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#!/bin/bash -l
#PBS -N nfrnaseq_QC
#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'

nextflow run nf-core/rnaseq \
      -profile singularity \
      -r 3.12.0 \
      --input samplesheet.csv \
      --outdir results \
      --genome GRCh38 \
      --skip_trimming \
      --skip_alignment \
      --skip_pseudo_alignment

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. Version 3.12.0 allows running the pipeline to do quality assessment only, without any alignment, read counting or trimming. To execute that option, add the following flags to your nextflow run nf-core/rnaseq command: --skip_trimming, --skip_alignment and --skip_pseudo_alignment.

Submitting the job

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

Submit the run with this command

qsub launch.pbs

Monitoring the Run

You can use the command

qstat -u $USER

Alternatively, use the 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.

Assessing QC report

Evaluate the nucleotide distributions in the 5'-end and 3'-end of the sequenced reads (Read1 and Read 2). Define how many nucleotides should be trimmed from each region of the sequences. This will inform the parameter setting for Case 3 below.

Case 3: Run RNA-seq pipeline

Adjusting the Trim Galore options

When the initial trimming is done, verify if any more clipping needs to be done and run the nf-core/rnaseq pipeline that will perform all the steps. For example:

nextflow run nf-core/rnaseq --input samplesheet.csv \
        --outdir results \
        -r 3.12.0 \
        --genome GRCh38 \
        -profile singularity \
        --aligner star_salmon \
        --extra_trimgalore_args "--clip_r1 12 --clip_r2 12 --three_prime_clip_r1 2 --three_prime_clip_r2 2 "

Submitting the job

Once you have created the folder for the run, the samplesheet.csv 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

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