TCGA-BRCA Differential Expression Analysis (PAM50 Subtypes) Exploring genetic differences between Basal and Luminal A subtypes The primary objective is to identify and characterize the molecular differences, including differentially expressed genes (DEGs) and enriched biological pathways, between the aggressive Basal-like and the more common Luminal A (LumA) molecular subtypes, using the PAM50 classification system.

This repository contains the R script used to perform differential genetic expression analysis and functional enrichment analysis using publicly available data from the Cancer Genome Atlas Project.

The primary script, tcga_brca_de_analysis.R executes the following steps

1. Data Acquisition: Using the TCGABiolinks package to query and download gene counts and associated clinical metadata for the TCGA BRCA dataset
2. Data Preparation: Filters samples based on the availability of the paper_BRCA_Subtype_PAM50 annotation and removes genes with low overall counts.
3. Differential Expression: Performs Differential Genetic Expression Analysis (DGEA) using the DESeq2 package, comparing the Basal-like subtype against the Luminal A (LumA) subtype.
4. Gene Annotation: Maps Ensembl Gene IDs to HUGO Gene Symbols using the org.Hs.eg.db annotation package.
5. Visualization: Generates a Volcano Plot and a Heatmap to visualize the differentially expressed genes (DEGs).
6. Functional Enrichment: Performs Gene Ontology (GO) Biological Process and KEGG Pathway enrichment analysis on the significant DEGs using clusterProfiler.

Prerequisites and Installation The script requires a combination of Bioconductor and CRAN packages.

R Version Tested with R version 4.5.2.

To save time and prevent redownload of the dataset anytime the dataset is run, the TCGA data was saved locally on the device to enable faster loading on next analysis using this: (save(data, file = "data/TCGA_BRCA_SE.rda")

Expected Outputs The script generates several high-quality plots essential for interpreting the differential expression results. These plots are saved in the plots/ directory.

1. Volcano Plot Visualizes the significance ($\text{-Log}_{10}$ adjusted p-value) versus the magnitude ($\text{Log}_2$ Fold Change) of all expressed genes in the Basal vs. LumA contrast. File: plots/volcano_plot_basal_vs_luma.png Cutoffs: |Log2FC| > 1.5 and p-adjusted < 0.05.

2. Heatmap Displays the expression of the top 50 differentially expressed genes across samples, clustered by gene expression similarity. The top annotation bar shows the PAM50 subtype for each sample. File: plots/heatmap_top50_basal_vs_luma.png Data: Variance-stabilizing transformed (vst) counts, scaled by row.

3. Functional Enrichment Plots

A. GO Biological Process Dot Plot

File: plots/GO_dotplot_basal_vs_luma.png

Shows the top 15 enriched Gene Ontology Biological Process terms.

B. KEGG Pathway Dot Plot

File: plots/KEGG_dotplot_basal_vs_luma.png

Shows the top 15 enriched KEGG pathways.

C. Combined Summary Bubble Plot (GO & KEGG)

File: plots/combined_enrichment_basal_vs_luma.png

Combines the top 10 most significant terms from both GO-BP and KEGG into a single bubble plot for easy comparison. The bubble size represents the number of genes found (Count), and the position represents the Gene Ratio (proportion of DEGs in the pathway).