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R script for summarising paramater / channel intensity from flow or mass cytometry data

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CytoBars

This script will generate one (or more) bar charts of the intensity of each parameter / channel from a flow or mass cytometry dataset (FCS file).

e.g.

Requirements:

  • R
  • An interface such as RStudio
  • some flow or mass FCS data!

In addition, you will need to install the relevant packages (FlowCore, ggplot2, svdialogs, tidyverse) using the install.packages("packagename") command, which is not included in the script.

UPDATE - Added installing (if required) and loading libraries to top of script.

This script will:

  1. Read in a specified FCS file opened with a dialogue window.
  2. Determine an approximate "cutoff" value for mass cytometry data - this is very roughly the point at which we consider values to be potentially lost in the noise of irrlevant markers - e.g. common contaminants such as I 127 / Pb 208. This is considered to be around 20k DC (dual counts) in solution mode (e.g. 1 second acquisition).
  3. Plot a single graph of all markers.
  4. If there are markers below the cutoff, it will show a second. If there are markers above the cutoff, it will plot a third.
  5. Colour-code the bars according to the CV of the intensity. e.g. if the intensity is highly varied (spread around from low to high), the bars will show as purple / red.
  6. Place a frequency above the bar. e.g. if all events include that parameter / channel, this will be 100%.
  7. Title the chart with the filename, the cutoff value used and the number of markers.

How I learned to do this:

I'm totally new to programming in R and very new to flow / mass cytometry.

The following links were incredibly useful for getting the code up and running:

  • A very simple script to import some FCS data and plot it
  • An incredibly complex CyTOF workflow with some snippets I could understand and implement
  • A lot of this!

Feedback / suggestions appreciated.

See also

https://github.com/JimboMahoney/CytobankGraphs

https://github.com/JimboMahoney/CyTOF-PlotViewer

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R script for summarising paramater / channel intensity from flow or mass cytometry data

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