🧬 End-to-End Automated RNA-Seq Pipeline & Differential Gene Expression Analysis

(Type 2 Diabetes Mellitus vs. Control Case Study)

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📌 Executive Summary

This repository contains a robust, scalable, and fully automated RNA-Seq data processing pipeline built using Nextflow (DSL2), coupled with downstream Differential Expression (DGE) and Functional Pathway Enrichment Analysis in R/Bioconductor.

The pipeline was executed on paired-end transcriptomic datasets comparing Type 2 Diabetes (T2D) patients against healthy controls, successfully identifying key biological pathways, dysregulated genes, and mitochondrial metabolic disruptions associated with T2D progression.

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🛠️ Pipeline Architecture & Workflow

The workflow integrates industry-standard bioinformatics utilities in a modular Nextflow DAG (Directed Acyclic Graph) architecture with checkpoint caching (-resume enabled):

       ┌────────────────────────┐
       │   Raw Paired-End FASTQ │
       └───────────┬────────────┘
                   │
                   ▼
       ┌────────────────────────┐
       │     FastQC (QC)        │
       └───────────┬────────────┘
                   │
                   ▼
       ┌────────────────────────┐
       │     fastp (Trimming)   │
       └───────────┬────────────┘
                   │
                   ▼
       ┌────────────────────────┐
       │ HISAT2 (Genome Align)  │ ◄─── GRCh38 Genome Index
       └───────────┬────────────┘
                   │
                   ▼
       ┌────────────────────────┐
       │ featureCounts (Matrix) │ ◄─── Ensembl GTF Annotation
       └───────────┬────────────┘
                   │
                   ▼
       ┌────────────────────────┐
       │ DESeq2 & Enrichment    │ ───► Volcano Plots, GO & KEGG Pathways
       └────────────────────────┘