fGSEA¶

fgsea is a Bioconductor package for fast preranked gene set enrichment analysis (GSEA) which has been "wrapped" for use in the Galaxy framework. As all GSEA approaches, fgsea implement an algorithm for cumulative GSEA-statisti calculation. We will use it in a the standard way, ie basing our metrics on fold changes that computed using the DESeq2 Galaxy tool.

fgsea inputs¶

1. The collection of DESeq2 DE tables (with headers)¶

fgsea reauires first a two-column file containing a ranked list of genes. The first column must contain the gene identifiers and the second column the statistic used to rank. Gene identifiers must be unique (not repeated) within the file and must be the same type as the identifiers in the Gene Sets file.

Since what is expected is in the form of

Symbol	Ranked Stat
VDR	67.198
IL20RA	65.963
MPHOSPH10	51.353
RCAN1	50.269
HILPDA	50.015
TSC22D3	47.496
FAM107B	45.926

and that our DESeq tables contain only Ensembl identifiers, we will start from the DEseq2 collection and replace Ensembl identifiers by gene symbols (using a table generated in the previous section)

→ Thus, copy the collection DESeq2 Results Tables from the history PRJNA630433 DESeq2 analysis in a new history that you will name PRJNA630433 fgsea

2. The table `EnsemblID-GeneSymbol table`¶

As mentionned above.

Note that you have generated this table in the previous section.

It is also available in the data library IOC_bulk_RNAseq / Mouse reference files, as well as in your own data library (if you followed the instructions).

→ Copy ENTREZID-GeneSymbol table in your history PRJNA630433 fgsea

3. One or several GMT files¶

GMT (Gene Matrix Transposed) files are available at https://www.gsea-msigdb.org/gsea/msigdb/mouse/collections.jsp

They are tabular files looking like:

HALLMARK_APOPTOSIS	http://www.broadinstitute.org/gsea/msigdb/cards/HALLMARK_APOPTOSIS	CASP3	CASP9	...
HALLMARK_HYPOXIA	http://www.broadinstitute.org/gsea/msigdb/cards/HALLMARK_HYPOXIA	PGK1	PDK1	...

Note that in such a file, each line represents a gene set. The two first columns identify the gene set (its name and it description URL). For each line, the number of column is otherwise variable, with one column for each symbol of gene belonging to the gene set. Thus, the number of these extra columns starting at col-3 reflects the number of genes in the geneset.

We have downloaded several GMT files on purpose from https://www.gsea-msigdb.org/gsea/msigdb/mouse/collections.jsp and made this files available from the data library IOC_bulk_RNAseq / Mouse reference files.

→ Copy the following datasets in the history PRJNA630433 fgsea:

How did we generated the GMT files

These files were retrieved from a search on msigdb with the keyword(s) indicated in their title.

Do no hesitate to generate your own GMT files using msigdb

The `fgsea` workflow¶

The Galaxy workflow Galaxy-Workflow-fgsea.ga performs fgsea analysis from

The collection DESeq2 Results Tables (history PRJNA630433 DESeq2 analysis)
The dataset EnsemblID-GeneSymbol table (from the data library IOC_bulk_RNAseq / Mouse reference files) or where available
5 GMT files dendritic.gmt, glycolysis.gmt, monocyte_OR_macrophage.gmt, mouse_immune_AND_response.gmt,osteoclast.gmt.

Run this workflow in the dedicated history PRJNA630433 fgsea paying extra attention to select the appropriate input datasets (follow the workflow form instructions)