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LEGENDplex Analysis in R

Owned by Suat Dervish
Last updated: Dec 20, 2019
1 min read

Background

In mid 2019 Westmead Cytometry started providing LEGENDplex testing as a service to researchers. 

The protocol used to assay samples can be found here.

This page describes parameters to set to run the script used to generate the spreadsheet containing calculated values as well as QC/standard data PDFs.

The citation for the script is below. Note the version we use, and that is downloadable below has been adapted to work into our workflow/detectors by Brian Gloss and Suat Dervish.

"Stervbo U, Westhoff TH, Babel N. beadplexr: reproducible and automated analysis of multiplex bead assays. PeerJ. 2018 Nov 16;6:e5794. doi: 10.7717/peerj.5794. PMID: 30479885; PMCID: PMC6241392."

Analysis

  1. Create a folder to place generated data into. The script has been written to wrangle names generated when samples have been acquired in a 96 well plate (however this can be modified if needed). 
  2. Place the script and the panel in the directory (legendplex_analysisi_V5.R & human_th_cytokine_13plex.yml).
  3. Update the script with the number of samples (i.e. 2 samples in duplicate results in 4 sample .fcs files and 16 standard .fcs files, in this case samples = 2).
  4. You may need to change the cutoffs for data entry (i.e. lower and upper bounds on FSC, SSC & the R660 detector). 
    Parts in bold.
    ssam<-lapply(fnam, function(x) read_fcs(x,.filter=list("FSC-A"=c(5e4,2.5e5),"SSC-A"=c(5e4,Inf),"R660_20C-A"= c(6,Inf)), .bead_channels = c("R660_20C-A", "B585_42D-A"),.fsc_ssc = c("FSC-A", "SSC-A"),))
    names(ssam)<-gsub("Specimen_|.fcs","",fnam)
  5. In R set the current working directory.
  6. Run the script. 
  7. There should be 3 pdfs generated along with a .xlsx file where all the data is outputted. 

Example data

16 controls, 2 samples & script

DOWNLOAD HERE