There are two methods for uploading your scRNA-seq data:
Upload a counts matrix and metadata file
If there are multiple samples, there should be a column labeled Condition.
Metadata should always be a .CSV file. Counts can either be a .CSV file or a .TXT file; if it is a .TXT file, the delimitor argument should be specified.
Upload a Seurat Object (Recommended)
Recommended data format, as large Counts data files can take a long time to be read in and processed.
The meta.data in the Seurat Object should still have a column labeled Condition.
Additionally, for comparison analysis, a Differentially Expressed Genes (DEG) .CSV file is needed. If multiple samples are detected and a DEG file is not provided, FlyPhone will generate its own DEG object to use for comparison analysis. Any user-uploaded file will override this. The required columns are listed below:
Gene - Gene name
avg_log2FC - Log 2 Fold Change value
cell_type - The cell type associated with a given gene
Required Parameters
These variables are required, regardless of the data uploaded:
Knowledgebase Version - Specifies which version of FlyPhone's knowledge base (Ligand-Receptor pairs & Core components data) to use when running the pipeline. Valid options include:
Version 1 - Utilizes FlyPhone V1's version
Version 2 All - Utilizes FlyPhoneDB2's version
Version 2 High - Filters the L-R pairs to include those with a "High" rank
Version 2 High/Moderate - Filters the L-R pairs to include those with either a "High" or "Moderate" rank
These variables are required if there are multiple samples present:
Control Sample Condition Name - The control condition name; value should match that in the Condition column in your metadata.
Mutant Sample Condition Name - The mutant condition name; value should match that in the Condition column in your metadata.
Optional Parameters
The remaining arguments are optional and have default values:
Percentage Cutoff - Any genes with a percent expression across all cells below this threshold will be ignored in the analysis. Default value is 10% (0.1).
Permutation Times - The number of times to run the permutation test for generating the p-value scores that determine the significance of a Ligand-Receptor signaling pair for any given celltypes. Larger values may result in more accurate results, but will take longer to run. Default value is 1,000.
Example Uploads
#1: Example of a Counts & Metadata file pair upload. Since the counts is a text file separated by spaces, the appropriate delimitor option is also selected. No DEG file was provided for this multi-sample analysis.
#2: Example uploading of a Seurat Object. A DEG file has also been uploaded, which FlyPhoneDB2 will use instead of generating a DEG object.
Example Status Pages
Example status page after a FlyPhoneDB2 submission has successfully been uploaded and started.
Example status page after a FlyPhoneDB2 run has finished.
