Exercise 3: Run nf-core/sarek using a liver samples
What is NAFLD?
Non-alcoholic fatty liver disease (NAFLD) is a condition characterized by an accumulation of fat in liver cells (hepatocytes). Excess fat in the liver can lead to significant damage over the years. There are two types of NAFLD:
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STEP1: 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 $HOME/workshop/sarek/runs/run3_liver Al |
Note: you could replace ‘$HOME/workshop/sarek/runs/liver’ 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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cp $HOME/workshop/sarek/scripts/launch_nf-core_sarek_liver.pbs $HOME/workshop/sarek/runs/run3_liver cd $HOME/workshop/sarek/runs/run3_liver |
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_liver #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 results as follows:
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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Evaluate the nucleotide distributions in the 5'-end and 3'-end of the sequenced reads (Read1 and Read2). Look into the “MultiQC” folder and open the provided HTML report.
During execution of the workflow two output folders are generated:
work - where all intermediate results and tasks are run
results - where all final results for all stages of the pipeline are copied
Let’s browse the results of the pipeline:
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results/ ├── annotation │ └── haplotypecaller ├── csv │ ├── markduplicates.csv │ ├── markduplicates_no_table.csv │ ├── recalibrated.csv │ └── variantcalled.csv ├── multiqc │ ├── multiqc_data │ ├── multiqc_plots │ └── multiqc_report.html ├── pipeline_info │ ├── execution_report_2024-05-11_20-17-13.html │ ├── execution_timeline_2024-05-11_20-17-13.html │ ├── execution_trace_2024-05-11_20-17-13.txt │ ├── params_2024-05-11_23-31-28.json │ ├── pipeline_dag_2024-05-11_20-17-13.html │ └── software_versions.yml ├── preprocessing │ ├── markduplicates │ ├── recalibrated │ └── recal_table ├── reports │ ├── bcftools │ ├── EnsemblVEP │ ├── fastp │ ├── fastqc │ ├── markduplicates │ ├── mosdepth │ ├── samtools │ ├── snpeff │ └── vcftools ├── tabix │ ├── wgs_calling_regions_noseconds.hg38.bed.gz │ └── wgs_calling_regions_noseconds.hg38.bed.gz.tbi └── variant_calling └── haplotypecaller |