RNA-Seq Analysis Pipeline with READemption and Docker

This project provides an automated pipeline for RNA-Seq analysis using READemption and Docker. It handles everything from environment setup and data downloading to differential gene expression analysis and visualization.

Project Structure

.
├── docker_installation_linux.sh  # Script to install Docker on Linux Mint/Ubuntu
├── rnaseq.sh                     # Main analysis pipeline script
└── READemption_analysis/         # Output directory (created during runtime)
    ├── input/                    # Raw reads and reference sequences
    └── output/                   # Analysis results and plots

Manuscript

The RNA-seq data and analysis pipeline in this repository are associated with the accompanying manuscript:

• File: manuscript.pdf
• Context: This pipeline replicates/tests the data analysis presented in the study. Please refer to the manuscript for detailed biological context and experimental design.

Prerequisites

• OS: Linux (Tested on Linux Mint 22.3 / Ubuntu Noble)
• Permissions: Sudo privileges are required for installation and running Docker.

Installation

1. Install Docker

If you haven't installed Docker yet, use the provided setup script. This script installs Docker Engine, CLI, and necessary plugins.

chmod +x docker_installation_linux.sh
./docker_installation_linux.sh

Note: You may need to log out and log back in for group permission changes to take effect.

Usage

Run the Analysis Pipeline

The rnaseq.sh script automates the entire analytical workflow.

chmod +x rnaseq.sh
./rnaseq.sh

Workflow Details

The pipeline performs the following steps automatically:

1. Setup: Creates the project directory structure for READemption.
2. Data Acquisition:
   • Downloads Methanosarcina mazei reference genome (FASTA) and annotations (GFF) from NCBI.
   • Downloads raw RNA-seq reads (SRR4018514 - SRR4018517) using sra-tools via Docker.
3. Preprocessing:
   • Compresses FASTQ files.
   • Renames files to sample names and replicates (single ended reads) (e.g., wt_R1, mut_R1).
4. Analysis (via READemption Docker container):
   • Alignment: Maps reads to the reference genome using segemehl.
   • Coverage: Calculates nucleotide-wise coverage.
   • Gene Quantification: Counts reads per feature (CDS, tRNA, rRNA).
   • Differential Expression: Performs DE analysis using DESeq2 comparing Mutant (mut) vs Wild Type (wt).
5. Visualization: Generates plots for alignment statistics, gene quantification, and differential expression.

Docker Images Used

• SRA Tools: ncbi/sra-tools (for downloading reads)
• READemption: tillsauerwein/reademption:latest (for analysis)

Output

Results will be located in the READemption_analysis folder:

• Coverage tracks: output/coverage/
• Gene counts: output/gene_quanti/
• DESeq2 results: output/deseq/
• Plots: output/viz_align/, output/viz_gene_quanti/, output/viz_deseq/

References

• READemption Documentation
• Docker Documentation