**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 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 .. **Test 2 – Running Enrichment Analysis Multiple Gene Lists** .. --------------------------------------------------------------- .. This test demonstrates how auto-Enrich can be run to perform .. **g:Profiler and PANTHER Over-Representation analysis** using .. one or more pre-prepared gene ID lists. .. - A **single gene list** can be run by specifying its name in the ``gene_list`` .. variable in config0. .. - **Multiple gene lists** can be run together by placing all the desired .. gene lists in a directory named ``/prepared_gene_lists`` within your data directory (/data). .. In this test, **four gene ID lists** are included, all from *Mus .. musculus*. .. The test files are provided in test2.zip: .. - `test2.zip _` **INSERIR URL** .. **Contents inside the inputs directory of test2.zip:** .. - **config0**: Pipeline configuration file, setup to map input gene IDs .. (Module 2) and run over-representation analysis (Modules 3 and 4) .. - **/prepared_gene_lists**: Contains the input gene ID lists. .. **Workflow (tools=”2,3,4”):** .. 1. All Gene IDs lists inside /prepared_gene_lists are mapped (Module 2) .. 2. Over-Representation Enrichment Analysis of all mapped Gene IDs lists .. are run using g:Profiler (Module 3) .. 3. Over-Representation Enrichment Analysis of all mapped Gene IDs lists .. are run using PANTHER (Module 4) .. **Output directories after the run:** .. - **/mapped_gene_lists** → mapped gene identifier list by **Module 2**. .. - **/gprofiler** → enrichment results from **Module 3** (g:Profiler .. plus). .. - **/panther** → enrichment results from **Module 4** (PANTHER plus). .. - **/annotations** → consolidated mapping and term annotation files from .. Modules 2–4, enabling easy cross-referencing of enriched terms and .. gene memberships. .. After running Over-Representation analysis with multiple gene lists, results are organized by **tool** and **gene list**: .. - /gprofiler/ .. - /gprofiler/gene_list1/results/ – Enrichment results for ``gene_list1`` using ``g:Profiler`` .. - /gprofiler/gene_list2/results/ – Enrichment results for ``gene_list2`` using ``g:Profiler`` .. - /panther/ .. - /panther/gene_list1/results/ – Enrichment results for ``gene_list1`` using ``PANTHER`` .. - /panther/gene_list2/results/ – Enrichment results for ``gene_list2`` using ``PANTHER`` .. Each **results** directory contains: .. - **Short and long result tables**: ``short_results.tsv`` and ``long_results.tsv`` .. - **Term annotation files**: ``terms_annotations_results.tsv`` .. - **Subdirectories** for each source, containing respective results files, and enriched .. terms annotations. .. **Test 3 – Preparing and Running GSEA Preranked** .. ---------------------------------------------------- .. This test demonstrates how auto-Enrich can be used prepare GSEA .. Preranked inputs from an expression matrix and run GSEA. The input .. includes an expression matrix with pre-calculated averages from *Mus .. musculus* gene IDs, the necessary configuration files and a gene set .. file from the `Mouse Collections of .. MSigDB `__. .. In this test two Preranked gene lists are generated, and two runs of .. GSEA are performed. .. The test files are provided in test3.zip: .. - `test3.zip _` **INSERIR URL** .. **Contents inside the inputs directory of test3.zip:** .. - **config0**: Pipeline configuration file, set up to run preparation of .. pre-ranked gene lists (Module 5) and GSEA Preranked run (Module 6) .. - **config5**: Configuration file to coordinate Module 5, to transform .. the input expression matrix TWO pre-ranked genes lists (.rnk) .. - **expression_matrix.tsv**: Gene expression data matrix (Gene ID and .. gene expression averages data) .. - **gsea_parameters**: GSEA run settings .. - **m2.all.v2024.Mm.entrez.gmt**: Gene Set file (GMT) containing the .. gene sets to run GSEA .. **Workflow (tools=”5,6”):** .. 1. Two preranked gene lists (RNK) for GSEA are generated (**Module 5**), .. following the module configuration file (**config5**). .. 2. Gene Set Enrichment Analysis (**GSEA**) is executed using the prepared .. inputs and **gsea_parameters** (**Module 6**). .. **Output directory after the run:** .. - **/gsea**: GSEA inputs and results .. The results of the GSEA input preparation and run, are stored inside the **gsea** .. directory, which contains: .. - **/preranked_gene_lists**: Contains the preranked gene lists generated .. by Module 5 .. - **/results**: Stores the GSEA results of both runs of the two .. preranked gene lists in separate directories .. - /m2.all.logFC_M_naive_A_naive.GseaPreranked .. - /m2.all.logFC_M_SCI_A_SCI.GseaPreranked .. - **gsea_parameters** files: The GSEA settings files used in the runs .. - **m2.all.v2024.Mm.entrez.gmt**: Gene Set file (GMT) containing the .. gene sets to run GSEA .. Each **results** directory contains: .. - **Short and long result tables**: ``short_results.tsv`` and ``long_results.tsv`` .. - **Term annotation files**: ``terms_annotations_results.tsv`` .. - **Subdirectories** for each source, containing respective results files, and enriched .. terms annotations.