable number of columns for X_PCA values, UMAP X and Y coordinates, and TSNE X and Y coordinates. false
Xenium
Process

Process Xenium data

1 Navigate to the Xenium process menu
  1. From the main menu select Xenium
  2. Select Process
2 Select the Xenium files
  1. Select the cell feature matrix .h5 file using Browse.
  2. Select the cells csv file using Browse.
  3. (Optional) select filter parameters or leave blank.
  4. Select the Run SVG Analysis toggle to perform spatial analysis (Moran Results)
  5. Select T-SNE & UMAP toggle.
  6. Click Process to confirm.

Selecting SVG Analysis and/or T-SNE/UMAP will increase processing time of the sample.

The processed data will be saved in the VR-Omics data folder. To Visualise the data select the complete folder as shown in the figure.

After clicking Process, the Python executable will be started. Don't close the pop up windows and wait until the pipeline has finished. This might take a couple of minutes.

Load
1 Navigate to the Xenium load menu
  1. From the main menu select Xenium
  2. Select Load
2 Select the Xenium sample folder
  1. Select Browse
  2. Using the explorer select the main folder of the processed Xenium folder.

Select the Xenium folder processed with the VR-Omics AW (highlighted in green in the picture) using Browse.

The data set can be found in the VR-Omics directory. Navigate from there to VR-Omics/Assets/PythonFiles/sample select the whole folder as shown in the picture

3 Confirm selection

Click Visualise to continue to the VR-Omics Visualiser.

Ensure the data has been processed before visualising it using the Process function.

Tomo-Seq
Load

Load Tomo-Seq data

1 Navigate to the Tomo-Seq load menu
  1. From the main menu select Tomo-Seq
  2. Select Load
2 Select the Tomo-Seq folder
  1. Select Browse
  2. Choose the main folder of the Tomo-Seq data using the Explorer.
  3. Click Visualise to confirm

* AP = anterior to posterior, VD = ventral to dorsal, and LR = left to right

The 3D reconstruction of the gene expression is explained in Asp et al. in Supplementary Data S2.

3 Select the Tomo-Seq files manually
  1. Click Manual File-Selection
  2. Select the AP gene expression .csv file in AP* cutting direction using Browse.
  3. Select the LR gene expression .csv file in VD* cutting direction using Browse.
  4. Select the VD gene expression .csv file in LR* cutting direction using Browse.
  5. (Optional) Select a bitmask.txt file using Browse.
  6. Select the folder containing the reconstructed gene expression .txt files using Browse.
  7. Click Visualise to confirm.

* AP = anterior to posterior, VD = ventral to dorsal, and LR = left to right

The 3D reconstruction of the gene expression is explained in Asp et al. in Supplementary Data S2.

STOmics
Process

Process STOmics data

1 Navigate to the STOmics process menu
  1. From the main menu select STOmics
  2. Select Load
2 Select the STOmics file

Select the gene expression matrix .h5 file of the STOmics data using Browse.

The dataset can be downloaded from STOmics Database.

3 Confirm selection

Click Process to process the data.

The processed data will be saved locally.

The process step can take couple of minutes depending on the size of the dataset and the computational power of the machine.

Once the data was processed this step can be skipped and the data can be visualised using the STOmics load option.

The processed data will be saved in the VR-Omics data folder. To Visualise the data select the complete folder as shown in the figure.

Load
1 Navigate to the STOmics load menu
  1. From the main menu select STOmics
  2. Select Load
2 Select the STOmics files

Select the processed gene expression STOmics .h5 file using Browse.

Ensure the data was processed using the Process function.

The processed data can be found in the VR-Omics data folder. To Visualise the data select the complete folder as shown in the figure.

MERFISH
Process
1 Navigate to the MERFISH process menu
  1. From the main menu select MERFISH
  2. Select Process
2 Select the MERFISH files
  1. Select the Vizgen counts file .csv using Browse
  2. Select the Vizgen cell_metadata .csv file using Browse
  3. Select the Vizgen detected_transcripts .csv file using Browse
  4. (Optional) Enter filter parameters or leave fields blank.
  5. (Optional) Select the Run SVG Analysis toggle to include spatial analysis (Moran Results)
  6. (Optional) Select the T-SNE & UMAP toggle
  7. Click Process to confirm

The MERFISH data we used is from Vizgen, where there data is publicly available under their data release program.

