Bio De Results

---
name: bio-de-results
description: Extract, filter, annotate, and export differential expression results from DESeq2 or edgeR. Use for identifying significant genes, applying multiple testing corrections, adding gene annotations, and preparing results for downstream analysis. Use when filtering and exporting DE analysis results.
tool_type: r
primary_tool: DESeq2
---

## Version Compatibility

Reference examples tested with: DESeq2 1.42+, edgeR 4.0+

Before using code patterns, verify installed versions match. If versions differ:
- R: `packageVersion('<pkg>')` then `?function_name` to verify parameters

If code throws ImportError, AttributeError, or TypeError, introspect the installed
package and adapt the example to match the actual API rather than retrying.

# DE Results

Extract, filter, and export differential expression results.

## Required Libraries

```r
library(DESeq2)  # or library(edgeR)
library(dplyr)   # For data manipulation
```

## Extracting DESeq2 Results

**Goal:** Retrieve DE statistics from a fitted DESeq2 model as a usable data frame.

**Approach:** Call results() with optional shrinkage, then convert to a data frame with gene identifiers.

```r
# Basic results
res <- results(dds)

# With specific alpha (adjusted p-value threshold)
res <- results(dds, alpha = 0.05)

# With log fold change shrinkage
res <- lfcShrink(dds, coef = 'condition_treated_vs_control', type = 'apeglm')

# Convert to data frame
res_df <- as.data.frame(res)
res_df$gene <- rownames(res_df)
```

## Extracting edgeR Results

**Goal:** Retrieve DE statistics from a fitted edgeR model as a data frame.

**Approach:** Use topTags with n=Inf to extract all gene-level results.

```r
# Get all results
results <- topTags(qlf, n = Inf)$table

# Add gene column
results$gene <- rownames(results)
```

## Filtering Significant Genes

**Goal:** Identify genes meeting statistical significance and biological effect size criteria.

**Approach:** Subset results by adjusted p-value, fold change magnitude, and expression level thresholds.

**"Get the significant differentially expressed genes"** → Filter DE results by adjusted p-value and fold change cutoffs to produce up- and down-regulated gene lists.

### By Adjusted P-value

```r
# DESeq2
sig_genes <- subset(res, padj < 0.05)

# edgeR
sig_genes <- subset(results, FDR < 0.05)

# Using dplyr
sig_genes <- res_df %>%
    filter(padj < 0.05) %>%
    arrange(padj)
```

### By Fold Change

```r
# Absolute log2 fold change > 1 (2-fold change)
sig_genes <- subset(res, padj < 0.05 & abs(log2FoldChange) > 1)

# Up-regulated only
up_genes <- subset(res, padj < 0.05 & log2FoldChange > 1)

# Down-regulated only
down_genes <- subset(res, padj < 0.05 & log2FoldChange < -1)
```

### Combined Filters

```r
# Stringent filtering
sig_genes <- res_df %>%
    filter(padj < 0.01,
           abs(log2FoldChange) > 1,
           baseMean > 10) %>%
    arrange(padj)
```

## Ordering Results

**Goal:** Rank DE genes by statistical significance or biological effect size.

**Approach:** Sort results by adjusted p-value, absolute fold change, or mean expression.

```r
# By adjusted p-value (most significant first)
res_ordered <- res[order(res$padj), ]

# By absolute fold change (largest changes first)
res_ordered <- res[order(abs(res$log2FoldChange), decreasing = TRUE), ]

# By base mean expression
res_ordered <- res[order(res$baseMean, decreasing = TRUE), ]

# Combined: significant genes ordered by fold change
sig_ordered <- res_df %>%
    filter(padj < 0.05) %>%
    arrange(desc(abs(log2FoldChange)))
```

