Overview of Summary plots generation using the Illustration Editor

Background 

The Illustration Editor is where figures (also known as Illustrations) are dynamically designed, created, modified, and exported in a Cytobank experiment. Illustrations can be used throughout your analysis process to visualize and ultimately communicate your findings.  

In the Cytobank platform, we have two types of plots available: Event-level plots and Summary plots (statistical plots). In this article, we cover the Summary plots that include Heatmap, Box plot, Violin plot, Bar chart, Line chart, and Summary dot. Heatmap is mentioned briefly here and in more detail in this article How to create and configure a Heatmap. To view the details of plot settings for Event-level plots, please check out Overview of figure generation using the Illustration Editor. 

The Illustration Editor works optimally with data after you have annotated files with Sample tags to designate experimental variables, such as individual, conditions, sample typeor any custom variable, termed a “dimension”. Sample tags become variables for configuring an Illustration. These variables can be toggled on or off and rearranged dynamically to build and modify a plot layout. This allows one to work above the level of FCS file names and instead use the scientific variables that were present in the experiment, to build a layout. The Summary plots will display those variables or dimensions in one unique box, violin, bar, etc. Therefore, allowing for a statistical comparison across those groups of samples. Thus, Sample tags acquire new importance when the goal of the Illustration is to demonstrate statistical significance of specific biomarkers.

Cytobank Summary plots with Statistical Inference Video

 

Outline

 

How to create Summary plots 

To create a new Summary plot, navigate to the Illustration Editor menu and select New illustration. On the new illustration click on the Plots menu and under the Plot type option, select the preferred Summary plot. 

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Summary plots available on the Plots menu of the Illustration Editor. 

 

General considerations when working with Summary plots 

The Summary plots, opposite to the Event-level plotsare designed to display a group of samples in one unique box, violin, bar, etc. Typically, the goal is to represent one category of an experimental variable per box, violin, bar, etc. Hence, annotating files with Sample tags to designate experimental variables and the different categories for each variable, will be essential to generate meaningful Summary plots. 

If you don’t want to display a group of samples together, just select FCS files as one of the Layout dimensions and exclude any Sample tags. 

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Summary plots displaying independent samples (top) or displaying samples grouped by conditions (bottom). 

 

When working with Box plots, Violin plots, Bar charts, Line charts or Summary dots, the typical Layout positions Rows and Columns will be replaced by X-axis groups and Subgroups. The Subgroups dimension indicates how the samples will be arranged in the box, bar, violin... while the X-axis groups dimension indicates how the Subgroups are gathered in the X-axis.  

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The subgroups conditions unstimulated and stimulated point to how the individual samples are assembled in the box plots. The X-axis groups channels will organize the samples so IFNg signal on CD+ cells is shown first for unstimulated and stimulated, and TNFa signal is displayed after. (orders of the variables within the Channels and Conditions are rearrangeable) 

 

You can render a maximum of 50 summary charts tables. If you configure the layout with more than 50 charts, an error message will appear saying “The maximum number of summary charts supported is 50. Please adjust your layout”. 

 

Summary plots 

The Summary plots in the Illustration Editor may show the same differences with different types of plot. If you want to follow along generating these examples, find the public experiment named “Illustration editor example” in the Experiment Manager on whichever Cytobank you are registered on and fully clone it. We will generate an illustration to visualize how stimulation of PBMCs is changing the expression of IFNg or TNFa in CD4+ T cells. 

Heatmap 

Please read the article on How to create and configure heatmaps to create meaningful heatmap illustrations. 

Box plots 

After creating a new illustration in the Illustration Editor, go to the Plots menu and select Box as the plot type. As X-axis groups select the Channels IFNg and TNFaMake sure that you use the Sample tags to group the samples by the Conditions, unstimulated or stimulatedunder the subgroups dimension. Finally, select the CD4+ population as the dimension for Table 1. The resulting Box plots show the median expression of IFNg and TNFa of CD4+ T cells between the stimulated and unstimulated groups for the 8 samples (4 per condition) analyzed on the experiment. 

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Box plots showing the effect of the stimulation of CD4+ T cells in the levels of IFNg and TNFa. The dots represent the independent samples (unstimulated in blue and stimulated in orange). 

 

When selecting Box as the Plot type the following settings will be available: 

  • Show dots: when toggled in dots representing the independent samples will appear.
    • Dot size: to make the dots bigger or smaller.
    • Show lines: lines connecting dots of paired samples will be displayed. Below the plot a sentence will appear with the information of how the samples were paired. For instance: Samples were paired up by referencing the following: Individuals.
    • Gray shading: will turn the box plots gray keeping the dots colored.
  • Jitter: controls the alignment of the dots. 0 will display all the dos in vertical while 1 will display them with maximum separation.
  • Y axis scales: controls the minimum and maximum values of the Y axis. It can be set as Local (per table), Global or Custom (user will input the values).
  • Widthsets the width of the boxes. There are three options to set up the width: Fixed, auto-expand or Custom.
  • Statisticthe parameter used to display on the Y axis. Please check the article on Statistics and fold change for a complete description of the options available. 

