Visualizer: Volcano Plot

Tutorial
Written By Melissa LaRocca, PhD

The Volcano plot in the Trovomics Visualizer produces a type of scatter plot that allows you to visually identify statistically significant and differentially expressed genes. The most differentially expressed genes will be on the outer right (upregulated) and left (downregulated) with the most significant genes towards the top of the plot.

Objective: Identify significantly, differentially, and highly expressed genes in your samples

Pre-requisites: To access the visualizer, you must have an analysis completed in one of your experiments

 

 

Customization Settings

All customization settings for each visualizer are accessed in the left-hand menu (Image 1).

  1. Global Controls

    • The Global Controls tab can be accessed by clicking the globe icon. Note: Any changes made here will apply to all graphs in the visualizer.

      • Help Icons: You can choose to toggle help icons on or off. Hovering over a help icon will reveal a tooltip containing more information about its respective parameter

      • Plot Font: You can select what font will be used in your plots.

  2. Explore the Data

    • The Explore the Data tab can be accessed by clicking the graph icon. These settings allow you to customize the data visualized in your graph. Changes made here are specific to the plot and will not affect other graphs in the visualizer

  3. Adjust the Look of the Plot

    • The Adjust the look of the plot tab can be accessed by clicking the brush icon. These settings allow you to customize the aesthetics of your graph. Changes made here are specific to the plot and will not affect other graphs in the visualizer

  4. All Downloads

    1. The All Downloads tab can be accessed by clicking the download icon. Here you can download plot-specific files, such as methods and data.

Image 1. Overview of the left-hand menu.

 

TIP: The tab you are currently in will be highlighted in our signature pink and purple gradient.

 

Explore the Data

 

Select a comparison to plot

Pick a comparison (e.g., female vs male) you want to visualize on the volcano plot by selecting a comparison in the dropdown menu under Select a Comparison (Image 2). The comparisons available for plotting were predetermined by your selections during analysis creation.

Image 2. A comparison must be selected before visualizing the volcano plot.

 

Plot elements

Title - A plot title is at the top of the graph. It will display the name of the comparison it is showing.

Plot - Once a comparison is selected, one volcano plot (scatter plot) will populate. If more than one comparison is selected, they will appear side-by-side.

Axes - The volcano plot shows the log2FoldChange on the X axis, plotted against the -log10 of the P value (pvalue) or adjusted P Value (padj) on the Y axis. This flips the Y axis and allows you to see a wide range of small P values, with smaller, more significant values at the top of the plot.

Legend - The legend on the right of the plot will show you how many differentially expressed genes are in each group. If more than one comparison is selected, the number of differentially expressed genes in each comparison will be separated by a comma in the order they were selected in Select a Comparison.

Data table - The data table below the plot includes a list of genes that are currently plotted.

Image 3. Overview of the main elements in a volcano plot in Trovomics.

 

Show significance by P value or adjusted P value and set fold change cutoff

The volcano plot can show P values or adjusted P values for significance of genes.

In the text box under Cutoff Value, you can set the P value cutoff/threshold to determine what may be considered significant (Image 4). For example, if the cutoff is set to “0.05”, then only genes that have a P value below “0.05” will be colored as differentially expressed. In other words, this cutoff highlights genes that may potentially be significantly up- or downregulated, or normal. The cutoff you enter will be represented on the plots as a black, horizontal dashed line on the Y axis (Image 4).  

Image 4. Significance is shown through P values as opposed to adjusted P values (the default) on the Y axis. The cutoff value is represented by the position of the black, horizontal dashed line on the Y axis.

 

View different gene groups

The color-coded gene groups visible on the plots can be customized. You can refer to the legend for what each color represents. Clicking a label on the legend will remove that group from the plot and the label will be grayed out (Image 5). Clicking on the label again will show the group again. Double-clicking a label will show only that group on the plot (Image 6). These functions allow you to either remove normally expressed or non-significant genes from the view or highlight one of the differentially expressed gene groups (upregulated or downregulated).

Image 5. All “Upregulated” genes were removed from the plot (dashed box), evidenced by the label being grayed out (arrow)

 

Image 6. Only “Downregulated” genes are shown on the plot (arrow), evidenced by all other groups being grayed out.

 

Identify gene, gene expression, fold change, and significance of a point

Hovering over a point in the volcano will allow you to see the following information about the gene the point represents:

  • Gene name (by gene symbol)

  • Selected comparison

  • Average gene count (Average Count)

  • Log2FoldChange

  • Fold change (FoldChange)

  • P value and source (pvalue or padj)

Image 8. The cursor is hovered over a point that represents COL20A1, and revealed a text box that contains information about the gene name, comparison, average count, Log2FoldChange, FoldChange, P value and source.

 

Adjust fold change cutoff

The fold change can be adjusted on the slider bar under FCcut Size (Image 9). The default value is “2”, and is reflected by two black, vertical dashed lines at -1 and +1 on the X axis on the volcano plot. Genes with a fold change beyond the cutoff will be colored as differentially expressed (e.g., greater than 1 for upregulated and more negative than -1 for downregulated for the default settings). Higher fold change values will move the dashed lines further out to the right and left on the volcano plot.

