🍪ChIP-Seq Part2: Peak Calling & Annotation in ChIP-Seq Analysis🍪

Peak calling is a crucial step in ChIP-seq analysis to identify enriched genomic regions due to protein binding. Peak calling and annotation enhance ChIP-seq data analysis, providing insights into protein binding and gene regulation mechanisms The normR Bioconductor package, utilizing a binomial mixture model, is employed for peak detection and normalization across ChIP and Input samples.

🍱Types of ChIP-seq Experiments:

🦪Sharp Peaks:

Short regions, typically from transcription factors or localized histone modifications like H3K4me3

🥟Broad Peaks:

Wide genomic domains, often linked with histone modifications like H3K36me3.

🍛Mixed Signals:

A combination of sharp and broad regions, often from RNA Polymerase 2.

🍲Peak Calling for Sharp and Broad Regions:

🦪Sharp Peaks (CTCF):

ChIP vs. Input samples compared, and regions visualized for enrichment.

🥟Broad Peaks (H3K36me3):

Tiling window size adjusted for broad domains, with resulting signal visualized.

✂️Quality Control:

Peak authenticity is confirmed by checking read percentages in peaks and verifying alignment with known transcription factor binding motifs.

📍Peak Annotation:

To assign identified peaks to functional genomic regions, adding context to their biological significance.

📁Download Gene Models:

Human gene models are downloaded to reference genomic regions like exons, introns, and promoters.

📍Peak Annotation Process:

Disjoint Peak Regions: Each peak is treated independently.

📚Overlapping Annotations: Nearest functional annotation like promoter, exon, intron, is assigned.

🔗Hierarchical Prioritization: Overlapping annotations are resolved.

📝Summary Statistics: Distribution of peaks across genomic regions is calculated.

📌Annotation Function Application: The annotatePeaks() function is applied to CTCF and H3K36me3 peaks using the lapply() function.

🧮Combining and Visualizing Results: Results are combined using dplyr::bind_rows() and visualized as a bar plot showing H3K36me3 peaks in gene bodies and CTCF peaks near promoters.

Insights:

H3K36me3 peaks’ localization in gene bodies suggests a role in transcriptional regulation, while CTCF’s enrichment near promoters highlights its involvement in chromatin architecture and transcription regulation.

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