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Software that we may utilise in different workflows (or access ARGUS for a desktop pre-installed with software)

1. Download and install R (latest stable version, or a specific version if needed) from here - https://www.r-project.org/
2. Download and install RStudio Desktop from here - https://www.rstudio.com/products/rstudio/#Desktop
3. Download and install Rtools (for package building from GitHub repositories) - https://cran.r-project.org/bin/windows/Rtools/
4. Download and install FlowJo from here (and organise a licence if you haven't already got one)
5. Download and install SeqGeq from here (and organise a licence if you haven't already got one)
6. Download and install Matlab

Excellent links for information that was used for R/Rstudio/flowCore

1. Basic intro to R - https://cran.r-project.org/doc/contrib/Torfs+Brauer-Short-R-Intro.pdf
2. Introduction to flowCore - https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-10-106

Data pre-processing in FlowJo using R plugins

FlowJo check sample quality

In FlowJo, after importing your cytometry data, click the 'Check sample quality' button in the tools workspace. This will flag samples that should be checked. FlowJo plots the median values for each parameter over time and flags any that are outside of 2 standard deviations. Green is good. Any thing else should be reviewed. 

Active sample quality check - FlowAI in FlowJo

FlowJo also provides access to 2 active cytometry data quality plugins. FlowClean & FlowAI

To enable FlowAI

• Have R installed (version >3.4)
• Install BioConductor & flowCore
  • source("https://bioconductor.org/biocLite.R")
  • biocLite("flowCore")
• Install PNG package
  • install.packages("png")
• Install flowAI
  • biocLite("flowAI")
• Install stringi (I required this package additionally)
  • biocLite("stringi")
• Have FlowJo installed with the correct R path in the preferences
  
• If not already present, download and move FlowAI.jar file into the FlowJo plugins folder (available from the FlowJo exchange)
• Restart FlowJo
• Select sample and navigate to Plugins, FlowAI. Select parameters and compute. Example output is shown below including layout depiction.
  
  

Active sample quality check - FlowClean

To enable FlowClean

• Have R installed (version >3.4)
• Install BioConductor
  • source("https://bioconductor.org/biocLite.R")
  • biocLite("flowCore")
  • biocLite("flowClean")
• Have FlowJo installed with the correct R path in the preferences
  
• If not already present, download and move FlowClean.jar file into the FlowJo plugins folder (available from the FlowJo exchange)
• Restart FlowJo
• Select sample and navigate to Plugins, FlowClean. Select parameters and compute. Example output is shown below.
  

Reading FCS files in RStudio

1. Open RStudio (ensure you run as administrator, otherwise yo umay run into permission errors).

2. Install Bioconductor and additional packages that are used for flow cytometry data analysis

   ## try http:// if https:// URLs are not supported
   source("https://bioconductor.org/biocLite.R")
   biocLite()
   biocLite("flowCore",dependencies=TRUE)
   biocLite("flowViz")
   biocLite("flowUtils")
   biocLite("geneplotter")
   install.packages("stringi")

3. Determine your working directory by entering 

   getwd()
   
   

4. Set your working directory by entering - for example

   setwd('C:/Users/utopi/Desktop/testdata')

5. Set the FCS folder as a variable

   ##file.name <- system.file("extdata","0877408774.B08", package="flowCore")
   read.FCS('A1.fcs')

6. Still in progress...

Example:

source("https://bioconductor.org/biocLite.R")

biocLite()

biocLite("flowCore",dependencies=TRUE)

install.packages("devtools").