Violin plots 

To switch to a violin plot just change the Plot type on the Plots menu to Violin. 

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Violin plots showing the increase of IFNg and TNFs after stimulation of CD4+ T cells. The dots represent the independent samples (unstimulated in blue and stimulated in orange). 

 

When selecting Violin as the Plot type the following settings will be available: 

  • Show dots: when toggled in dots representing the independent samples will appear.
    • Dot size: to make the dots bigger or smaller.
    • Show lines: lines connecting dots of paired samples will be displayed. Below the plot a sentence will appear with the information of how the samples were paired. For instance: Samples were paired up by referencing the following: Individuals.
    • Gray shading: will turn the violin plots gray keeping the dots colored.
  • Jitter: controls the alignment of the dots. 0 will display all the dos in vertical while 1 will display them with maximum separation.
  • Show boxes
  • Y axis scales: controls the minimum and maximum values of the Y axis. It can be set as Local (per table), Global or Custom (user will input the values).
  • Width: sets the width of the boxes. There are three options to set up the width: Fixed, auto-expand or Custom.
  • Statistic: the parameter used to display on the Y axis. Please check the article on Statistics and fold change for a complete description of the options available.

Bar charts 

To switch to a bar chart, change the Plot type on the Plots menu to Bar. 

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Bar charts showing the effect of the stimulation (orange) of CD4+ T cells in the levels of IFNg and TNFa respect to the unstimulated control (blue). 

 

When selecting Bar as the Plot type the following settings will be available: 

  • Stacked bar: toggle on this option to display stacked bars.
    • Stacked method: absolute or relative. 
  • Cut off: select a value to display a cut off dotted line. 
  • Y axis scales: controls the minimum and maximum values of the Y axis. It can be set as Local (per table), Global or Custom (user will input the values). 
  • Width: sets the width of the boxes. There are three options to set up the width: Fixed, auto-expand or Custom.
  • Error bars: Select between not showing error bars (none) or show them as the standard deviation, standard error or 95thconfidence interval.
  • Summary method: the bar can represent the mean or the median of the statistic parameter among the samples.
  • Statistic: the parameter used to display on the Y axis. Please check the article on Statistics and fold change for a complete description of the options available.

Line chart and stain index 

Switch to line chart selecting the line plot type in the Plots menu. To obtain the Illustration on Fig 7 go to the Layout menu and switch positions between X-axis group and Subgroups. 

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Line chart showing the Median of Median expression of IFNg (orange) and TNFa (green) for CD4+ T cells unstimulated or stimulated (left panel; error bars indicate the standard deviation between individuals). The Layout configuration is shown on the right.

 

When selecting Line as the Plot type the following settings will be available: 

  • Stain index: To learn how to create stain index please read the article on How to create stain index charts for antibody titration. 
  • Show dots: when toggled in dots representing the summary method will appear. 
    • Dot size: to make the dots bigger or smaller. 
  • Y axis scales: controls the minimum and maximum values of the Y axis. It can be set as Local (per table), Global or Custom (user will input the values). 
  • Width: sets the width of the chart. There are three options to set up the width: Fixed, auto-expand or Custom. 
  • Summary method: the line may represent the mean or the median of the statistic parameter among the samples. 
  • Statistic: the parameter used to display on the Y axis. Please check the article on Statistics and fold change for a complete description of the options available. 

Summary dots 

Switch to summary dot selecting the Summary dots plot type in the Plots menu. 

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Summary dots showing the effect of the stimulation (green) of CD4+ T cells in the levels of IFNg and TNFa respect to the unstimulated control (orange). 

 

When selecting Summary dots as the Plot type the following settings will be available: 

  • Dot size: to make the dots bigger or smaller. 
  • Swarm 
  • Jitter: controls the alignment of the dots. 0 will display all the dos in vertical while 1 will display them with maximum separation. 
  • Mean/median line: shows a line for the mean or the median. 
  • Y axis scales: controls the minimum and maximum values of the Y axis. It can be set as Local (per table), Global or Custom (user will input the values). 
  • Width: sets the width of the chart. There are three options to set up the width: Fixed, auto-expand or Custom. 
  • Summary method: the line may represent the mean or the median of the statistic parameter among the samples. 
  • Statistic: the parameter used to display on the Y axis. Please check the article on Statistics and fold change for a complete description of the options available. 

 

Statistical inference on Summary plots 

To run a Statistical inference analysis on Summary plots, go to the Plots menu and select the Significant test that better suits your data set. Please check out the Overview of statistical inference method for a detailed description of the Significant tests available on the Cytobank platform. 

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Run a Significance test on the Summary plots by selecting the test on the Plots menu. 

 

How to export Summary plots 

When you finish creating the Summary plot illustration you can save the illustration in your Cytobank experiment and export a copy as PDF, PNG, or SVG. You can also separately export the table of statistics and the Statistical inference table below the figure.   

 

 



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