t-SNE is a visualization method commonly used analyze single-cell RNA-Seq data. Each cell is shown as a point on the plot and each cell is positioned so that it is close to cells with similar overall gene expression. When working with multiple samples, a t-SNE plot can be drawn for each sample or all samples can be combined into a single plot. Viewing samples individually is the default in Partek Flow because sample to sample variation and outlier samples can obscure cell type differences if all samples are plotted together. However, as you will see in this tutorial, in some data sets, cell type differences can be visualized even when samples are combined.
Using the t-SNE plot, cells can be classified based on clustering results or differences in gene and pathway expression.
Multiple single-sample t-SNE plots
By default, each sample in a multi-sample data set is plotted on its own t-SNE.
- Click the Filtered counts node
- Select t-SNE from the Exploratory analysis section of the task menu (Figure 1)
- Click Finish from the t-SNE dialog to run t-SNE with the default settings
A t-SNE task node will be generated (Figure 2).
Once the t-SNE task has completed, we can view the t-SNE plot.
- Click the t-SNE node
- Click Task report from the task menu or double click the t-SNE node
The t-SNE plot will open to the first sample in the data set, MGH36 (Figure 3). Please note that the appearance of the t-SNE plot will differ each time it is drawn so your t-SNE plots will look different than those shown in this tutorial; however, the cell-to-cell relationships indicated will be the same.
The t-SNE plot is in 3D by default. You can rotate the 3D plot by lef-clicking and dragging your mouse. You can zoom in and out using your mouse wheel. The 2D t-SNE is also calculated and you can switch between the 2D and 3D plots using the Plot style radio buttons.
Each sample has its own plot. We can switch between samples using the Back and Next buttons on the upper left.
- Select Next
The t-SNE plot has switched to show the next sample, MGH42 (Figure 4).
The goal of this analysis is to compare malignant cells from two different glioma subtypes, astrocytoma and oligodendroglioma. To do this, we need to identify which cells are the malignant cells we want to include and which cell are the normal cells we want to exclude.
The t-SNE plot in Partek Flow offers several options for identifying, selecting, and classifying cells. In this tutorial, we will use expression of known marker genes to identify normal cells.
To visualize expression of a marker gene, we can color cells on the t-SNE plot by their expression level.
- Open the Color by drop-down menu
- Select Gene expression from the drop-down menu (Figure 5)
The cells will turn black and a text box Gene ID will open below the drop-down box.
- Type BCAN in the Gene ID text box
- Select BCAN from the list of genes in the data set (Figure 6)
The cells will be colored from black to green based on their expression level of BCAN, with cells expressing higher levels more green (Figure 7). BCAN is highly expressed in glioma cells.
In Partek Flow, we can color cells more than one gene. We will now add a second glioma marker gene, GPM6A.
- Select the icon next to BCAN
- Type GPM6A in the new Gene ID box
- Select GPM6A from the list of genes in the data set
Cells expressing GPM6A are now colored red and cells expressing BCAN are colored green. Cells expressing both genes are colored yellow, while cells expressing neither are colored black (Figure 8).
Relative expression of the two genes for selected cells can be visualized on the legend.
- Activate the 3D lasso tool by selecting
- Draw a circle around the cluster of yellow cells (Figure 9)
Selected cells are shown in bold and unselected cells are dimmed.
The relative expression of the two genes for the selected cells will be shown on the legend as dots (Figure 10).
Numerical expression levels for each gene can be viewed for individual cells.
- Switch modes by selecting
- Select a cell by pointing and clicking
The expression level for that cell is displayed on the legend for each gene (Figure 11).
- Deselect the cell by clicking on any black space on the plot
Expression values can also be viewing by selecting Gene Expression from the Label by drop-down menu and mousing over a cell.
Now that cells are colored by expression of two glioma cell markers, we can classify any cell that expresses these genes as glioma cells. Because t-SNE groups cells that are similar across the high-dimensional gene expression data, we will consider cells that form a group with BCAN or GPM6A-expressing cells as same cell type, even if they do not express the marker gene.
- Click anywhere on the t-SNE plot without a cell to clear the selection
- Activate the 3D lasso tool by selecting
- Draw the lasso around the cluster of yellow cells and click the circle to close the lasso. You may need to switch to selection mode and rotate the 3D plot to select only cells from the yellow cluster
The number of selected cells is indicated in the Selection section of the menu.
