How to combine events from different files into one plot in the Illustration Editor(Virtual Concatenation)

Background

The Cytobank Illustration Editor allows you to concatenate the plots of multiple files.  The virtual concatenation feature enables you to aggregate plots from multiple FCS files into a single plot. The plots grid can be collapsed by row, column or all FCS files in the same layout dimension. Using virtual concatenation, you can merge files based on groups to compare plots between categories of files. The concatenated image will have information representative of all files selected for merging. However, concatenation does not work on populations and channels, or tables. The process of concatenation takes place virtually (no need to download the file), therefore, the actual files remain unaltered. Like that, you can study the overall picture as a combination of files, based on your desired groups or individually, while working on the same experiment. This feature works on dot plot, histogram and contour plots.

 

How to perform virtual concatenation in the Illustration Editor

The virtual concatenation process is accomplished inside the Illustration Editor, and it works for Dot, Histogram and Contour plot types. The virtual concatenation can be used on dimensionality reduction plots too. To perform virtual concatenation, navigate to the Illustration Editor either by creating a new Illustration or accessing an existing one.

Check this article to read more about how to use the Illustration Editor.

 

 

Virtual Concatenation using the Plots menu

The virtual concatenation feature can be accessed using the Plots menu on the top left corner of the screen. The virtual concatenation option can be identified as the third option from the top in the Plots menu. Since the concatenate step works on multiple files selected previously, set this option that shows All multi-file positions. Make sure the Overlaid option is switched off to ensure that the concatenation option is available.

 

Alternatively, you can select the Rows or Columns option from the Concatenate option. Switching to Rows or Columns will virtually concatenate all the files displayed on the rows or columns, respectively.

 

This will concatenate the events from all files into a single plot that represents the events from each of the files used. Please see the animation below that illustrates the common steps taken to concatenate the files for Plots. These steps can also be repeated for Histogram and Contour Plots

 

  GifForConcatenationDotPlot2.gif

(Animation representing the steps to concatenate the dot plots created from a population generated using Plots in the Cytobank Gating Editor. The process starts from accessing Histogram from the Plots menu in the Illustration Editor. Later merging can be achieved from the File section in the Layout menu. You can get the same results from the Plots or Layout menu.)

 

Please also see the animation below that represents the common steps taken to concatenate the files for Histogram

 

 

 

 GifForConcatenationHistogram2.gif

(Animation representing the steps to take to concatenate the Histogram created from a population generated using the Plots menu in the Cytobank Gating Editor. The process starts from accessing Histogram from the Plot option in the Illustration Editor. Later merging can be achieved from the File section in the Layout option.)

 

Please see the animation below that represents the common steps taken to concatenate the files for Contour Plot.

 

 GifForConcatenationContourPlot2.gif

(Animation representing the steps to take to concatenate the Contour created from a population generated using the Cytobank Gating Editor. The process starts from accessing Contour from the Plot option in the Illustration Editor. Later merging can be achieved from the File section in the Layout option.)

 

Virtual Concatenation using the Layout menu

The Layout menu in the Illustration Editor can also be utilized for creating concatenated plots. To use this tool on a Dot Plot, first create a new illustration in the Illustration Editor. Make sure the Overlaid option is switched off to ensure that the concatenation option is available.

 

 

 

Next, click on the Layout menu to populate the visualization options from the drop-down menu. 

 

In the drop-down menu you can select the file names you wish to concatenate selecting FCS files as the Rows or Columns dimension. 

 

 

 

Next select the Populations that you wish to highlight when you create a concatenated image.

 

In addition to this you can also select markers that you wish to visualize by adding the Channel under the Layout menu.

 

Finally click on the icon that shows vertical bi-arrow symbol. This will show the horizontal trident symbol for Virtual Concatenate. Click on this trident symbol to concatenate the files.

 

This will generate the concatenated image merging the information from each of the files.  

 

Please note, when selecting the concatenation option, it’s concatenated by Columns or Rows. For example, if you have the Condition, untreated and treated, in the Columns position, selecting concatenation by Columns will combine all files from untreated and treated into one plot. If you wish to combine all files from all untreated samples into one plot and all files from treated samples into another, then you should use the All multi-file positions under the Plots menu. Alternatively, hover over the plot and switch on the Concatenate option or access the Concatenate option under the Plot settings menu as shown in the picture below.

 

Tables can’t be concatenated. You will need to switch the dimensions to either Rows or Columns if you wish to concatenate by certain dimensions. Also Channels and Populations dimensions can’t be concatenated.

Please see the animation below that represents the common steps taken to concatenate the files for Dot Plot.

 

 ConcatenateFromPlots.gif

(Animation representing the steps to create a concatenated dot plot from manually gated populations. The process starts from accessing Dot from the Plots menu in the Illustration Editor. Merging can be achieved from the FCS Files dimension in the Layout menu.)

 

Concatenating the files in Dimensionality Reduction Experiments

You may want to use virtual concatenation to combine FCS files into the same dimensionality reduction plot. Please check these articles to learn more about analysis and interpretation of DR results and how to virtually concatenate DR plots.

 

Note

In the Cytobank platform, a maximum number of 25,000,000 total events per plot can be concatenated; if you exceed this number, the following warning message will display.

 Error_virtual_concat.png

You can either adjust the layout setting to reduce the number of events per plot, or turn off concatenation.

 

 

*For Research Use Only. Not for use in diagnostic procedures.

 

 



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