4  Module list

List and explanation of the different Shiny actions available in the app, along with their functions and usage.

Table 4.1:

Current available actions in app, see file type

icon action description prerequite_type output_type

correct batch effects 2: fb 2: fb

extract features from FCS concat with clustering 3: sce 5: fe

import OMIQ FE 0: none 5: fe

import FCS concat 0: none 5: fe

control quality of features 5: fe 6: html

analyse abundancy of clusters - univar 5: fe 6: html

analyse intensity of clusters - univar 5: fe 6: html

import .rds object 0: none 5: fe

export to phantasus 5: fe 4: files

4.1 Batch Correction

mod_correct_batch

tbd

4.2 Extract Clustering Features

mod_extract_sce

Functionality Overview: This panel allows users to extract features from a specified cell set. Users can define State Markers and choose their preferred clustering method.

Instructions:

  1. Specify State Markers:
    • Use the provided input field to specify State Markers.
  2. Select Clustering Method:
    • Choose a clustering method from the dropdown menu. This selection determines how the cells will be grouped.
  3. Run Feature Extraction:
    • Once you have specified the State Markers and selected a clustering method, click on the "Extract Features" button to initiate the process.

Additional Information:

  • Accurate specification of State Markers and the appropriate choice of clustering method are essential for effective feature extraction.

4.3 Import Features

mod_import_feature

Functionality Overview: The "Import from Feature" panel allows users to import various data tables such as abundance, MFI (Mean Fluorescence Intensity), and metadata tables. Users also have the option to load a marker table. All these tables should be linked by a common ID.

Instructions:

  1. Browse and Select Files:
    • Click on the ‘Browse’ button to select the data files you wish to import.
    • You can import files for abundance data, MFI data, and metadata.
    • Optionally, you can also load a marker table. (not available yet)
  2. Ensure Common ID Column:
    • Make sure that each table has a common ID column. This ID is essential for creating the bound between the different tables.
  3. Upload Files:
    • After selecting the files, complete widgets.

Additional Information:

  • This import functionality is designed especially for OMIQ exported features.

4.4 Import Concatenated FCS

mod_import_concat

Functionality Overview: This panel allows users to import a concatenated Flow Cytometry Standard (FCS) file representing a complete experiment. Each FCS file within the concatenated file should be annotated and assigned to specific biological samples, studied conditions, etc. Additionally, users must provide a metadata file (in .xlsx or .csv format) containing an FCS ID to identify the origin of cells.

Instructions:

  1. Uploading Files:
    • If using this application on a server, first upload your files.
    • Click on the ‘Browse’ button (max file size: 500MB) to select and upload your concatenated FCS file and metadata file.
  2. Selecting FCS Directory:
    • After uploading, select the ‘uploaded_fcs’ directory containing the fcs file by clicking on ‘Select your FCS directory’.
  3. Metadata File Upload:
    • Upload the metadata file that includes the FCS IDs. This file should be in either .xlsx or .csv format.
    • Ensure that the metadata file correctly maps each FCS ID to its respective sample and condition.

File Requirements:

  • Concatenated FCS file must include properly annotated data with clustering and origine fcs id of each cells.
  • Metadata file in .xlsx or .csv format, containing FCS IDs for cell origin identification.

Marker Selection:

  • Once the concatenated FCS is loaded, manually select the marker of interest by clicking in the table.

4.5 Quality Control

mod_qc

Functionality Overview: The Quality Control Module provides an interactive interface for users to generate customized quality control reports. Users can set parameters, define thresholds, and choose various options for their report.

Instructions:

  1. General Parameters:
    • In the ‘General Parameters’ tabset, you can use the widgets to choose general annotations for your report.
  2. Threshold Settings:
    • Navigate to the ‘Threshold’ tabset to set cutoffs for your analysis, like the minimum number of cells for each cluster.
  3. ON/OFF Options:
    • In the ‘ON/OFF’ tabset, use boolean widgets to customize your report.
    • Options may include showing the code used for analysis or ordering the graphs in a specific manner.
  4. Run the Report:
    • After setting all parameters and options, click on the "RUN THE REPORT" button located at the top of the page.
    • This will initiate the generation of your report.
    • The report will take some time to generate so be patient, it will open in a new window.

4.6 Differential Abundance

mod_da

Functionality Overview: The Differential Analysis Panel is designed for users to conduct comprehensive differential analyses. It includes advanced settings for thresholds, like p-value and fold change, and a dedicated tab for setting univariate parameters to define the differential analysis design.

Instructions:

  1. General Parameters:
    • Define the Condition column on which the differential comparison is to be performed.
  2. Threshold Settings:
    • In the ‘Thresholds’ tabset, set critical values for your analysis.
    • This includes specifying a p-value threshold for statistical significance and a fold change threshold to identify meaningful differences.
  3. Univariate Parameters:
    • Navigate to the new ‘Univariate Parameters’ tab to configure the design of your differential analysis. You will need to confirm the condition the ‘Design columns’ selector.
  4. Run Analysis:
    • After setting all thresholds and univariate parameters, initiate the differential analysis by clicking the "RUN ANALYSIS" button.

4.7 Differential State

mod_ds

tbd

4.8 Import R Object

mod_import_rds

Functionality Overview: This panel enables users to import an existing .rds file that corresponds to a previously processed Flow Cytometry Standard (FCS) file.

Instructions:

  1. Browse File: Click on the ‘Browse’ button to navigate through your local directory.
  2. Select File: Look for the .rds file you wish to import. These files represent pre-processed FCS data.
  3. Confirmation: Once the file is successfully uploaded, a confirmation message will be displayed.
  4. Next Steps: After importing, you can proceed to new actions in the "Select an action" box.

Additional Information:

  • This panel is designed to streamline the workflow by allowing easy access to pre-processed data.

4.9 Export to Phantasus

mod_export_ph

Functionality Overview: This panel facilitates the export of your data to Excel format for advanced analysis in Phantasus.

Downloadable Excel Files:

  • MFI Split by Markers: Export the MFI data, providing detailed insights split by each marker.
  • Median of MFI: Obtain the median MFI values for each FCS file and marker.
  • Abundance Matrix: Generate an abundance matrix displaying the percentage of cells in each cluster for each FCS file.