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name: bio-chipseq-peak-annotation description: Annotate ChIP-seq peaks to genomic features and genes using ChIPseeker. Assign peaks to promoters, exons, introns, and intergenic regions. Find nearest genes and calculate distance to TSS. Generate annotation plots and statistics. Use when annotating ChIP-seq peaks to genomic features. tool_type: r primary_tool: ChIPseeker

Peak Annotation with ChIPseeker

Load Peaks and Annotations

Copylibrary(ChIPseeker)
library(TxDb.Hsapiens.UCSC.hg38.knownGene)
library(org.Hs.eg.db)

txdb <- TxDb.Hsapiens.UCSC.hg38.knownGene

# Read peaks from MACS3
peaks <- readPeakFile('sample_peaks.narrowPeak')

Annotate Peaks

Copy# Annotate with default settings
peak_anno <- annotatePeak(
    peaks,
    TxDb = txdb,
    annoDb = 'org.Hs.eg.db'
)

# View annotation summary
peak_anno

Custom Promoter Definition

Copy# Define promoter region (-3kb to +3kb from TSS)
peak_anno <- annotatePeak(
    peaks,
    TxDb = txdb,
    tssRegion = c(-3000, 3000),  # Promoter definition
    annoDb = 'org.Hs.eg.db'
)

Extract Annotated Data Frame

Copy# Convert to data frame
anno_df <- as.data.frame(peak_anno)

# Key columns: seqnames, start, end, annotation, distanceToTSS, SYMBOL, GENENAME
head(anno_df)

# Export to CSV
write.csv(anno_df, 'annotated_peaks.csv', row.names = FALSE)

Get Genes with Peaks in Promoter

Copy# Filter for promoter peaks
promoter_peaks <- anno_df[grep('Promoter', anno_df$annotation), ]

# Get unique genes
promoter_genes <- unique(promoter_peaks$SYMBOL)

Annotation Pie Chart

Copy# Pie chart of genomic feature distribution
plotAnnoPie(peak_anno)

# Bar plot alternative
plotAnnoBar(peak_anno)

Distance to TSS Plot

Copy# Distribution of peaks relative to TSS
plotDistToTSS(peak_anno, title = 'Distribution of peaks relative to TSS')

Compare Multiple Peak Sets

Copy# Read multiple peak files
peak_files <- list(
    H3K4me3 = 'H3K4me3_peaks.narrowPeak',
    H3K27ac = 'H3K27ac_peaks.narrowPeak',
    H3K27me3 = 'H3K27me3_peaks.broadPeak'
)

peak_list <- lapply(peak_files, readPeakFile)

# Annotate all
anno_list <- lapply(peak_list, annotatePeak, TxDb = txdb, annoDb = 'org.Hs.eg.db')

# Compare annotations
plotAnnoBar(anno_list)
plotDistToTSS(anno_list)

Venn Diagram of Peak Overlap

Copy# Find overlapping peaks
genes_list <- lapply(anno_list, function(x) as.data.frame(x)$SYMBOL)
vennplot(genes_list)

Coverage Plot

Copy# Plot peak coverage around TSS
covplot(peaks, weightCol = 'V5')  # V5 is score column in narrowPeak

Profile Heatmap Around TSS

Copy# Get promoter coordinates
promoter <- getPromoters(TxDb = txdb, upstream = 3000, downstream = 3000)

# Get tag matrix
tagMatrix <- getTagMatrix(peaks, windows = promoter)

# Plot heatmap
tagHeatmap(tagMatrix, xlim = c(-3000, 3000), color = 'red')

# Average profile
plotAvgProf(tagMatrix, xlim = c(-3000, 3000), xlab = 'Distance from TSS')

Functional Enrichment of Peak Genes

Copylibrary(clusterProfiler)

# Get genes from peaks
genes <- unique(anno_df$ENTREZID)

# GO enrichment
ego <- enrichGO(
    gene = genes,
    OrgDb = org.Hs.eg.db,
    ont = 'BP',
    pAdjustMethod = 'BH',
    pvalueCutoff = 0.05
)

Seq2Gene - All Genes in Peak Regions

Copy# Find all genes overlapping peak regions (not just nearest)
genes_in_peaks <- seq2gene(peaks, tssRegion = c(-1000, 1000), flankDistance = 3000, TxDb = txdb)

Different Organisms

Copy# Mouse
library(TxDb.Mmusculus.UCSC.mm10.knownGene)
library(org.Mm.eg.db)
peak_anno_mm <- annotatePeak(peaks, TxDb = TxDb.Mmusculus.UCSC.mm10.knownGene, annoDb = 'org.Mm.eg.db')

# Zebrafish
library(TxDb.Drerio.UCSC.danRer11.refGene)
library(org.Dr.eg.db)

Key Functions

Annotation Categories

Related Skills

• peak-calling - Generate peak files with MACS3
• differential-binding - Find differential peaks
• pathway-analysis - Functional enrichment
• chipseq-visualization - Additional visualizations