RNA-seq Analysis Pipeline

A modular, scalable RNA-seq pipeline for quantifying and analyzing gene expression data. Built using Nextflow, Salmon, and DESeq2, with reproducibility and clarity in mind.

🧪 Features

• Pseudo-alignment using Salmon
• Quantification to transcript- and gene-level
• Differential expression analysis with DESeq2
• Modular structure using Nextflow
• Easy setup via Conda environments
• Generates QC reports, PCA plots, and volcano plots

🔧 Requirements

• Nextflow
• Conda / Mamba
• Git
• Internet access (for pulling reference or demo data)

📁 Repo Structure

| Folder | Description | |----------------|-----------------------------------------------| | data/ | Input FASTQ files or sample accessions | | results/ | All output files: quant, stats, plots | | workflow/ | Nextflow scripts (main.nf, modules, etc.) | | scripts/ | Custom R and Python scripts for plotting, QC | | envs/ | Conda YAMLs for reproducibility | | config/ | Sample sheet, pipeline settings |

▶️ Quick Start

```bash

clone the repo

git clone https://github.com/Cslaer2025/rnaseqpipeline.git cd rnaseqpipeline

run pipeline (demo mode)

nextflow run workflow/main.nf -profile conda

rnaseqpipeline

Modular RNA-seq pipeline using Nextflow, Salmon, and DESeq2

f6ec3f3659f13f73b855f193dcad2c5ce5b0522a

🚀 How to Run the Pipeline

Make sure you have Nextflow and Conda installed.

📁 Project Structure

rnaseqpipeline/ ├── config/ │ └── samplesheet.csv # Sample metadata ├── data/ │ └── transcriptome.fa # Reference transcriptome ├── scripts/ │ └── deseq2.R # DESeq2 analysis script ├── results/ ├── main.nf # Nextflow workflow └── .gitignore

▶️ Command to Run

```bash nextflow run main.nf -profile standard

By default, this will:

Build a Salmon index

Run quantification on paired-end reads

Perform DESeq2 differential expression analysis

Output: results/deseq2results.csv 🧪 Sample Sheet Format sampleid condition fastq1 fastq2 sample1 control data/sample1R1.fastq.gz data/sample1R2.fastq.gz sample2 treatment data/sample2R1.fastq.gz data/sample2R2.fastq.gz yaml Copy Edit

🔹 3. Save and exit nano:

• Press Ctrl + O → then Enter to save
• Press Ctrl + X to exit

✅ Git Commands to Commit and Push the README Update

```bash cd ~/rnaseqpipeline git add README.md git commit -m "Add usage instructions and project layout to README" git push