The processed data will be saved in the VR-Omics data folder. To Visualise the data select the complete folder as shown in the figure.

After clicking Process, the Python executable will be started. Don't close the pop up windows and wait until the pipeline has finished. This might take a couple of minutes.

Load
1 Navigate to the MERFISH load menu
  1. From the main menu select MERFISH
  2. Select Load
2 Select the MERFISH files
  1. Select Browse
  2. Select the MERFISH sample folder processed using the VR-Omics AW
  3. Select Visualise to confirm.

The processed data can be found in the VR-Omics data folder. To Visualise the data select the complete folder as shown in the figure.

The MERFISH data used was provided by Vizgen where there data is publicly available under their data release program.

Custom data
Load
1 Navigate to the Custom data load menu
  1. From the main menu select Custom data
  2. Select Load
2 Select the Custom data files
  • Select the custom gene expression* .csv file using Browse
  • Select the custom meta data** .csv file using Browse
  • Optional: If the dataset contains three dimensions (X,Y, and Z) move the slider with the 2D label to the right.
  • * Ensure the data is stored in a gene per location data file using columns for locations and rows for gene expressions

    ** The meta data file stores the X,Y and optional Z coordinates of the dataset. Each row needs to represent one location where the locations need to be in same order as in the gene expression file.

    3 Specify columns
  • For the location information uploaded in 2. provide the column that stores the respective information.
  • Select the Column for the X coordinates. For the first column enter 0 for the second column enter 1 etc.
  • Select the Column for the Y coordinates. For the first column enter 0 for the second column enter 1 etc.
  • Select the Column for the Z coordinates. For the first column enter 0 for the second column enter 1 etc.*
  • Select the Column for the Location IDs. For the first column enter 0 for the second column enter 1 etc.**
  • Tick the No CSV header box if the CSV files contain no header information.***
  • * Leave blank if no three dimensional dataset

    ** Leave blank if no IDs available

    *** The header is the first row in a csv file usually used to store the headers of each column.

    3D Object
    Load
    1 Navigate to the 3D object menu

    From the main menu select 3D object

    2 Select the 3D object file
  • Select the 3D object* using Browse
  • Enter the X, Y, and Z Coordinates where the object should be rendered**
  • Enter the X, Y, and Z Rotation of the 3D object***
  • * Only 3D object supported by Unity can be uploaded. More information can be found using the Unity documentation. This feature was tested with .fbx and .obj files.

    ** The coordinates are relative to the dataset, the dataset will always be rendered in the origin X,Y,Z → 0,0,0. If no coordinates available use 0,0,0. The 3d object can easily be moved within the VR-Omics Visualiser.

    *** The rotation is relative to the dataset, the dataset will always be rendered in the origin X,Y,Z → 0,0,0 facing the user with 0-rotation. If no rotation values are available use 0,0,0. The 3d object can easily be rotated within the VR-Omics Visualiser.

    Ensure the 3D object has the right measurements, the object can be resized within the VR-Omics Visualiser but if the object was uploaded top big, or too small compared to the rendered dataset this might lead to issues. In this case please adapt the dimensions of the object prior.

    3 Submit the object

    Store the selected 3D object by clicking Submit

    Ensure the object was submitted before uploading a spatial dataset. This can't be done after entering the VR-Omics Visualiser.

    4 Removing an object

    Remove all selected objects from the selection using Delete All

    Multiple objects can be uploaded, although this feature is only tested with one object.

    AW Packages used
    Name of package Description Platforms used Link
    Scanpy

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

  • Visium
  • Xenium
  • Merfish
  • STOmics
  • Scanpy
    Squidpy

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

  • Xenium
  • Merfish
  • Squidpy
    Stereopy

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

  • STOmics
  • Stereopy
    Matplotlib

    Matplotlib is a Python 2D plotting library.

  • Visium
  • Xenium
  • Merfish
  • STOmics
  • Matplotlib
    Seaborn

    Seaborn is a data visualization library based on Matplotlib.

  • Visium
  • Xenium
  • Seaborn
    SpatialDE

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

  • Visium
  • SpatialDE
    LeidenAlg

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

  • Visium
  • LeidenAlg
    Pandas

    Pandas is a data manipulation and analysis library for Python.

  • Visium
  • Xenium
  • Pandas
    NumPy

    NumPy is a fundamental package for scientific computing with Python.

