Ozette Resolve
Spectral Unmixing

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Multicolor File Unmixing

Once you have generated satisfactory  control unmixing, you may proceed to unmixing your multicolor files.

Setting Up Multicolor Unmixing Runs

  1. Create new run
    • Use the breadcrumb header to navigate back to your Panel Home Page.
    • Navigate to the “Resolve Runs” card
    • Click “Add Run”
    • Select the appropriate control set from the dropdown menu
      • Only control sets where the single stain samples have been unmixed will be available.
    • Provide unique run name
    • Click “Next”
  2. Upload experimental sample files
    • Drag-and-drop to upload raw multicolor files in the “Files To Unmix” card
    • Each file will appear as a line item under the upload box.
      • A spinning circle on the far right indicates an uploading file.
      • When a file has completed upload, there will be a black checkmark on the far right.
      • Each line item will remain red until metadata has been mapped.
  3. Optional: Upload sample-specific Source of Background files
    • Ozette Resolve executes event-level autofluorescence adjustment. In order to execute this, Resolve requires the user to specify an unstained file that should be used as an autofluorescent reference. You can use either a universal unstained file, or sample-matched unstained files (“sample-specific Source of Background” files).
    • If you choose to use a universal unstained file, you do not need to upload another unstained file here.
      • Ozette Resolve will match unstained files from the control set to your multicolor files based on material type.
      • Resolve matches multicolor and unstained files by material type in order to ensure the unstained file has the most accurate autofluorescence profile relative to the multicolor file.
    • For optimal results, you should upload sample-matched unstained files: for each multicolor file, you upload an unstained file generated on the same sample.
      • This ensures optimal autofluorescence matching.
  4. Map file metadata
    • After all files have completed uploading, you can either manually map metadata, or again use a .csv to map file metadata.
      • You can download a template .csv file from the upload page.
      • The .csv file you upload to the platform should have one line for each multicolor file to be unmixed and all unstained files to be used as a source of background.
      • The required information is:
        • File Name: Name of each multicolor or unstained file
        • File Type: “TARGET” for multicolor files or “BACKGROUND” for sample-specific unstained files
        • Material Type: material type used to generate each file. Must be one of the material types you specified during Resolve panel creation.
        • Source of background file name: Name of the unstained file to be used as the source of background estimation for the corresponding multicolor file. This can be an unstained file in the control set or a newly uploaded sample-specific-source-of-background file.
    • If the metadata mapping was successful, the line items will no longer be red.
  5. Optional: Gate sample-specific source of background files
    • If you uploaded sample-specific-source-of-background files, you will need to gate those files to remove unwanted material such as debris and doublets.
    • After uploading all FCS files and mapping metadata, a “Gate Source of Background” button will become available. Click this button to enter a gating experience similar to that used to gate the control set, except that you only need to perform cleanup gating.
      • It is suggested to at least gate out non-target cell lineages, debris, doublets.
  6. Click “Unmix” to unmix the multicolor samples.
    • This will take several minutes
  7. Optional: Archive Resolve Runs
    • Should you want to hide a multicolor run within a panel, you can archive completed Resolve Runs. To un-archive please contact support@ozette.com

Unmixed Multicolor Samples

Once unmixing of the multicolor samples is complete, the View Results page will populate with metadata, downloadables, and visuals that can be used to QC the results.

  1. Downloadables:
    • Cofactor file: This file contains estimated transformation parameters for each fluorochrome in the panel. For downstream analysis, we recommend that each fluor in output Resolve data be transformed by the hyperbolic arcsine function with the cofactor value listed in this file.
    • Bulk export: All unmixed control files
    • Individual files: Single control downloads
  2. Nx1 Plots:
    • Click on individual file names on the View Results page to view the Nx1 plots for that file.
      • These Nx1 plots show unmixing of the root population. These visuals can be used for basic QC review of the results, but do keep in mind that these plots include dead cells and debris that may be gated out in your final downstream analysis.
    • You can toggle between samples using the arrows at the top left of the page.
    • You can change the X axis using the “X” menu at the top left of the page.
    • You can change the axes settings between the range of the data and the range of the instrument using the “Display Range” menu.

If you identify QC concerns with the unmixed results, you may create a new multicolor run and use a different control set for the unmixing until you are satisfied with the results.