Differential gene expression for single-cell data using pseudo-bulk counts with edgeR

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flowchart TD
  0["ℹ️ Input Dataset\nSource AnnData file"];
  style 0 stroke:#2c3143,stroke-width:4px;
  1["ℹ️ Input Parameter\nPseudo-bulk: Fields to merge"];
  style 1 fill:#ded,stroke:#393,stroke-width:4px;
  2["ℹ️ Input Parameter\nGroup by column"];
  style 2 fill:#ded,stroke:#393,stroke-width:4px;
  3["ℹ️ Input Parameter\nSample key column"];
  style 3 fill:#ded,stroke:#393,stroke-width:4px;
  4["ℹ️ Input Parameter\nName Your Raw Counts Layer"];
  style 4 fill:#ded,stroke:#393,stroke-width:4px;
  5["ℹ️ Input Parameter\nFactor fields"];
  style 5 fill:#ded,stroke:#393,stroke-width:4px;
  6["ℹ️ Input Parameter\nFormula"];
  style 6 fill:#ded,stroke:#393,stroke-width:4px;
  7["ℹ️ Input Parameter\nGene symbol column"];
  style 7 fill:#ded,stroke:#393,stroke-width:4px;
  8["Decoupler pseudo-bulk"];
  1 -->|output| 8;
  5 -->|output| 8;
  2 -->|output| 8;
  0 -->|output| 8;
  4 -->|output| 8;
  3 -->|output| 8;
  b28fbf89-5621-459e-9321-6cd0a72261ea["Output\nPseudobulk count matrix"];
  8 --> b28fbf89-5621-459e-9321-6cd0a72261ea;
  style b28fbf89-5621-459e-9321-6cd0a72261ea stroke:#2c3143,stroke-width:4px;
  6e8b4090-3ab7-4158-805a-2d757fd5e0fb["Output\nPseudobulk Plot"];
  8 --> 6e8b4090-3ab7-4158-805a-2d757fd5e0fb;
  style 6e8b4090-3ab7-4158-805a-2d757fd5e0fb stroke:#2c3143,stroke-width:4px;
  93e42181-49b2-47cf-ab73-98373376caf9["Output\nFiltered by expression"];
  8 --> 93e42181-49b2-47cf-ab73-98373376caf9;
  style 93e42181-49b2-47cf-ab73-98373376caf9 stroke:#2c3143,stroke-width:4px;
  9["Sanitize matrix"];
  8 -->|count_matrix| 9;
  10["Sanitize factors"];
  8 -->|samples_metadata| 10;
  11["Remove start, end, width"];
  8 -->|genes_metadata| 11;
  12["Sanitize first factor for leading digits"];
  10 -->|outfile| 12;
  13["Text reformatting"];
  12 -->|outfile| 13;
  14["edgeR"];
  11 -->|output_tabular| 14;
  13 -->|outfile| 14;
  6 -->|output| 14;
  9 -->|outfile| 14;
  12 -->|outfile| 14;
  851ac5bc-9c57-4f36-b469-33a2e8dde894["Output\nTables: DEG"];
  14 --> 851ac5bc-9c57-4f36-b469-33a2e8dde894;
  style 851ac5bc-9c57-4f36-b469-33a2e8dde894 stroke:#2c3143,stroke-width:4px;
  5ad48faa-d0a7-4bb1-b307-7861f540ec29["Output\nReport Results: HTML File"];
  14 --> 5ad48faa-d0a7-4bb1-b307-7861f540ec29;
  style 5ad48faa-d0a7-4bb1-b307-7861f540ec29 stroke:#2c3143,stroke-width:4px;
  15["Get contrast labels"];
  14 -->|outTables| 15;
  16["Select gene symbols, logFC, PValue and FDR"];
  7 -->|output| 16;
  14 -->|outTables| 16;
  647df402-93cb-4f9e-9bde-7afcbcffad5b["Output\nTables for volcano plot"];
  16 --> 647df402-93cb-4f9e-9bde-7afcbcffad5b;
  style 647df402-93cb-4f9e-9bde-7afcbcffad5b stroke:#2c3143,stroke-width:4px;
  17["Replace Text"];
  15 -->|output| 17;
  18["Split contrasts"];
  17 -->|outfile| 18;
  19["Contrast as parameters"];
  18 -->|list_output_txt| 19;
  20["Volcano Plot"];
  16 -->|output_tabular| 20;
  19 -->|text_param| 20;
  02cc64ea-d34d-4081-8a2b-2dc59e102c66["Output\nVolcano Plot on input dataset(s): PDF"];
  20 --> 02cc64ea-d34d-4081-8a2b-2dc59e102c66;
  style 02cc64ea-d34d-4081-8a2b-2dc59e102c66 stroke:#2c3143,stroke-width:4px;

Inputs

Outputs

Tools

To use these workflows in Galaxy you can either click the links to download the workflows, or you can right-click and copy the link to the workflow which can be used in the Galaxy form to import workflows.

Importing into Galaxy

Hands On: Importing a workflow

1. Click on galaxy-workflows-activity Workflows in the Galaxy activity bar (on the left side of the screen, or in the top menu bar of older Galaxy instances). You will see a list of all your workflows
2. Click on galaxy-upload Import at the top-right of the screen
3. Provide your workflow
   • Option 1: Paste the URL of the workflow into the box labelled “Archived Workflow URL”
   • Option 2: Upload the workflow file in the box labelled “Archived Workflow File”
4. Click the Import workflow button

Below is a short video demonstrating how to import a workflow from GitHub using this procedure:

Version History

For Admins

Installing the workflow tools

wget https://training.galaxyproject.org/training-material/topics/single-cell/tutorials/pseudobulk-analysis/workflows/pseudo-bulk_edgeR.ga -O workflow.ga
workflow-to-tools -w workflow.ga -o tools.yaml
shed-tools install -g GALAXY -a API_KEY -t tools.yaml
workflow-install -g GALAXY -a API_KEY -w workflow.ga --publish-workflows