Exercise 3 - liver case study
- Roberto Barrero Gumiel
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:
Non-alcoholic fatty liver (NAFL)
Non-alcoholic steatohepatitis (NASH)
NAFL (Non-alcoholic fatty liver) - It is a benign condition and in this fat is accumulated in the liver that can cause inflammation of liver but not a progressive damage or complications of the liver. This may result in abdominal discomfort or pain due to an enlarged liver (hepatomegaly).
NASH (Non-alcoholic steatohepatitis) – It is a malignant condition and in this fat is accumulated in the liver can cause scarring of liver, liver fibrosis and liver damage further it can cause cirrhosis of liver (also called as end-stage liver disease), further cirrhosis of the liver may lead to liver cancer.
Location of raw data:
/work/training/sarek/data/WES/liver├── liver
│ ├── Control1_C1_L001_R1_001.fastq.gz
│ ├── Control1_C1_L001_R2_001.fastq.gz
│ ├── Control2_C2_L001_R1_001.fastq.gz
│ ├── Control2_C2_L001_R2_001.fastq.gz
│ ├── Control3_C3_L001_R1_001.fastq.gz
│ ├── Control3_C3_L001_R2_001.fastq.gz
│ ├── Control4_C4_L001_R1_001.fastq.gz
│ ├── Control4_C4_L001_R2_001.fastq.gz
│ ├── NAFLD1_P1_L001_R1_001.fastq.gz
│ ├── NAFLD1_P1_L001_R2_001.fastq.gz
│ ├── NAFLD2_P2_L001_R1_001.fastq.gz
│ ├── NAFLD2_P2_L001_R2_001.fastq.gz
│ ├── NAFLD3_P3_L001_R1_001.fastq.gz
│ ├── NAFLD3_P3_L001_R2_001.fastq.gz
│ ├── NAFLD4_P4_L001_R1_001.fastq.gz
│ ├── NAFLD4_P4_L001_R2_001.fastq.gz
│ └── samplesheet.csvSTEP1: Create the metadata file (samplesheet.csv):
Change to the data folder directory:
cd $HOME/workshop/sarek/runs/run3_liver
pwdCopy the python script “create_samplesheet_nf-core_sarek.py" to the working folder
Note: you could replace ‘$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:
usage: create_samplesheet_nf-core_sarek.py [-h] [--dir DIR] [--read1_extension READ1_EXTENSION] [--read2_extension READ2_EXTENSION] [--out OUT] Extract metadata from fastq files in a directory. optional arguments: -h, --help show this help message and exit --dir DIR Directory to search for files (default: current directory) --read1_extension READ1_EXTENSION Extension for fastq_1 files (default: R1_001.fastq.gz) --read2_extension READ2_EXTENSION Extension for fastq_2 files (default: R2_001.fastq.gz) --out OUT Output metadata CSV file |
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Let’s generate the metadata file by running the following command:
Check the newly created samplesheet.csv file:
patient,sample,lane,fastq_1,fastq_2 Control1,C1,L001,/sarek/data/WES/liver/Control1_C1_L001_R1_001.fastq.gz,/sarek/data/WES/liver/Control1_C1_L001_R2_001.fastq.gz Control2,C2,L001,/sarek/data/WES/liver/Control2_C2_L001_R1_001.fastq.gz,/sarek/data/WES/liver/Control2_C2_L001_R2_001.fastq.gz Control3,C3,L001,/sarek/data/WES/liver/Control3_C3_L001_R1_001.fastq.gz,/sarek/data/WES/liver/Control3_C3_L001_R2_001.fastq.gz Control4,C4,L001,/sarek/data/WES/liver/Control4_C4_L001_R1_001.fastq.gz,/sarek/data/WES/liver/Control4_C4_L001_R2_001.fastq.gz NAFLD1,P1,L001,/sarek/data/WES/liver/NAFLD1_P1_L001_R1_001.fastq.gz,/sarek/data/WES/liver/NAFLD1_P1_L001_R2_001.fastq.gz NAFLD2,P2,L001,/sarek/data/WES/liver/NAFLD2_P2_L001_R1_001.fastq.gz,/sarek/data/WES/liver/NAFLD2_P2_L001_R2_001.fastq.gz NAFLD3,P3,L001,/sarek/data/WES/liver/NAFLD3_P3_L001_R1_001.fastq.gz,/sarek/data/WES/liver/NAFLD3_P3_L001_R2_001.fastq.gz NAFLD4,P4,L001,/sarek/data/WES/liver/NAFLD4_P4_L001_R1_001.fastq.gz,/sarek/data/WES/liver/NAFLD4_P4_L001_R2_001.fastq.gz |
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Alternatively copy the samplesheet.csv file:
STEP2 - Run the nf-core/sarek pipeline
Copy and paste the code below to the terminal:
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:
#!/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:
Monitoring the Run
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.
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:
Windows PC
Mac
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: