10x Genomics
Visium Spatial Gene Expression
Space Ranger6.0, printed on 11/22/2024
Navigating the Loupe Browser Interface
To learn how to navigate the Loupe Browser interface, a pre-loaded mouse brain dataset is included and described to demonstrate the interactive functionality.
NOTE: Spatial Enrichment is new with Loupe Browser 5.1! Click here for a complete list of features new to Loupe Browser 5.1.
Components of the Interface
The following image provides an overview of the key components of the Loupe Browser interface. Each of these components are described in more detail below.
Mouse Selector
The Mouse Selector has three options from left to right:
| Tool | Description |
|---|---|
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Pan - Move the image up and down, or left and right. |
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Drag Selection - Select and label an image area within a region of your choosing. Hovering over the button will allow you to choose either a freehand or rectangular selection. |
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Draw Selection - Select and label individual spots using a brush tool. |
Toolbox
The Toolbox is on the left side of the window. Move your mouse over the toolbox icons to see an explanation of what each tool does. The tools in the toolbox perform the functions listed in the table below.
| Tool | Description |
|---|---|
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Zoom In and Zoom Out - Click on + to Zoom In, click on - to Zoom Out, or use the slide bar to zoom in and out. In Spatial mode, the current magnification level is shown. |
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Autoscale Autoscale to fit the screen after zooming in or out. |
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Save/Save As Save changes to a .cloupe file. |
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Open File Open a new .cloupe file. |
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Export Plot Click to export a spatial plot as .PNG, or a scatter plot as an SVG or PNG. |
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Marker Settings Marker Settings can be used with t-SNE, UMAP and Feature Plot views to scale marker size. Uncheck the Auto-scale box to choose a percentage. |
To return to the home screen and the **Recent Files** list, click the 10x Genomics button in the top left corner.
Data Panel Selector
The Data Panel Selector tools are on the bottom left of the window. They are used to change the information located in the Data Panel at the bottom of the window. Move your mouse over the icons to see an explanation of what each tool does. The tools perform the functions listed in the table below.
| Tool | Description |
|---|---|
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Heat Map |
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Feature Table |
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Spatial Enrichment |
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Violin Plots |
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Hide Bottom Panel |
To expand the viewable area of the data panel, use your cursor to drag the top border of the panel up. Drag it down to reduce the size.
View Selector and View-Dependent Controls
The View Selector controls what is displayed in the View Panel. There are three different types of views that can be selected:
- t-SNE/UMAP View
- Spatial View
- Feature Plot View
Multiple concurrent views of the same data can be viewed via linked windows on the desktop. For example, a Spatial projection can also be opened alongside a t-SNE, UMAP, or Feature Plot view. Changes made in any of the linked windows are seen in all open linked windows.
Linked windows can be opened through the View Selector. Icons shown in the following table explain how to use linked windows.
| Icon | Description |
|---|---|
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Click on the icon to see a list of linked windows for the current projection. |
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This icon is seen in the View Selector to indicate what projections can be opened from the current view. It is also seen in new projection windows. |
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Toggle Mini-Mode - Linked windows open in a small size. Use this icon to open the window to full size or toggle back to the smaller size. |
A few navigational elements are now view-dependent. The following sections underline the differences between views.
Spatial View
When a Spatial Gene Expression .cloupe file is opened, the initial workspace is centered around the Spatial View, which shows the tissue image overlayed by the tissue covered spots on the slide. The spots have already been aligned to the correct positions in the image. Each spot is color-coded based on the active legend in the Mode Panel.
In the Spatial view, drag the mouse over the image to reposition it, and use the mouse wheel or track pad to smoothly zoom in and out. The cluster label associated with a spot is shown when you hover your mouse over that spot in the image.
Spatial Image Settings
Spatial Image Settings, located in the top left corner, provides a number of options to view the image and gene expression data including:
- The Fiducial Display, Scale Display, and the Tissue Display can be toggled on or off with the buttons.
- The Brightness, Contrast, Saturation, and Opacity of the image colors can be increased or decreased.
