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Scalable RNAseq pipeline in Snakemake

This Snakemake pipeline processes and analyzes RNA sequencing data using various tools such as fastp, hisat2, samtools, fastqc, and htseq-count. The pipeline is configured using the config.yaml file, which contains the necessary parameters for running the pipeline. If more than 3 samples per group are provided the pipeline will also automatically run differential gene expression analysis using DESeq2.

Prerequisites

Make sure you have Mamba installed on your system. If you don't have Mamba installed, you can follow the instructions at mamba.io to install Mamba.

Pipeline Setup

  1. Clone or download the pipeline code from the repository.
  2. Ensure that the config.yaml file is present in the main directory of the repository, along with the Snakefile.

Creating the Conda Environment

  1. Create a new environment named "RNAseq" and install the dependencies from the environment.yaml file by running the following command:
    mamba env create -f envs/RNAseq.yaml
    

This will create a new environment called "RNAseq" and install all the required dependencies.

Activating the Conda Environment and run the pipeline

conda activate RNAseq
snakemake --cores <num_cores>

Docker Integration

Prefer Docker? Utilize our Dockerfile to build the image and run the pipeline within a Docker container. The built image includes all dependencies and the GRCh38 reference genome. The configfile can be downaloded from the repository. If using Docker the only paramenters to modify in the config file are the samples, computing_threads, and tertiary.

Build and run the pipeline using Docker:

docker build -t rnaseq .

docker run \
  -v /path/to/config.yaml:/app/config.yaml \
  -v /path/to/raw_data/:/data \
  -v /path/to/results/:/results \
  rnaseq \
  conda run -n RNAseq snakemake --cores 20


Output directory structure:

output/
├── alignment/
│   └── hisat2/
│       ├── simulated1.bam
│       ├── simulated1.bam.bai
│       ├── simulated1.sam
│       ├── simulated2.bam
│       ├── simulated2.bam.bai
│       └── simulated2.sam
├── counts/
│   ├── simulated1_count.txt
│   └── simulated2_count.txt
├── qc/
│   ├── fastp/
│   │   ├── simulated1_R1_fastp.fastq.gz
│   │   ├── simulated1_R2_fastp.fastq.gz
│   │   ├── simulated2_R1_fastp.fastq.gz
│   │   └── simulated2_R2_fastp.fastq.gz
│   ├── fastqc/
│   │   ├── simulated1_fastqc.html
│   │   └── simulated2_fastqc.html
│   └── qualimap/
│       ├── simulated1_qualimap.html
│       └── simulated2_qualimap.html
├── logs/
│   ├── simulated1.fastp.log
│   ├── simulated1.Hisat2.log
│   ├── simulated1.samtools_sort.log
│   ├── simulated1_fastqc.log
│   ├── simulated2.fastp.log
│   ├── simulated2.Hisat2.log
│   ├── simulated2.samtools_sort.log
│   └── simulated2_fastqc.log
├── benchmarks/
│   ├── fastp/
│   │   ├── simulated1.tsv
│   │   └── simulated2.tsv
│   ├── Hisat2/
│   │   ├── Hisat2_index.tsv
│   │   ├── simulated1.tsv
│   │   └── simulated2.tsv
│   └── samtools_sort/
├── dex/
│   ├── DE_analysis.csv
│   ├── Normalized_counts.csv
│   ├── Heatmap.pdf
│   └── Volcano_Plot.pdf
└── multiqc/
    └── multiqc_report.html

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