## Summary Statistics

**Goal:** Quantify the number of up- and down-regulated genes at chosen thresholds.

**Approach:** Count genes passing significance filters and report directional breakdown.

```r
# DESeq2 summary
summary(res)

# Manual counts
n_tested <- sum(!is.na(res$padj))
n_sig <- sum(res$padj < 0.05, na.rm = TRUE)
n_up <- sum(res$padj < 0.05 & res$log2FoldChange > 0, na.rm = TRUE)
n_down <- sum(res$padj < 0.05 & res$log2FoldChange < 0, na.rm = TRUE)

cat(sprintf('Tested: %d genes\n', n_tested))
cat(sprintf('Significant (padj < 0.05): %d genes\n', n_sig))
cat(sprintf('Up-regulated: %d genes\n', n_up))
cat(sprintf('Down-regulated: %d genes\n', n_down))

# edgeR summary
summary(decideTests(qlf))
```

## Adding Gene Annotations

**Goal:** Enrich DE results with gene symbols, descriptions, and cross-database identifiers.

**Approach:** Map Ensembl or Entrez IDs to human-readable annotations using org.db, biomaRt, or custom files.

**"Add gene names to my DE results"** → Map gene identifiers to symbols and descriptions using annotation databases, then merge with the results table.

### From Bioconductor Annotation Package

```r
library(org.Hs.eg.db)  # Human; use org.Mm.eg.db for mouse

# If gene IDs are Ensembl
res_df$symbol <- mapIds(org.Hs.eg.db,
                         keys = rownames(res_df),
                         column = 'SYMBOL',
                         keytype = 'ENSEMBL',
                         multiVals = 'first')

res_df$entrez <- mapIds(org.Hs.eg.db,
                         keys = rownames(res_df),
                         column = 'ENTREZID',
                         keytype = 'ENSEMBL',
                         multiVals = 'first')

res_df$description <- mapIds(org.Hs.eg.db,
                              keys = rownames(res_df),
                              column = 'GENENAME',
                              keytype = 'ENSEMBL',
                              multiVals = 'first')
```

### From BioMart

```r
library(biomaRt)

mart <- useMart('ensembl', dataset = 'hsapiens_gene_ensembl')

annotations <- getBM(
    attributes = c('ensembl_gene_id', 'external_gene_name', 'description'),
    filters = 'ensembl_gene_id',
    values = rownames(res_df),
    mart = mart
)

# Merge with results
res_annotated <- merge(res_df, annotations,
                        by.x = 'row.names', by.y = 'ensembl_gene_id',
                        all.x = TRUE)
```

### From Custom File

```r
# Load annotation file
gene_info <- read.csv('gene_annotations.csv')

# Merge with results
res_annotated <- merge(res_df, gene_info, by = 'gene', all.x = TRUE)
```

## Exporting Results

**Goal:** Save DE results in formats suitable for sharing, publication, or downstream tools.

**Approach:** Write filtered and annotated results to CSV, Excel workbooks, or ranked gene lists for pathway analysis.

### To CSV

```r
# All results
write.csv(res_df, file = 'deseq2_all_results.csv', row.names = FALSE)

# Significant only
sig_genes <- res_df %>% filter(padj < 0.05)
write.csv(sig_genes, file = 'deseq2_significant.csv', row.names = FALSE)
```

### To Excel

```r
library(openxlsx)

# Create workbook with multiple sheets
wb <- createWorkbook()

addWorksheet(wb, 'All Results')
writeData(wb, 'All Results', res_df)

addWorksheet(wb, 'Significant')
writeData(wb, 'Significant', sig_genes)

addWorksheet(wb, 'Up-regulated')
writeData(wb, 'Up-regulated', up_genes)

addWorksheet(wb, 'Down-regulated')
writeData(wb, 'Down-regulated', down_genes)

saveWorkbook(wb, 'de_results.xlsx', overwrite = TRUE)
```