- The image can be rotated by sliding the button to a specific degree, by using the up and down arrows in the rotation field, or by entering a number in the rotation field.
- The image can also be flipped horizontally by toggling the button on.
There are more controls for multi-channel fluorescent images:
- Channels can be shown or hidden by selecting the checkbox next to an image.
- Channel settings can be modified by pressing the name of a channel.
- The color, brightness, opacity, and saturation of each channel can be modified through channel settings.
- Channels may also be renamed by clicking on the pencil icon in the channel settings menu.
UMAP and t-SNE Views
Space Ranger and Cell Ranger pipelines calculate t-SNE and UMAP projections over expression data. Loupe Browser shows these scatter plots. When a UMAP, t-SNE or custom coordinate projection is shown, a Split View selector is visible in the upper-left hand corner of the interface. Like the Spatial View, each spot is color-coded based on the active legend in the Mode Panel.
In t-SNE and UMAP views, dragging the mouse over the display area repositions the projection, and the mouse wheel or track pad can be used to smoothly zoom in and out. The cluster label associated with a spot is shown when you hover your mouse over that spot in the image.
Feature Plot View
The Feature Plot view allows you to visualize the expression levels of one or two genes for each spot. This view makes it easy to threshold sets of spots based on the level of expression of one or two genes. Features, in this case genes, can be entered in the text box at the top of the Y axis or on the right side of the X axis. These selectors also contain a control to switch the scale of the axis between linear and log scale.
Import Projections
There are many scripts and third-party tools that can be used to generate different projections of the data including but not limited to the following:
- Alternate clustering and projection algorithms, for example, trajectory analysis.
- Data filtered for unwanted artifacts, for example, dead or dying cells.
If you generate alternate projection coordinates from customer analysis, you can input that projection into Loupe Browser. To do this, click on the three vertical dots in the View Selector to import a csv file to use a custom projection for a dataset.
The csv format for importing projections is as follows:
- A header is required.
- The first column is barcodes that are at least a subset of the barcodes in the .cloupe file.
- The second column is numbers that represent the X coordinates.
- The third column is numbers that represent the Y coordinates.
To import new projections, click on Import Projection, select the csv file, and click on Import Projection. Once a custom projection is uploaded, click on Import Projection again to edit the name of the projection, or to delete it.
Mode Selector and Mode Panel
Use the mode selector in the top right corner of the workspace to switch between Loupe Browser's different modes. Switching between modes applies mode-specific coloring to the View Panel, and changes mode-specific functionality to the Mode Panel. There are four modes in Loupe Browser:
- Categories
- Gene/Feature Expression
- Filters
- V(D)J Clonotypes
Controlling the Categories Mode
When you are in Categories Mode, you can control the content in the Mode Panel by clicking on the three vertical dots next to the drop-down menu.
New Category
Use this option to create a custom category and associated custom clusters.
Edit Category Name
This option is only applicable to custom categories.
Delete Category
This option is only applicable to custom categories.
Import Categories
Use this option to upload a csv file that contains customer cluster definitions on a per-barcode basis. The format of the csv file is as follows:
- The first row must be a header, such as Barcode, Cell Type, etc.
- The first column must be barcode and the column’s contents must match at least a subset of the barcodes in the .cloupe file.
- After the barcode column there may be an arbitrary number of columns corresponding to different types of categories.
- Each additional column must have a unique category name and the column’s contents are cluster labels.
Export [Name of Category]
For this option, the [Name of Category] reflects the name of the current Category that you are viewing. This option exports a complete list of barcodes for the current Category and the associated cluster name that is associated with that Category. For example, if you are currently viewing the Graph-Based category, then this option in the drop-down menu is Export Graph-based. When you select this option, you can download a csv file that contains a list of all barcodes in the first column and their associated graph-based cluster label in the second column. See the example below.
Show All Clusters
This option automatically selects all clusters in the Mode Panel.
Hide All Clusters
This option automatically deselects all clusters in the Mode Panel.