  • Visium
  • Xenium
  • NumPy
    Scanorama

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

  • Visium
  • Scanorama

    VR-Omics Visualisation
    Gene Search Features
    Feature Description Icon Image
    Gene search - Heatmap
    All
    VR Demo

    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.
    Binary search - Gene is on
    Visium Xenium STOmics Merfish Custom
    VR Demo

    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.
    Change if gene search contains or starts with term
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Threshold - Minimum
    Visium Xenium STOmics Tomo-Seq Merfish Custom
    VR Demo

    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.
    The minimum threshold is set to 0% including every location of the dataset with expression levels 0 or Lower.
    The minimum threshold is increased to around 9%, clearing all locations with expression levels below 9% of the normalised values.
    Location Interaction Features
    Feature Description Icon Image
    Selecting Regions of Interest (ROI)
    Visium Xenium STOmics Merfish Custom
    VR Demo

    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.

    Remove a single location
    Visium Xenium STOmics Merfish Custom
    VR Demo

    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.

    Remove all selections
    Visium Xenium STOmics Merfish Custom
    VR Demo

    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.

    Export ROI selections
    Visium Xenium STOmics Merfish Custom
    VR

    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.
    Import ROI selections
    Visium
    VR

    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
    3D ROI selection
    Visium Xenium STOmics Merfish Custom
    VR Demo

    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.

    Dimensionality Reduction Features
    Feature Description Icon Image
    Visualising UMAP
    Visium Xenium STOmics Merfish
    VR
    1. From the menu select the dropdown menu spatial
    2. Select UMAP

    Visualising t-SNE
    Visium Xenium STOmics Merfish
    VR
    1. From the menu select the dropdown menu spatial
    2. Select t-SNE

    Clusters can be visualised regardless of the visualisation selected.
    Visualising spatial coordinates
    Visium Xenium STOmics Merfish
    VR
    1. From the menu select the dropdown menu UMAP or t-SNE
    2. Select Spatial

    Clusters can be visualised regardless of the visualisation selected.
    Cluster Visualisation Features
    Feature Description Icon Image
    Show Leiden Cluster
    Visium Xenium STOmics Merfish Custom
    VR Demo

    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.
    Show Single Clusters
    Visium Xenium STOmics Merfish Custom
    VR Demo
    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.
    Visualisation of only cluster 0
    Tissue Overlay Features
    Feature Description Icon Image
    Tissue Overlay
    Visium
    VR

    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.

    More information about the tissue alignment can be found in the 10X Genomics Spatial Outputs section.

    Tissue Opacity
    Visium
    VR

    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.

    Low tissue image opacity.
    High tissue image opacity.
    Explorative Comparison Features
    Feature Description Icon Image
    Side-by-Side comparison
    Visium Xenium STOmics Merfish Custom
    VR Demo

    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.

    Comparison of two gene expression patterns
    Visium Xenium STOmics Merfish Custom
    VR Demo

    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.

    Showing spatially variable genes (SVGs) results
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.

    Browse SVG results in the Visualiser
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Export Features
    Feature Description Icon Image
    Continue Session
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.

    Screenshot
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Export ROI selections
    Visium
    VR

    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.
    Import ROI selections
    Visium
    VR

    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
    Output plots
    Visium
    VR

    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

    Sample overview of the spatial plots.
    Switch VR and Desktop Application
    Feature Description Icon Image
    Switching between desktop and VR application
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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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    Additional Parameters to Explore
    Feature Description Icon Image
    Access spatial plots
    Visium
    VR

    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.
    Show all available plots:
    3D Object Alignment
    Feature Description Icon Image
    Change 3D Object Opacity
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Slider left, the 3D object becomes invisible
    Slider further to the right, the 3D object becomes non transparent.
    Move 3D object
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Rotate 3D Object
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Resize 3D Object
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Customisation
    Feature Description Icon Image
    Change the Symbol
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Symbol changed to Cubes.
    Change the Symbol Size
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Size
    Small symbols.
    Big symbols.
    Reset the view
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

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

    The slide is twisted.
    Clicking the view reset button adjusts the view back to the origin.
    Change Background Colour
    Visium Xenium STOmics Merfish Tomo-Seq Custom
    VR Demo

    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.
    Bright mode - background set to white.
    Address

    Murdoch Children's Research Institute, Parkville, Melbourne

    Email

    denis.bienroth@mcri.edu.au


    Website

    MCRI website

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