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Prepared by the eResearch Office, QUT.

This page provides a guide to QUT users on how to install and run the nextflow nf-core/rnaseq workflow on the HPC.

Pre-requisites

Install Nextflow

The nf-core/rnaseq workflow requires Nextflow to be installed in your account on the HPC. Find details on how to install and test Nextflow here. Prepare a nextflow.config file and run a PBS pro submission script for Nextflow pipelines.

Additional information is available here: https://nf-co.re/usage/installation

Additional details on the workflow can be found at:

Overview: https://nf-co.re/rnaseq/3.10.1

Usage: https://nf-co.re/rnaseq/3.10.1/usage

GitHub: https://github.com/nf-core/rnaseq

Pipeline Summary

Getting Started

Download and run the workflow using minimal data provided by nf-core/rnaseq. We recommend using singularity as the profile for QUT’s HPC. Another profile option can be ‘conda.’ Note: the profile option ‘docker’ is unavailable on the HPC.

nextflow run nf-core/rnaseq -profile test,singularity --outdir results -r 3.10.1

Preparing a ‘samplesheet.csv’ file

Prepare a sample sheet file that specifies the input files to be used. To do this, we use an nf-core script to generate the ‘samplesheet.csv’ file as follows:

#load python 3.7
module load phyton3.7

#download script and make executable
wget -L https://raw.githubusercontent.com/nf-core/rnaseq/master/bin/fastq_dir_to_samplesheet.py
chmod +x fastq_dir_to_samplesheet.py

#generate the samplesheet.csv file
fastq_dir_to_samplesheet.py /path/to/directory/containing/fastq_files/ samplesheet.csv \
    --strandedness unstranded \
    --read1_extension R1.fastq.gz \
    --read2_extension R2.fastq.gz

Example index.csv (Version 3.10.1):

sample,fastq_1,fastq_2,strandedness
control_1,/path/to/fastq/control-1_R1.fastq.gz,/path/to/fastq/control-1_R2.fastq.gz,unstranded
control_2,/path/to/fastq/control-2_R1.fastq.gz,/path/to/fastq/control-2_R2.fastq.gz,unstranded
control_3,/path/to/fastq/control-3_R1.fastq.gz,/path/to/fastq/control-3_R2.fastq.gz,unstranded
infected_1,/path/to/fastq/infected-1_R1.fastq.gz,/path/to/fastq/infected-1_R2.fastq.gz,unstranded
infected_2,/path/to/fastq/infected-1_R1.fastq.gz,/path/to/fastq/infected-2_R2.fastq.gz,unstranded
infected_3,/path/to/fastq/infected-1_R1.fastq.gz,/path/to/fastq/infected-3_R2.fastq.gz,unstranded

Preparing to run on the HPC

To run this on the HPC a PBS submission script needs to be created using a text editor. For example, create a file called launch.pbs using a text editor of choice (i.e., vi or nano) and then copy and paste the code below:

#!/bin/bash -l
#PBS -N nfrna2
#PBS -l select=1:ncpus=2:mem=4gb
#PBS -l walltime=24:00:00

cd $PBS_O_WORKDIR
module load java
NXF_OPTS='-Xms1g -Xmx4g'

nextflow run nf-core/rnaseq -profile singularity -r 3.10.1 --input samplesheet.csv --genome GRCm38 --aligner star_salmon

We recommend running the nextflow nf-core/rnaseq pipeline once and then assessing the fastqc results folder to assess if sequence biases are present in the 5'-end and 3'-end ends of the sequences. Then, we can use the PBS script below to tell the pipeline to remove a defined number of bases from the 5'-end (--clip_r1 or --clip_r2) or 3'-end (--three_prime_clip_r1 or --three_prime_clip_r2). Also, we can specify to remove ribosomal RNA as these sets of sequences are non-informative.

#!/bin/bash -l
#PBS -N nfrna2
#PBS -l select=1:ncpus=2:mem=4gb
#PBS -l walltime=24:00:00

#work on current directory (folder)
cd $PBS_O_WORKDIR

#load java and set up memory settings to run nextflow
module load java
NXF_OPTS='-Xms1g -Xmx4g'

#run the rnaseq pipeline
nextflow run nf-core/rnaseq --input samplesheet.csv \
        --outdir results \
        -r 3.10.1 \
        --genome GRCh38 \
        -profile singularity \
        --aligner star_rsem \
        --clip_r1 10 \
        --clip_r2 10 \
        --three_prime_clip_r1 2 \
        --three_prime_clip_r2 2

Submitting the job

Once you have created the folder for the run, the input.tsv file, nextflow.config, and launch.pbs, you are ready to submit.

Submit the run with this command (On Lyra)

qsub launch.pbs

Monitoring the Run

You can use the command

qstat -u $USER

Alternatively, use the following command:

qjobs

To check on the jobs, you are running. Nextflow will launch additional jobs during the run.

You can also check the .nextflow.log file for details on what is going on.

Finally, if you have configured the connection to the NFTower, you can log on and check your run.

Troubleshooting

  1. I have been using version 3.3. and now, when I run version 3.10.1, I get an error that the asset is corrupted. What should I do?

#delete the existing assests associated with the RNAseq pipeline:
cd ~/.nextflow/assets/nf-core
rm -r rnaseq/

#run again a test with the new version that you are testing, for example, version 3.10.1. See details on how to run a test above (under 'Getting Started')

Add output folders/files

Running the pipeline using custom data

Example of a typical command to run an RNA-seq analysis for mouse samples: 

nextflow run nf-core/rnaseq --input samplesheet.csv \
        --outdir results \
        -r 3.10.1 \
        --genome GRCh38 \
        -profile singularity \
        --aligner star_rsem \
        --clip_r1 10 \
        --clip_r2 10 \
        --three_prime_clip_r1 2 \
        --three_prime_clip_r2 2

Note, if the running was interrupted or you did not complete a particular step, or you want to modify a parameter for a particular step, instead of re-running all processes again, nextflow enables you to “-resume” the workflow.

nextflow run nf-core/rnaseq --input samplesheet.csv \
        --outdir results \
        -r 3.10.1 \
        --genome GRCh38 \
        -profile singularity \
        --aligner star_rsem \
        --clip_r1 10 \
        --clip_r2 10 \
        --three_prime_clip_r1 2 \
        --three_prime_clip_r2 2 \
      -resume
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