After successfully setting up and running PeacoQC, you can download a zip folder with run info and plots. You can also inspect the resulting FCS files on the Cytobank platform. This article outlines the content of the output files from the PeacoQC process, how to inspect the resulting FCS files, and how to assess the anomaly and clean populations before performing downstream data analysis. Click the links below to jump to the relevant section:
- Analysis complete page, run info and plots
- Inspect the resulting FCS files in the Cytobank platform
Analysis complete page
Once the run finishes you can access the Analysis complete page to visualize the PeacoQC report and to download the run info and plots. Check this article to learn all the ways to navigate to the PeacoQC results page.
(Analysis complete page showing the PeacoQC report and the option to download run info and plots)
Run info and plots
The PeacoQC report contains information connected to the advanced settings selected and the results. This information will be plotted as a heatmap where each row corresponds to one FCS file and every column corresponds to an aspect of the settings and results. The legend below the heatmap will help you visualize the results. It shows the advanced settings selected during the setup (consecutive bins, IT limit and MAD).The following results will be included on the heatmap:
- Percentage of events removed from the margins
- Percentage of events removed from the full analysis (sum of IT analysis, MAD analysis and in consecutive cells)
- Percentage of events removed IT analysis
- Percentage of events removed MAD analysis
- Percentage of events removed in consecutive cells
- Presence of increasing or decreasing effects
The PeacoQC process will look for a monotonic increase or decrease in the signal of the markers selected for analysis and indicate if such an effect is present in a file. Events are not identified as anomaly based on those effects. Note that files with a low number of events are more prone to be flagged as having increasing or decreasing effects.
Click on Download run info and plots to download the experimentID-analysisID-PeacoQC run name-PeacoQC.zip folder:
(Run info and plots PeacoQC folder)
This folder contains the PeacoQC heatmap report (the same that appears on the Analysis complete page), a log with run information, a TSV report with data cleaning results and a plots folder. Inside the plots folder there is one PNG per FCS file included on the PeacoQC run named as the FCS file. These PNGs show the density peaks identified for the selected markers for all events ordered according to the acquisition time. Density peaks with good values are shown without a background color, removed density peak are colored in pink (identified in the IT), purple (MAD) or light pink (in consecutive bins).
(Example PeacoQC plot showing density peaks per selected marker for all events ordered on time colored according to their good -no color- or anomaly value. Removed density peaks are shown in purple -IT-, pink -MAD- or light pink -in consecutive bins-)
Inspect the resulting FCS files in the platform
The PeacoQC process will generate new FCS files, two new populations and two illustrations to help you inspect the results in the Cytobank experiment.
The new FCS files are named (original FCS file name)_QC.fcs. These files contain a new parameter named Anomaly and belong to a new panel. Go to the Assign panels option under the Sample tags menu to check the new panel and files. The original files belong now to the (Original pre-QC files) panel(s)and they are marked as not visible, while the QC after-PeacoQC files are put into the (Cleaned files) panel(s) and they are visible. Only visible files will be available for further analysis. Check this article to learn how to make panels visible or not.
(original pre-QC files panel is not visible and cleaned files are placed into a new visible panel automatically)
After running the PeacoQC process, the output FCS files present a new channel called Anomaly added to indicate whether an event is Clean (the value is zero on the Anomaly channel) or Anomaly (the value is one on the Anomaly channel). Cluster gates are drawn around the Clean events and the Anomaly events automatically, and the Populations are named as such.
(The two populations Anomaly and Clean visualized on the Gating editor)
You may use the Clean population as the starting point for further analysis using manual gates or other advanced analysis.
Two Quality Check illustrations are automatically generated after a PeacoQC run to help you compare the Clean and original populations directly. One is called PeacoQC Quality Check - Event count ratio heatmap and displays a heatmap with the Event counts as a ratio of the Clean to the Ungated populations. Each row represents an FCS_QC file. Learn more about working with heatmaps on the Cytobank platform in this article.
(PeacoQC Quality Check - Event count ratio heatmap illustration)
The second illustration is called PeacoQC Quality Check - Cleaned and Anomaly events comparison and shows density dot plots of the first channel on the Y axis and Time on the X axis, where rows are the FCS_QC files and the columns the Ungated, Anomaly and Clean populations. This illustration allows you to visually compare and inspect PeacoQC results before moving to the next step of your analysis. Learn how to work in the Illustration Editor in this article.
(PeacoQC Quality Check - Cleaned and Anomaly events comparison illustration)