### Gene Lists for Pathway Analysis

```r
# Just gene IDs for GO/KEGG analysis
sig_gene_list <- rownames(subset(res, padj < 0.05))
write.table(sig_gene_list, file = 'significant_genes.txt',
            quote = FALSE, row.names = FALSE, col.names = FALSE)

# With fold changes for GSEA
gsea_input <- res_df %>%
    filter(!is.na(log2FoldChange)) %>%
    select(gene, log2FoldChange) %>%
    arrange(desc(log2FoldChange))
write.table(gsea_input, file = 'gsea_input.rnk',
            sep = '\t', quote = FALSE, row.names = FALSE, col.names = FALSE)
```

## Comparing Results Between Methods

**Goal:** Assess concordance between DESeq2 and edgeR results to identify robust DE genes.

**Approach:** Compute set overlaps and visualize with a Venn diagram.

```r
# Get significant genes from both methods
deseq2_sig <- rownames(subset(deseq2_res, padj < 0.05))
edger_sig <- rownames(subset(edger_results, FDR < 0.05))

# Overlap
common <- intersect(deseq2_sig, edger_sig)
deseq2_only <- setdiff(deseq2_sig, edger_sig)
edger_only <- setdiff(edger_sig, deseq2_sig)

cat(sprintf('DESeq2 significant: %d\n', length(deseq2_sig)))
cat(sprintf('edgeR significant: %d\n', length(edger_sig)))
cat(sprintf('Common: %d\n', length(common)))
cat(sprintf('DESeq2 only: %d\n', length(deseq2_only)))
cat(sprintf('edgeR only: %d\n', length(edger_only)))

# Venn diagram
library(VennDiagram)
venn.diagram(
    x = list(DESeq2 = deseq2_sig, edgeR = edger_sig),
    filename = 'de_overlap.png',
    fill = c('steelblue', 'coral')
)
```

## Multiple Testing Correction

**Goal:** Apply or compare multiple testing correction methods for DE p-values.

**Approach:** Use Benjamini-Hochberg (default), Bonferroni, or IHW for adjusted p-values.

```r
# DESeq2 uses Benjamini-Hochberg by default
# To use different methods:

# Independent Hypothesis Weighting (more powerful)
library(IHW)
res_ihw <- results(dds, filterFun = ihw)

# Manual p-value adjustment
res_df$padj_bonferroni <- p.adjust(res_df$pvalue, method = 'bonferroni')
res_df$padj_bh <- p.adjust(res_df$pvalue, method = 'BH')
res_df$padj_fdr <- p.adjust(res_df$pvalue, method = 'fdr')
```

## Handling NA Values

**Goal:** Understand and handle missing values in DE results caused by filtering or outlier detection.

**Approach:** Identify the source of NAs (zero counts, independent filtering, outliers) and remove or investigate them.

```r
# Count NAs
sum(is.na(res$padj))

# Remove genes with NA padj
res_complete <- res[!is.na(res$padj), ]

# Understand why NAs occur
# - baseMean = 0: No counts
# - NA only in padj: Outlier or low count filtered by independent filtering

# Check outliers
res[which(is.na(res$pvalue) & res$baseMean > 0), ]
```

## Quick Reference: Result Columns

### DESeq2

| Column | Description |
|--------|-------------|
| `baseMean` | Mean normalized counts |
| `log2FoldChange` | Log2 fold change |
| `lfcSE` | Standard error of LFC |
| `stat` | Wald statistic |
| `pvalue` | Raw p-value |
| `padj` | Adjusted p-value (BH) |

### edgeR

| Column | Description |
|--------|-------------|
| `logFC` | Log2 fold change |
| `logCPM` | Average log2 CPM |
| `F` | Quasi-likelihood F-statistic |
| `PValue` | Raw p-value |
| `FDR` | False discovery rate |

## Related Skills

- deseq2-basics - Run DESeq2 analysis
- edger-basics - Run edgeR analysis
- de-visualization - Visualize results
- pathway-analysis - Use gene lists for enrichment