- Select Classify selection (Figure 12)
- Name the classification Glioma
- Select Save (Figure 13)
Once cells have been classified, the classification is added to the Classifications section of the panel. The number of cells belonging to the classiciation is listed; in MGH42, there are 413 glioma cells (Figure 14).
- Select Next to move to the next sample, MGH45
- Rotate the 3D t-SNE plot to allow you to select only cells from the yellow cluster
- Activate the 3D lasso tool by selecting
- Draw the lasso around the cluster of black cells and click the circle to close the lasso (Figure 14).
- Select Classify selection
- Type Glioma or select Glioma from the prompt (Figure 15)
- Select Save
- Repeat these steps for each of the 6 remaining samples
Once all samples have been classified, it is useful to check the number of cells in each sample assigned to each classification.
- Select Summary (Figure 16)
The classifications summary lists every sample, the number of cells in the sample, the number of cells in each classification, and the percentage of cells in each sample that belong to each classification (Figure 17).
With the malignant cells in every sample classified, it is time to save the classifications.
- Click Apply classifications
- Select Apply when asked to confirm
The pipeline view will open and the Classify cells tasks will run, generating a Classified groups data node (Figure 18).
One multi-sample t-SNE plot
For some data sets, cell types can be distinguished when all samples can be visualized together on one t-SNE plot. We will use a t-SNE plot of all samples to classify microglia/macrophage and oligodendrocyte cell types.
- Select the Single cell data data node
- Select t-SNE from the Exploratory anlaysis section of the task menu
- Select Configure on the t-SNE dialog (Figure 21)
- Click the Split cells by sample option under Misc to uncheck it
- Click Apply (Figure 22)
- Click Finish to run the t-SNE task
The t-SNE task will be added as a green layer in the analysis tab (Figure 23).
Once the task has completed, we can view the plot.
- Select the green t-SNE plot task node
- Select Task Report from the task menu
In the multi-sample t-SNE plot, each cell is initially colored by its sample (Figure 24).
- Select 2D from the Plot style section
Viewing the 2D t-SNE plot, while most cells cluster by sample, there are a few clusters with cells from multiple samples (Figure 25).
Using the known maker genes, CD14 and MOBP, we can assess whether these multi-sample clusters belong to our known cell types.
- Select Gene expression from the Color by drop-down menu
- Type CD14 in the new Gene ID box
- Select CD14 from the list of genes in the data set
- Select the icon next to CD14
- Type MOBP in the new Gene ID box
- Select MOBP from the list of genes in the data set
After coloring by CD14, a microglia/macrophage marker, and MOBP, a oligodendrocyte maker, these two cell populations are clearly visible (Figure 26).
- Activate the 3D lasso tool by selecting
- Draw the lasso around the cluster of red cells and click the circle to close the lasso (Figure 27)
- Select Save classifications
These red cells are MOBP positive, indicating that they are the oligodendrocytes from every sample.
- Name the classification Oligodendrocytes
- Select Save
To clearly see the CD14 positive population, clear the current selection.
- Switch modes by selecting
- Deselect by clicking on any black space on the plot
Green CD14 positive cells are the microglia/macrophages from every sample.
- Activate the 3D lasso tool by selecting
- Draw the lasso around the cluster of green cells and click the circle to close the lasso (Figure 28)
- Select Save classifications
- Name the classification Microglia
- Select Save
- Switch modes by selecting
- Deselect by clicking on any black space on the plot
Finally, we will classify all unclassified cells on the plot as the malignant cells from every sample.
- Select Classifications from the Color by drop-down menu
Cells are now colored by classification, with Oligodendrocytes in blue, Microglia in red, and unclassified cells in grey.
- Activate the 3D lasso tool by selecting
- Draw the lasso around the grey cells and click the circle to close the lasso (Figure 29)
- Select Save classifications
- Name the classification Malignant
- Select Save
With every cell from every sample classified, we can proceed to save classifications.
- Select Save classifications
- Select Save when asked to confirm
The pipeline view will open and the Classify cells tasks will run, generating a new green-layer Classified groups data node (Figure 30).
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