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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 measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes. allowed-tools:

• read_file
• run_shell_command

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Peak Annotation with ChIPseeker

Load Peaks and Annotations

library(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

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

# View annotation summary
peak_anno

Custom Promoter Definition

# 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

# 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

# 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

# Pie chart of genomic feature distribution
plotAnnoPie(peak_anno)

# Bar plot alternative
plotAnnoBar(peak_anno)

Distance to TSS Plot

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

Compare Multiple Peak Sets

# 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

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

Coverage Plot

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

Profile Heatmap Around TSS

# 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

library(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

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

Different Organisms

# 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

Function	Purpose
readPeakFile	Read peak file (BED, narrowPeak)
annotatePeak	Annotate peaks to genes
plotAnnoPie	Pie chart of annotations
plotAnnoBar	Bar plot of annotations
plotDistToTSS	Distance to TSS distribution
getPromoters	Get promoter regions
getTagMatrix	Coverage matrix around regions
tagHeatmap	Heatmap of signal
plotAvgProf	Average profile plot
seq2gene	Map peaks to all overlapping genes

Annotation Categories

Category	Description
Promoter	Within tssRegion of TSS
5' UTR	5' untranslated region
3' UTR	3' untranslated region
Exon	Coding exon
Intron	Intronic region
Downstream	Within 3kb downstream
Distal Intergenic	Beyond gene regions

Related Skills

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

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