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Steps for SIV data processing

This is a description of the manual steps to create a report of the biologically relevant output of the SIV use case gating.

From FlowJo workspaces to a Excel document showing the % of cells showing cytokine induction over the timecourse of the study for each target population for each primate. This project took 5 hours for the Summer Intern's analysis when working with a system that had all the files and software required AND I had the interns export their tables of the stats required. Run5 had strange compensation issues and was not used in this round of gate-a-thoning.

For the ApScis Summer analysis, there are 29 workspaces.

Steps

  1. Open each workspace in FlowJo (due to file names being identical for runs 2-6, and workspaces not being in same folder as FCS files, need to check the loading of the files each time a workspace is opened to make sure the right fcs files are being loaded, otherwise strange results are shown, exported, and propagated. Best accomplished by shutting down FlowJo in between opening each workspace to force FlowJo to ask for FCS file locations)
  2. Open the Table Editor
  3. Select the "freq of parent" table
  4. Click the Batch Button (now in 7.5.5 or 7.6 you have to use the output menu and that will bring up the Table General Options Dialog)
  5. Select to export as .csv
  6. Select Group = Test Samples
  7. Select the destination folder
  8. Re-name the exported file to include the ApSci name and Run # for file organization.
  9. Click Okay
  10. Do steps 1-9 for all 29 workspaces... takes over an hour just to export all needed files. thought about setting up as a batch, but would have to do that for each workspace taking the same amount of time.
  11. Open each of the 30 exported tables in spreadsheet program (Excel prefered, but Open Office has been acceptable)
  12. Sort the order of the tables to be the same for each of the 30 tables.
  13. Subtract the % parent value of the unstimulated from the %freq of parent of the peptide stimulated samples (monkey by monkey AND timepoint by timepoint) Copy and paste once you have the tables sorted and the format of the subtraction done on the first table.
  14. Save each table as .xls so you don't have to do this step again. If using Open Office, this saving as .xls requires more steps in the save process.
  15. Transform the data for each expert to show the 6 populations for each monkey and each timepoint such as (3 tables for each expert):
    Monkey X CD4 IFN CD4 IL2 CD4 TNF CD8 IFN CD8 IL2 CD8 TNF
    Run 1            
    Run 2            
    Run 3            
    Run 4            
    Run 6            
  16. Transform the data by copy and pasting the values to create separate sheets organized by each target population for each monkey and then showing a table as such (total of 18 tables):
    Monkey X CD4 IFN CD4 IL2 CD4 TNF CD8 IFN CD8 IL2 CD8 TNF
    Run 1            
    Run 2            
    Run 3            
    Run 4            
    Run 6            

Graph the results. Each graph only shows one primate and one target population, but shows the timecourse for all gater's results. These graphs will show whether a primate responded to the treatment by demonstrating a change in the amount of cytokine response over the time-course and the graph will show the inter-gater variability.example graph