  • Log2FoldChange vs Fold Change explained: In a volcano plot, the X axis is “Log2FoldChange”. Gene expression is often described in “log2 space” because it makes it easier to see expression across a large magnitude of expression level changes and also allows us to see up- and downregulated genes symmetrically on the X axis (If gene A has twice the expression level of gene B, there is a 2-fold change when comparing gene A/gene B. On the other hand, gene B/gene A is equal to a fold change of 0.5! In log2 space, the Log2FoldChanges would be 1 and -1, respectively. However, it tends to be easier for us to understand “Fold Change” instead of “Log2FoldChange”. Therefore, we have our slider bar set for you to adjust “Fold Change”. For the volcano plot, we then convert your selected “Fold Change” (FC) to “Log2FoldChange” (LFC) using the formula:

    LFC = log2(FC)

    We can also write this equation as:

    FC = 2^(LFC)

    Therefore, a “Fold Change” of “2” (as seen on the slider bar as a default) converts to a Log2FoldChange of 1. Reasons for using this convention are sometimes more obvious when discussing greater changes. For example, a Log2FoldChange of 2 is actually equal to a Fold Change of 4 (2^2) while a Log2FoldChange of 3 is equal to a Fold Change of 8 (2^3). When discussing even higher Fold Changes, the differences in scale become more obvious. A Log2FoldChange of 5 is equal to a Fold Change of 32! (2^5). Due to the differences in scale, it is more practical to plot Log2FoldChange in volcano plots.

Image 9. The fold change was set to “4.3” (box) under FC Cut Size, which translates to a Log2FoldChange of 2.1. The FC Cut Size value is represented by position of the two black, vertical dashed lines on the volcano plots that are visible at -2.1 and +2.1 on the X axis (arrows).

 

Adjust Y axis

The maximum (max) value of the Y axis on the volcano plot can be adjusted by using the slider bar under Y max size. Genes that have a Y axis value beyond the max value will automatically be adjusted to sit at the max value and be designated by a triangle symbol on the plot. 

Image 10. The max value for the Y axis was set to “100” under Y Max Size (box), resulting in the “-log10(pvalue)” on the Y axis to go up to 100. All genes have a value below 100 and therefore no points were represented as triangles in this example.

 

Zoom in on points

To zoom in on specific points, select the magnifying glass in the top left corner above the plot, then click and drag to create a selection box around the desired area. Double click to zoom out.

Image 11. A section of the volcano plot was zoomed in on.

Select a subset of points

To select a group of points, click on the Square Select tool and click and drag to create a selection box around the desired points as you would for zooming in (Image 12). You can also select points using the Lasso Select Tool, to manually draw around the desired points (Image 12). Once selected, the data table will be updated to show only those points. Double click on the plot to deselect the points.

You can also select a single point by clicking on it and the data table will show only that point. For the data table to show all points again, you need to click on the select or lasso tool at the top.

Image 12. The Lasso Select Tool and the Square Select Tool are found at the top of left corner of the plot.

 

Save a subset of points as a new gene list

You can save a subset of points as a new gene list for subsequent subsetting (Filters) or visualization (PCA, GO, Heatmap, Comparison) in Trovomics. Once you have selected points on your plot, open the Data Table and check the genes you want to be saved (click the checkbox in the header row to select all). A button will appear above the Data Table. Clicking it will prompt you to enter a name for the new gene list before saving.

 

Reset Volcano settings

Clicking RESET ADJUSTMENTS TO DEFAULT will restore all settings back to their default.

Back to top

 

Adjust the Look of the Plot

 

Adjust size of points

The size of the points that represent genes can be adjusted using the slider bar under Mark size (Image 13). Sliding it to the left will reduce the point size and sliding it to the right will increase the point size.

Image 13. The point size was set to “8” on the slider bar under Mark size (box), which increased the size of all points on the plot.

 

Change color of points

The colors that designate upregulated, downregulated, not significant, and normal genes can be adjusted. Click the color boxes to pick a new color or enter a hex (Image 14).

Image 14. Colors for each group were selected using the associated color pickers (box).

 

Change plot title and font size

To change the plot title, enter a new title in the text box under Plot Title (Image 15). This will replace the default “Volcano Plot” title. To change the font size, enter the desired size under Title Font Size.

Image 15. The plot title and font size has changed to ‘Volcano Plot Tutorial’ and ‘17’, respectively.

 

Adjust height and width of plot

Click to expand Plot layout. Slider bars to adjust plot width and height can be used to change how wide and tall your volcano plot appears (Image 16). These settings do not affect the Y axis values.

Image 16. The plot is enlarged by increasing the width and height.

All Downloads

 

Download methods and data

Within the All Downloads tab (Image 17), Volcano methods can be downloaded as a .TXT file to your computer by clicking Volcano Methods (Image 18). This file will detail how the volcano plot was generated exactly as the data was visualized at the time of download. You can use this as a guide for writing your methods during publication.

You can also download the data visualized in your Volcano plot by clicking Download Volcano Data. It is the same data found in the data table below the plot.

Image 17. Volcano plot methods and data can be downloaded within All Downloads.

 

Image 18. Sample Volcano Methods .TXT file.

 

Download plot image

To download your plot as it is shown in the visualizer, click the download icon in the top right corner of the plot area (Image 19). You can choose from .JPEG, .PNG, or .SVG image file formats and designate the image size. Note: The download icon is visible regardless of what tab you are in.

Image 19. The arrow is pointing to the download icon. The plot is being downloaded as a JPEG image.

 

Ready to make your Volcano plot?

Melissa LaRocca, PhD
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