Background

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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. 

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