RNA-seq pipeline

This repository contains a reproducibile RNA-seq data processing pipeline implemented using Snakemake. The pipeline automates the essential steps of an RNA-seq analysis, including Quality control (FastQC, FastQ Screen, Trimmomatic, Alignment (STAR), Quantification (FeatureCount), and BedGraph generation.

Prerequisites

Before running the pipeline, ensure that you have the following installed:

• Conda or Miniconda
• Snakemake v8.14.0

Getting started

1. Clone the repository

git clone https://github.com/krupas26/RNA-seq_pipeline.git
cd RNA-seq_pipeline

2. Create a Conda Environment

Use the provided env_rnaseq.yml to create the environment with all the required dependencies:

conda env create --file env_rnaseq.yml

Once the environment is created, activate it:

conda activate rnaseq

3. Configure the Pipeline

To keep sensitive data such as the file paths private, this project uses a configuration file that is excluded from version control. You can create a config.yml file in your favorite text editor and update the placeholder values with the paths and settings specific to your environment.

Example snippet of config.yml file

 workflow-profile: "/path/to/your/workflow/profile"
 metadata: "metadata.xlsx"
 chrom_sizes: "/path/to/chrom_sizes"
 adapter: "/path/to/adapter/file"
 fastq_dir: "/path/to/fastq/files"
 sample_column: "SampleID"
 samples_list: null
 run_id: "<your experiement identifier>"

4. Running the pipeline

Run the pipeline with Snakemake:

snakemake -s rnaseq_pipeline.smk --configfile <config.yml> --cores <number-of-cores>

To submit jobs on cluster as sbatch jobs

Create a generic profile_config.yml

  executor: "cluster-generic"
  cluster-generic-submit-cmd: "sbatch --output=logs/{rule}_{wildcards}_%j.out --error=logs/{rule}_{wildcards}_%j.err --mem={resources.mem_mb}"
  jobname: "{wildcards}_{rule}_{jobid}"
  default-resources: [mem_mb=4000, time='02:00:00', threads=1]
  use-conda: true
  jobs: 100
  rerun-incomplete: true
  latency-wait: 60

Then run the pipeline with Snakemake as:

snakemake -s rnaseq_pipeline.smk --profile <profile_config.yml> --configfile <config.yml> --cores <number-of-cores>