Significant Feature Comparison
The Significant Feature Comparison functionality determines what features in the dataset distinguish one or more clusters, and allows differential expression analysis between clusters. There are two types of controls for this functionality. Select options for these two controls using the drop-down menus in the bottom right-hand corner. Once options are selected, click on the calculator icon to execute the comparative analysis.
Significant Feature Comparison Method
There are two different methods available for conducting a Significant Feature Comparison analysis:
- Globally Distinguishing: For each selected cluster displayed in the Mode Panel, this option determines the features that distinguish each selected cluster from every other cell in the dataset.
- Locally Distinguishing: For each selected cluster displayed in the Mode Panel, this option determines the features that distinguish each cluster from the other selected clusters in that category.
Significant Feature Comparison Method
Use this drop-down menu to select which type of feature you want to use in the comparison. For Spatial Gene Expression dataset, the only option here is Gene. However, for other products, there may be more than one option.
Gene/Feature Expression Mode
The Gene/Feature Expression mode provides a graphical representation of gene or feature expression across a dataset. One or more features can be viewed at a time. You can search for a feature of interest, or upload and save lists of features.
Active Feature List
The Active Feature List contains a list of the features that you have either searched for, or uploaded. The expression patterns of those features are displayed in the View Panel. If more than one feature is in the Active Feature List, then the expression pattern corresponds to a combination of all the features. If only one feature is present or selected in the Active Feature List, then the expression pattern corresponds to that feature.
At the bottom of the Active Feature List there are a number of options that control how the data is visualized in the View Panel. The Scale & Attribute parameters control how the expression patterns are rendered in the View Panel. The top left menu sets which scale value to display. The top right menu sets how to combine values when there are multiple features in the Active Feature List.
The Select by Count controls how to filter the expression values displayed.
The color palette in the bottom right controls the color scale and range of values. You can also choose to manually set the min and max of the color scale by unchecking the Auto-scale checkbox, typing in a value, and clicking the Update Min/Max button. When setting manual min and max values, barcodes with values outside the range, less than the minimum or greater than the maximum, are colored gray. This is particularly useful if there is a lot of noise or ambient expression of a gene. Increasing the minimum value of the scale filters that noise. It is also useful to configure the scale to optimally highlight the expression of genes of interest.
Controlling the Active Feature List
In Gene/Feature Expression Mode, you can control the content in the Mode Panel under the Active Feature List by clicking on the three vertical dots next to the drop-down menu. This gives a number of options, as described below.
New List
Use this option to create a custom list of features to visualize.
Edit Name
Edit the name of a list.
Delete List
Delete a list.
Import Lists
Use this option to upload a csv file that contains a custom set of markers that results in one or more new lists. The format of the csv file is as follows:
The first row must be a header such as List Name, Gene Name, etc.
The first row can contain the name of one or more Feature Lists that will be created.
The second column is a list of the feature names, such as gene names associated with each list.
You can optionally include the Ensembl IDs in a third column.
The feature names and Ensembl IDs, if applicable, must match those that were in the reference file that was passed into Space Ranger.
Export Lists
If you have manually added features to a list, use this option to export the list of features as a csv file. This is useful if you want to reuse a list for other samples visualized in Loupe.
Filters Mode
In Filter mode, you can compose complex boolean filters to find barcodes which fulfill your criteria. You can create rules based on feature counts or cluster membership and combine these rules using boolean operators. You can then save and load filters and use them across multiple datasets. For more details on using the Filter Mode, see the following tutorial built around Single Cell Gene Expression data.
Data Selector and Data Panel
The data panel on the bottom of the workspace displays information about the features driving differences between clusters. By default, it enumerates the genes that drive differences between the current precomputed clustering selected in the Categories sidebar. It also displays the results of a Significant Features analysis. The mini-toolbar to the left of the data panel switches between a table and a hierarchical heat map of differentiating features. Clicking on a feature name in the table view allows you to add that feature to a list for future reference, or to show counts of that feature across the dataset in the active projection.