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snakemake-RNAseq

This pipeline performs a standard RNAseq analysis, including fastQC, STAR alignment, RSEM & salmon quantification.

Directory structure

.
├── config            # Contains sample sheet (samples.tsv) and config file (config.yaml)
├── rules             # Snakemake rules
├── scripts           # Scripts to run each step of RNAseq
├── README.md
└── Snakefile         # Snakemake workflow

Installation

Option 1: Download the package

  • Choose "Download ZIP"
Screenshot 2024-07-09 at 3 37 12 PM
  • The folder named snakemake-RNAseq-main is downloaded.
  • Transfer the folder to users' working directory on argos. 
    scp -r path/to/snakemake-RNAseq-main <USER_ID>@argos-stgw2.dfci.harvard.edu:/mnt/storage/home/<USER_ID>/
  • Log onto argos: 
    ssh USER_ID@argos.dfci.harvard.edu
  • Change the name of the folder to snakemake-RNAseq 
    mv $HOME/snakemake-RNAseq-main $HOME/snakemake-RNAseq

Option 2: git clone

Clone the pipeline using the following command

git clone https://github.com/SViswanathanLab/snakemake-RNAseq.git

Make sure there is a folder named snakemake-RNAseq in users' working directory.

Usage

Instructions for preparing sample sheet

  • Paired-end data is assumed.

  • 4 types of RNAseq data formats are accommodated: .fastq.gz, .fq.gz, .fastq, .fq

  • The config/samples.tsv file is an example sample sheet.

  • Users should modify config/samples.tsv to have the first column consisting of sample names, the second column consisting of fq1 file names, and the third column consisting of fq2 file names. Each column is separated by one space.

  • The fq1 & fq2 file names must contain the full sample names.

    For example:

293T-TFE3-1 293T-TFE3-1_R1_001.fastq.gz 293T-TFE3-1_R2_001.fastq.gz
293T-TFE3-2 293T-TFE3-2_R1_001.fastq.gz 293T-TFE3-2_R2_001.fastq.gz

Input files

  • The fq1 & fq2 files for analysis should be copied to data. 
    cp -r path/to/<fq_files_folder> $HOME/snakemake-RNAseq/
  • Users should change the name of the folder containing fq files into data. 
    mv $HOME/snakemake-RNAseq/<fq_files_folder> $HOME/snakemake-RNAseq/data

Run snakemake

  • Step 1: Change into the directory snakemake-RNAseq

    cd $HOME/snakemake-RNAseq

  • Step 2: Activate the environment with snakemake installed & install plugin for cluster submission

    source /mnt/storage/apps/Mambaforge-23.1.0-1/etc/profile.d/conda.sh
conda activate snakemake

pip install snakemake-executor-plugin-cluster-generic

  • Step 3: Run snakemake pipeline

    snakemake --unlock
snakemake --executor cluster-generic --jobs 50 --latency-wait 60 --cluster-generic-submit-cmd "qsub -l h_vmem=256G, -pe pvm 32 -o $HOME/snakemake-RNAseq/joblogs/ -e $HOME/snakemake-RNAseq/joblogs/"
    • This step might take long, depending on the sample sizes.
    • If the command execution is interrupted, users need to rerun Step 3 to generate all results expected.

  • Step 4: Deactivate the environment as needed

    conda deactivate

Output

  • The results are saved in the folder snakemake-RNAseq/results.
    • snakemake-RNAseq/results/fastqc_results contains the fastqc results.
    • snakemake-RNAseq/results/STAR_results contains the STAR results, and each subfolder is named by the sample name.
    • snakemake-RNAseq/results/salmon_results contains the salmon results, and each subfolder is named by the sample name.
    • snakemake-RNAseq/results/star_wide_countMatrix.csv and snakemake-RNAseq/results/star_wide_countMatrix.Rds contain the star count matrix, with genes as rows and samples as columns, in both .csv and .Rds format.
    • snakemake-RNAseq/results/salmon_wide_TPM_Matrix.csv and snakemake-RNAseq/results/salmon_wide_TPM_Matrix.Rds contain the salmon TPM matrix, with genes as rows and samples as columns, in both .csv and .Rds format.
    • snakemake-RNAseq/results/rsem_geneLevel_wide_TPM_Matrix.csv and snakemake-RNAseq/results/rsem_geneLevel_wide_TPM_Matrix.Rds contain the rsem gene-level TPM matrix, with genes as rows and samples as columns, in both .csv and .Rds format.
    • snakemake-RNAseq/results/rsem_isoformLevel_wide_TPM_Matrix.csv and snakemake-RNAseq/results/rsem_isoformLevel_wide_TPM_Matrix.Rds contain the rsem transcript-level TPM matrix, with genes as rows and samples as columns, in both .csv and .Rds format.
  • snakemake-RNAseq/rsem_ref contains the reference files generated for rsem quantification.
  • snakemake-RNAseq/logs contains the log files for running each step of this analysis, for debugging.
  • snakemake-RNAseq/joblogs contains the log files for job submission, for debugging.

config

config.yaml contains the information about versions of each tool used, reference file paths

  • Module versions (latest ones globally installed on argos):
    • fastqc: 0.11.7
    • star: 2.7.10a
    • rsem: 1.3.1
    • salmon: 1.10.1
    • snakemake: 8.15.2
    • snakemake-executor-plugin-cluster-generic: 1.0.9
  • Reference file directory: /mnt/storage/labs/sviswanathan/snakemake_RNAseq_2024/Human_genome_2024/
    • Can be modified as needed