Workflow Type: Galaxy
Frozen
Frozen
This workflow uses the decoupler tool in Galaxy to generate pseudobulk counts from an annotated AnnData file obtained from scRNA-seq analysis. Following the pseudobulk step, differential expression genes (DEG) are calculated using the edgeR tool. The workflow also includes data sanitation steps to ensure smooth operation of edgeR and minimizing potential issues. Additionally, a Volcano plot tool is used to visualize the results after the DEG analysis.
Inputs
ID | Name | Description | Type |
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Factor fields | Factor fields | The fields from Obs to be provided to EdgeR as factors. The first field should represent the main contrast for comparisons, while the subsequent fields will be used as covariates. |
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Formula | Formula | Example 1: ~ 0 + Factor_1 (Use this formula when you only want to account for one factor, Factor_1). Example 2 (With covariate adjustment): ~ 0 + Factor_1 + Factor_2 (Use this formula if you need to adjust for additional factors, such as Factor_2, which serves as a covariate). Note: Ensure that all factors (e.g., Factor_1, Factor_2) included in the formula are defined in your factor file. |
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Gene symbol column | Gene symbol column | Specify the name of the column containing your gene symbols. For example: gene_symbol, gene_name, x, etc. |
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Group by column | Group by column | Typically, the column in obs that you want to use for comparisons later (the main contrast field) should be specified here. This column will also be used for plotting the pseudo-bulk samples, showing the number of counts and cells. |
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Name Your Raw Counts Layer | Name Your Raw Counts Layer | Name of the layer containing your raw (non-normalized) counts. |
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Pseudo-bulk: Fields to merge | Pseudo-bulk: Fields to merge | Merge Obs fields before pseudo-bulk analysis to create new categories for grouping, such as 'sample,phase' or 'sample,louvain'. Ensure the fields exist in the Obs of the AnnData object. Multiple groups can be merged with a colon (':'), e.g., 'sample,phase ,phase' creates 'sample_phase' and 'louvain_phase'. |
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Sample key column | Sample key column | The field used to create the pseudo-bulk replicates is typically a combination of multiple Obs fields merged together. |
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Source AnnData file | Source AnnData file | Ensure your AnnData object contains all necessary layers before using the decoupler tool. The raw counts should be included in AnnData. If they are missing, create a new layer (e.g., 'raw_counts') and copy the raw counts into it. |
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Steps
ID | Name | Description |
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8 | Decoupler pseudo-bulk | toolshed.g2.bx.psu.edu/repos/ebi-gxa/decoupler_pseudobulk/decoupler_pseudobulk/1.4.0+galaxy8 |
9 | Sanitize matrix | toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_replace_in_line/9.3+galaxy1 |
10 | Sanitize factors | toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_replace_in_line/9.3+galaxy1 |
11 | Remove start, end, width | A column that may affect EdgeR and DESeq2. toolshed.g2.bx.psu.edu/repos/iuc/column_remove_by_header/column_remove_by_header/1.0 |
12 | Sanitize first factor for leading digits | toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_replace_in_column/9.3+galaxy1 |
13 | Text reformatting | toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_awk_tool/9.3+galaxy1 |
14 | edgeR | toolshed.g2.bx.psu.edu/repos/iuc/edger/edger/3.36.0+galaxy5 |
15 | Get contrast labels | toolshed.g2.bx.psu.edu/repos/iuc/collection_element_identifiers/collection_element_identifiers/0.0.2 |
16 | Select gene symbols, logFC, PValue and FDR | toolshed.g2.bx.psu.edu/repos/iuc/column_remove_by_header/column_remove_by_header/1.0 |
17 | Replace Text | toolshed.g2.bx.psu.edu/repos/bgruening/text_processing/tp_replace_in_line/9.3+galaxy1 |
18 | Split contrasts | toolshed.g2.bx.psu.edu/repos/bgruening/split_file_to_collection/split_file_to_collection/0.5.2 |
19 | Contrast as parameters | param_value_from_file |
20 | Volcano Plot | toolshed.g2.bx.psu.edu/repos/iuc/volcanoplot/volcanoplot/0.0.6 |
Outputs
ID | Name | Description | Type |
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Pseudobulk count matrix | Pseudobulk count matrix | n/a |
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Pseudobulk Plot | Pseudobulk Plot | n/a |
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Filtered by expression | Filtered by expression | n/a |
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Tables: DEG | Tables: DEG | n/a |
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Report Results: HTML File | Report Results: HTML File | n/a |
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Tables for volcano plot | Tables for volcano plot | n/a |
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Volcano Plot on input dataset(s): PDF | Volcano Plot on input dataset(s): PDF | n/a |
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Version History
v0.1.1 (latest) Created 22nd Nov 2024 at 03:02 by WorkflowHub Bot
Updated to v0.1.1
Frozen
v0.1.1
1a15aa4
v0.1 (earliest) Created 17th Nov 2024 at 03:03 by WorkflowHub Bot
Updated to v0.1
Frozen
v0.1
72f8bbd
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Views: 199 Downloads: 35 Runs: 2
Created: 17th Nov 2024 at 03:03
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