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"

• 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.

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

• The config/samples.csv file is an example sample sheet, modify it as needed so that

  • 1st column: sample names
  • 2nd column: fq1 file names (if using .bam as input, put the name of .fastq.gz files generated from .bam)
  • 3rd column: fq2 file names (if using .bam as input, put the name of .fastq.gz files generated from .bam)
  • 4th column: RNA-seq file format - choose between bam and fq (all inputs need to be in the same file format)

  Each column is separated by one comma. 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,bam
293T-TFE3-2,293T-TFE3-2_R1_001.fastq.gz,293T-TFE3-2_R2_001.fastq.gz,bam

Input files

• The RNAseq 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: Initiate a screen to run snakemake pipeline in the background without requiring terminal connection all the time

  screen -S snakemake-RNAseq
  

• Step 2: Change into the directory snakemake-RNAseq

  cd $HOME/snakemake-RNAseq
  

• Step 3: 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 4: 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 16 -o $HOME/snakemake-RNAseq/joblogs/ -e $HOME/snakemake-RNAseq/joblogs/"
  

  • This step might take long, depending on the sample sizes.

• Step 4: Detach the screen as needed

  • Press Ctrl+A and then D to detach the screen as needed. Now closing the terminal or losing connection to the cluster should not interrupt the snakemake pipeline.

  • Use screen -ls to see all detached screens.

  • Example:

    

    numbers like 27120 and 26844 are <SCREEN-ID> used to track the processes running in that screen

• Step 5 (Optional): Reattach to a screen

  screen -r <SCREEN-ID>
  

  • If the command execution in the screen is interrupted, users need to rerun Step 4 in the screen to generate all results expected.
  • Press Ctrl+A and then D to detach the screen as needed.

• Step 6: Delete the screen after done

  screen -S <SCREEN-ID> -X quit
  

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):
  • samtools: 1.9
  • 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