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Pre-requisites

• Nextflow installed → If not see: 1. Getting started with Nextflow Ask for help if you need assistance.

Overview:

• Create working directory folders for hands-on training exercises

• Copy data and scripts for today’s session

• (Optional ) run a test to verify Nextflow is properly installed and to assess the execution of the nf-core/rnaseq pipeline with example data provided by developers.

Before we start using the HPC, let’s start an interactive session:

qsub -I -S /bin/bash -l walltime=10:00:00 -l select=1:ncpus=12:mem=4gb

Get a copy of the scripts to be used in this module

Use the terminal to log into the HPC and create a /RNAseq/ folder to run the nf-core/rnaseq pipeline. For example:

mkdir -p

• where:

  • ‘walltime’ is amount of time requested to run the interactive session

  • ‘cpus’ number of CPUs to be used by the interactive session

  • ‘mem’ amount of memory assigned to the interactive session

Create working directories

Let’s create the working directories for today’s session:

mkdir -p $HOME/workshop/2024-2/session4_RNAseq
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/scripts
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/scripts/local
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/data
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/data/mouse
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/data/human
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/data/mydata
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/runs
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/runs/run1_QC
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/runs/run2_RNAseq
mkdir -p $HOME/workshop/2024-2/session4_RNAseq/runs/run3_RNAseq_T2T

The above code creates the following folder structure:

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Get a copy of the scripts

Now let’s copy the scripts necessary for today’s session:

cp /work/training/2024/rnaseq/scripts/* $HOME/workshop/2024-2/session4_RNAseq/scripts
ls -l $HOME/workshop/2024-2/rnaseqsession4_RNAseq/scripts
cp /work/training/2024/rnaseq/scripts/local/* $HOME/workshop/2024-2/rnaseqsession4_RNAseq/scripts/local
ls -l $HOME/workshop/2024-2/rnaseqsession4_RNAseq/scripts/local

• Line 1: The -p indicates create 'parental directories as required. Thus the line 1 command creates both /workshop/ and the subfolder /workshopCopies all files from /work/datasets/workshop/scripts/ as noted by an asterisk to the newly created folder $HOME/workshop/2024-2/session4_RNAseq/scripts/

• Line 2: List the files copied to the ‘script’ folder

• Line 3: Copies all files from /work/datasets/workshop/scripts/local as noted by an asterisk to the newly created folder $HOME/workshop/2024-2/session4_RNAseq/scripts/local

• Line 4: List the files copied to the ‘script’ folder

Copy public data to your $HOMEworking folder

mkdir -pcp /work/training/2024/rnaseq/data/mouse/* $HOME/workshop/2024-2/session4_RNAseq/data/mouse
ls -l $HOME/workshop/2024-2/rnaseqsession4_RNAseq/data/mouse
cp /work/training/2024/rnaseq/data/human/* $HOME/workshop/2024-2/rnaseqsession4_RNAseq/data/human
# list the content of thels -l $HOME/workshop/2024-2/rnaseqsession4_RNAseq/data/ human

• Line 1: The first command creates the folder /scripts/Copies mouse RNAseq folder to created ~/data/mouse folder

• Line 2: Copies all files from /work/datasets/workshop/scripts/ folder as noted by an asterisk to newly created $HOME/workshop/scripts/ folder

• Line 3: a quick challenge - see the previous section for hints

Create a folder for running the nf-RNA-seq pipeline

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• List the files copied to the ‘data’ folder

• Line 3: Copies human RNAseq folder to created ~/data/human folder

• Line 4: List the files copied to the ‘data’ folder

(Optional ): Running a test with nf-core sample data

First, let’s assess the execution of the nf-core/rnaseq pipeline . For exampleby running a test using sample data.

Create a folder for running the pipeline test:

mkdir -p $HOME/workshop/2024-2/rnaseqsession4_RNAseq/runs
mkdir $HOME/workshop/2024/rnaseq/runs/run1/run0_test
mkdircd $HOME/workshop/2024-2/rnaseqsession4_RNAseq/runs/run2_QC
mkdirrun0_test

• Line 1: Create folder using the make directory “mkdir” command. We use the flap -p (parental) to create parental folders if not yet created.

• Line 2: Move to the newly created folder.

Copy the launch_nf-core_RNAseq_test.pbs from the ‘scripts’ folder to the current folder

cp $HOME/workshop/2024-2/rnaseqsession4_RNAseq/runs/run3scripts/launch_nf-core_RNAseq
cd $HOME/workshop/2024/rnaseq/runs

...

Lines 1-4: create sub-folders for each exercise

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Line 5: change the directory to the folder “run1_test”

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_test.pbs .

View the content of the script as follows:

cat launch_nf-core_RNAseq_test.pbs

...

• nextflow command: nextflow run

• pipeline name: nf-core/rnaseq

• pipeline version (revision): -r 3.14.0

• container type and sample data: -profile test,singularity

• output directory: --outdir results

Submitting the job

Submit the test job to the HPC cluster as follows:

qsub launch_nf-core_RNAseq_test.pbs

Monitoring the Run

qjobs