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.
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.
- Clone or download the pipeline code from the repository.
- Ensure that the
config.yamlfile is present in the main directory of the repository, along with the Snakefile.
- Create a new environment named "RNAseq" and install the dependencies from the
environment.yamlfile 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.
conda activate RNAseq
snakemake --cores <num_cores>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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