Source: https://github.com/aipoch/medical-research-skills

Differential Expression Analysis

When to Read External Files

| Situation | File to Read | Purpose | |-----------|--------------|---------| | Need algorithm details | references/algorithm.md | Statistical methods, formulas, assumptions | | Need to run analysis | scripts/main.R | Execute: Rscript scripts/main.R --input_file ... --group_file ... | | Encounter errors | references/troubleshooting.md | Common errors and solutions | | Need CLI examples | references/cli-guide.md | Detailed CLI usage examples | | Need test data | tests/data/ | Sample input files for testing |

Usage

Rscript scripts/main.R \
  --input_file ./expression_matrix.csv \
  --group_file ./group_info.csv \
  --output_dir ./output/ \
  --diff_method limma \
  --p_threshold 0.05 \
  --logfc_threshold 0.1 \
  --seed 42

Arguments

| Short | Long | Type | Default | Description | |-------|------|------|---------|-------------| | -i | --input_file | character | required | Expression matrix file (genes as rows, samples as columns) | | -g | --group_file | character | required | Group information file (sample ID + group columns) | | -o | --output_dir | character | ./output/ | Output directory | | -m | --diff_method | character | limma | Method: limma, deseq2, edger, t, wilcox | | -n | --norm_method | character | TMM | Normalization for edgeR: TMM, RLE, upperquartile | | -p | --p_threshold | numeric | 0.05 | P-value threshold | | -f | --logfc_threshold | numeric | 0.1 | Log fold change threshold | | -s | --seed | integer | 42 | Random seed for reproducibility |

Input Format

Expression Matrix (input_file)

Genes as rows, samples as columns, CSV format with gene ID in first column.

"","GSM1442228","GSM1442229","GSM1442230"
"0610006L08Rik",3.438,3.237,3.265
"0610007P14Rik",6.734,7.017,6.807

Group File (group_file)

CSV with sample ID and group columns.

"ID","group"
"GSM1442228","Control"
"GSM1442229","Control"
"GSM1442230","DIC"

Output Files

| File | Description | |------|-------------| | Diffanalysis.csv | Complete DE results with gene_id, logFC, Pvalue, Padj | | volcano_plot.pdf | Volcano plot with significance thresholds | | heatmap.pdf | Heatmap of top upregulated/downregulated genes | | session_info.txt | R session and package version info | | temp/rdegs.csv | Significant differentially expressed genes | | temp/Diffanalysis_filtered.csv | Full results with group annotations |

Workflow

Step 1: Validate Input

Check file existence
Validate sample matching between expression matrix and group file
Verify at least 2 samples per group

Step 2: Run Differential Expression

Choose method: limma, DESeq2, edgeR, t-test, or Wilcoxon
Calculate logFC and p-values
Apply multiple testing correction (Benjamini-Hochberg)

Step 3: Filter Results

Filter by p-value and logFC thresholds
Classify genes as Up, Down, or Not significant

Step 4: Generate Visualizations

Volcano plot showing significance vs fold change
Heatmap of top differential genes

Methods

limma

Linear models for microarray and RNA-seq with empirical Bayes moderation. Recommended for normalized expression data (FPKM, TPM).

DESeq2

Negative binomial GLM with variance stabilization. Recommended for raw count data.

edgeR

Empirical Bayes methods with TMM normalization. Supports robust dispersion estimation.

t-test / Wilcoxon

Simple pairwise statistical tests. t-test for parametric, Wilcoxon for non-parametric.

Examples

Basic Usage (limma)

Rscript scripts/main.R \
  -i expression_matrix.csv \
  -g group_info.csv \
  -o ./output \
  -m limma

With DESeq2 for Count Data

Rscript scripts/main.R \
  -i count_matrix.csv \
  -g group_info.csv \
  -o ./output \
  -m deseq2

Custom Thresholds

Rscript scripts/main.R \
  -i expression_matrix.csv \
  -g group_info.csv \
  -o ./output \
  -p 0.01 \
  -f 0.5

Error Handling

Common Errors

| Error | Cause | Solution | |-------|-------|----------| | SKILL_FILE_NOT_FOUND | Input file doesn't exist | Check file path | | SKILL_SAMPLE_MISMATCH | Sample names don't match | Verify group file matches expression matrix columns | | SKILL_INVALID_DATA | Less than 2 groups or samples per group | Check group file | | SKILL_FILTER_ERROR | No significant genes found | Relax thresholds or check data quality | | SKILL_DEPENDENCY_MISSING | R package not installed | Install required packages |

IF error persists, READ: references/troubleshooting.md

Testing

Test with Sample Data

# Check help
Rscript scripts/main.R --help

# Run with sample data
Rscript scripts/main.R \
  -i tests/data/Combined_Datasets_Matrix_mus.csv \
  -g tests/data/Combined_Datasets_mus_Group.csv \
  -o tests/output/

Validation Commands

# Count lines in output
wc -l output/Diffanalysis.csv

# Check volcano plot exists
ls -la output/volcano_plot.pdf

Implementation Checklist

[x] CLI parsing with optparse
[x] set.seed() for reproducibility
[x] requireNamespace() dependency checks
[x] Session info recording
[x] Temp file cleanup
[x] File reading instructions in SKILL.md
[x] Modular script structure (<100 lines per file)
[x] Test data provided
[x] Error handling with SKILL_* codes
[x] Scripts in scripts/ directory
[x] References in references/ directory

Last updated: 2026-04-01 | Version: 2.0.0