Test data

Test 1 – Running Enrichment Analysis with Expression Dataset

This test demonstrates how the auto-Enrich pipeline can be run using all modules to perform Over-Representation Analysis (ORA), with g:Profiler and PANTHER, and Gene Set Enrichment Analysis (GSEA).

The inputs includes a Gene Expression matrix from Mus musculus, the necessary configuration files, and two gene set files from the Mouse Collections of MSigDB

The test files are provided in test_data.zip:

  • test_data.zip <inserir_evo6_url>_ INSERIR URL

Contents inside the inputs directory of test_data.zip:

  • expression_matrix.tsv: Gene expression data matrix, Gene IDs and respective gene expression samples data, from Nogueira-Rodrigues et al. (2022)

  • config: Pipeline configuration file, setup to run input file generation for ORA and GSEA methods (Modules 1, 2, and 5), enrichment analysis (Modules 3, 4, and 6) on pre-selected sources, and output processing (Module 7)

  • /gene_sets: Folder holding the Gene Sets from the MSigDB to be provided to the GSEA runs (includes the Reactome Pathways gene set: m2.cp.reactome.v2026.1.Mm.symbols.gmt; and the Gene Ontology gene set: m5.go.v2026.1.Mm.symbols.gmt)

Pipeline behaviour workflow (modules=”1,2,3,4,5,6,7”):

1. The gene expression matrix is processed to build a genes list (from column index set in the variable gene) of the pre-selected differentially expressed genes marked (1) at the column index (set in the variable selected) [Module 1]

2. The given input gene identifiers are mapped to GeneID, UniProtKBs, HUGO Gene Symbol and Full Name [Module 2]

3. Over-Representation Enrichment Analysis are perfomed, with the mapped genes list, using g:Profiler g:GOSt tool on selected annotations-sources (set in the variable gprofiler_dbs), outputs are processed and enriched terms annotations are mapped [Module 3]

4. Over-Representation Enrichment Analysis are perfomed, with the mapped genes list, using PANTHER Overrepresentation test on selected annotations-sources (set in the variable panther_dbs), outputs are processed and enriched terms annotations are mapped [Module 4]

5. The gene expression matrix is partitioned into scoring Preranked Genes lists (.rnk) by calculating the set fold-changes (in the variables preranked1; preranked2) between computed experimental groups averages (set with the variables number_groups, number_samples, samples, groups, calculate_averages, isoform) [Module 5]

6. Preranked Gene Set Enrichment Analysis (GSEA) are executed using the previously generated Preranked Genes lists with set paramters (method, gene_set, nperm, others set by default) [Module 6]

7. The enrichment results from the different tools are intersected to find common results (intersection boolean variable) and individual tool results are filtered (by set variable max_annot; maximum allowed enriched term size ) [Module 7]

Output directories after the run:

# Generated inputs for Enrichment Analysis
/data/
├── annotations/*                                   → Utilized sources to map gene identifiers and terms annotations
├── preranked_gene_lists/selected_genes_list        → Selected genes list
├── mapped_gene_lists/selected_genes_list_map       → Mapped selected genes list
└── gsea/
    ├── parameters_log2FC_A_SCI_A_Sham      → GSEA parameters run files (one per set run)
    └── preranked_lists/                    → Generated Preranked Gene lists
        ├── log2FC_A_SCI_A_Sham.rnk
        ├── log2FC_A_SCI_A_Sham.rnk
        └── log2FC_A_Sham_M_Sham.rnk

# Enrichment Analysis results
/data/
├── common-results/
│   ├── selected_genes_list_&&_log2FC_A_SCI_A_Sham.GseaPreranked/*
│   ├── selected_genes_list_&&_log2FC_M_SCI_M_Sham.GseaPreranked/*
│   └── selected_genes_list_&&_log2FC_A_Sham_M_Sham.GseaPreranked/
│       ├── report                            → Summary report of similar found terms across compared tools
│       ├── common_gprof_panther_gsea.txt     → Common enriched terms and genes across g:Profiler, PANTHER and GSEA
│       ├── common_gprof_panther.txt
│       ├── common_gprof_gsea.txt
|       └── common_panther_gsea.txt
├── gprofiler/
│   └── selected_genes_list/
│       ├── enrichment_fields.tsv             → Enrichment results fields provided by gProfiler
│       ├── enrichmed_terms_annotations.tsv   → Enrichmed terms annotations mapping summary
│       └── source/annotations/*              → Raw enriched terms annotations sources
├── panther/
│   └── selected_genes_list/
│       ├── enrichment_fields.tsv             → Enrichment results fields provided by PANTHER
│       ├── enrichmed_terms_annotations.tsv   → Enrichmed terms annotations mapping summary
│       └── source/annotations/*              → Raw enriched terms annotations sources
└── gsea/
    └── results/
        ├── log2FC_A_SCI_A_Sham.combined.GseaPreranked/*
        ├── log2FC_M_SCI_M_Sham.combined.GseaPreranked/*
        └── log2FC_A_Sham_M_Sham.combined.GseaPreranked/
            ├── enrichment_fields.tsv                     → Enrichment results fields provided by GSEA
            ├── enrichmed_terms_annotations.tsv           → Enrichmed terms annotations mapping summary
            ├── source/annotations/*                      → Raw enriched terms annotations sources
            └── raw_GSEA_output.zip                       → Raw GSEA output report files