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Prior running the nf-core/sarek pipeline
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Running a test to verify the execution of the pipeline
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Running the sarek variant calling pipeline with a HapMap trio data
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with real data, we will first run a test with sample data to make sure the pipeline runs properly.
Work in the HPC |
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Before we start using the HPC, let’s start an interactive session:
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mkdir -p $HOME/workshop/sarek/scripts cp /work/training/sarek/scripts/* $HOME/workshop/sarek/scripts/ ls -l $HOME/workshop/sarek/scripts/ |
Line 1: The -p indicates create 'parental directories as required. Thus the line 1 command creates both /workshop/ and the subfolder /workshop/scripts/
Line 2: Copies all files from /work/datasets/workshop/scripts/ as noted by an asterisk to the newly created folder $HOME/workshop/scripts/
Copy public data to your $HOME
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mkdir -p $HOME/workshop/sarek/data/WES/trio mkdir -p $HOME/workshop/sarek/data/WES/liver cp /work/training/sarek/data/WES/trio/* $HOME/workshop/sarek/data/WES/trio cp /work/training/sarek/data/WES/liver/* $HOME/workshop/sarek/data/WES/liver |
Lines 1 -2: Command creates the folders to copy data
Line 3: Copies all files from /work/datasets/workshop/sarek/data/WES/trio folder as noted by an asterisk to newly created $HOME/workshop/sarek/data/WES/trio folder.
Line 4: Copies all files from /work/datasets/workshop/sarek/data/WES/liver folder as noted by an asterisk to newly created $HOME/workshop/sarek/data/WES/liver folder.
Create folders for running the nf-core/sarek pipeline
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mkdir -p $HOME/workshop/sarek mkdir $HOME/workshop/sarek/run1_test mkdir $HOME/workshop/sarek/run2_trio mkdir $HOME/workshop/sarek/run3_liver cd $HOME/workshop/ |
Lines 1-4: create sub-folders for each exercise
Line 5: change the directory to the folder “run1_test”
Line 6: print the current working directory
Exercise 1: Running a test with nf-core sample data
First, let’s assess the execution of the nf-core/rnaseq pipeline by running a test using sample data.
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#!/bin/bash -l #PBS -N nfsarek_run1_test #PBS -l walltime=48:00:00 #PBS -l select=1:ncpus=1:mem=5gb cd $PBS_O_WORKDIR NXF_OPTS='-Xms1g -Xmx4g' module load java #specify the nextflow version to use to run the workflow export NXF_VER=23.10.1 #run the sarek pipeline nextflow run nf-core/sarek \ -r 3.3.2 \ -profile test,singularity \ --outdir ./results |
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nextflow command: nextflow run
pipeline name: nf-core/sarek
pipeline version: -r 3.3.2
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:
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results/
├── csv
│ ├── markduplicates.csv
│ ├── markduplicates_no_table.csv
│ ├── recalibrated.csv
│ └── variantcalled.csv
├── multiqc
│ ├── multiqc_data
│ ├── multiqc_plots
│ └── multiqc_report.html
├── pipeline_info
│ ├── execution_report_2024-05-08_15-28-38.html
│ ├── execution_timeline_2024-05-08_15-28-38.html
│ ├── execution_trace_2024-05-08_15-28-38.txt
│ ├── params_2024-05-08_15-41-30.json
│ ├── pipeline_dag_2024-05-08_15-28-38.html
│ └── software_versions.yml
├── preprocessing
│ ├── markduplicates
│ ├── recalibrated
│ └── recal_table
├── reports
│ ├── bcftools
│ ├── fastqc
│ ├── markduplicates
│ ├── mosdepth
│ ├── samtools
│ └── vcftools
├── tabix
│ ├── genome.bed.gz
│ └── genome.bed.gz.tbi
└── variant_calling
└── strelka |
Exercise 2: Run nf-core/sarek using trio data
The pipeline requires preparing at least 2 files:
Metadata file (samplesheet.csv) thatspecifies the following information:
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patient,sample,lane,fastq_1,fastq_2 ID1,S1,L002,/full/path/to/ID1_S1_L002_R1_001.fastq.gz,/full/path/to/ID1_S1_L002_R2_001.fastq.gz |
PBS Pro script (launch_nf-core_sarek_trio.pbs) with instructions to run the pipeline
Create the metadata file (samplesheet.csv):
Change to the data folder directory:
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cp /work/training/sarek/scripts/create_samplesheet_nf-core_sarek.py $HOME/workshop/sarek/data/trio |
Note: you could replace ‘$HOME/workshop/sarek/data’ with “.” A dot indicates ‘current directory’ and will copy the file to the directory where you are currently located
Check help option on how to run the script:
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Copy the PBS Pro script for running the nf-core/sarek pipeline (launch_nf-core_sarek_trio.pbs)
Copy and paste the code below to the terminal:
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cp $HOME/workshop/sarek/data/WES/trio/samplesheet.csv $HOME/workshop/sarek/runs/run2_sarek_trio cp $HOME/workshop/sarek/scripts/launch_nf-core_sarek_trio.pbs $HOME/workshop/sarek/runs/run2_trio cd $HOME/workshop/sarek/runs/run2_trio |
Line 1: Copy the samplesheet.csv file generated above to the working directory
Line 2: copy the launch_nf-core_sarek_trio.pbs submission script to the working directory
Line 3: move to the working directory
View the content of the launch_nf-core_RNAseq_QC.pbs script:
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#!/bin/bash -l #PBS -N nfsarek_run2_trio #PBS -l walltime=48:00:00 #PBS -l select=1:ncpus=1:mem=5gb cd $PBS_O_WORKDIR NXF_OPTS='-Xms1g -Xmx4g' module load java #specify the nextflow version to use to run the workflow export NXF_VER=23.10.1 #run the sarek pipeline nextflow run nf-core/sarek \ -r 3.3.2 \ -profile singularity \ --genome GATK.GRCh38 \ --input samplesheet.csv \ --wes \ --outdir ./results \ --step mapping \ --tools haplotypecaller,snpeff,vep \ --snpeff_cache /work/training/sarek/NXF_SINGULARITY_CACHEDIR/snpeff_cache \ --vep_cache /work/training/sarek/NXF_SINGULARITY_CACHEDIR/vep_cache \ -resume |
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The above script will screen for germline (inherited) mutations using GATK’s haplotypecaller and then annotate the identified variants using snpeff and VEP.
Version 3.3.2 allows running the pipeline to do quality assessment only, without any alignment, read counting, or trimming.
The pipeline enables use to start at distinct stages, we are commencing from the start “--step mapping”
Submitting the job
Once you have created the folder for the run, the samplesheet.csv file, and launch.pbs, you are ready to submit the job to the HPC scheduler:
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Once the pipeline has finished running - Assess the QC report:
NOTE: To proceed, you need to be on QUT’s WiFi network or signed via VPN.
To browse the working folder in the HPC type in the file finder:
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Assess QC reports (FastQC and MultiQC) to define how many nucleotides should be trimmed from the 5'-end and/or 3-end regions of the FASTQ reads (see Case 3 below).
Exercise 3: Healthy vs. disease liver samples
Copy and paste the code below to the terminal:
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cp $HOME/workshop/data/samplesheet.csv $HOME/workshop/RNAseq/run3_RNAseq cd $HOME/workshop/RNAseq/run3_RNAseq pwd |
Line 1: Copy the samplesheet.csv file to the working directory
Line 2: move to the working directory
Line 3: print working directory → verify the folder location
Copy the PBS Pro script to run the nf-core/rnaseq pipeline:
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