This guide provides a step-by-step guide to 1) convert BAM files (i.e., public) to paired-end FASTQ files; and 2) run the nextflow nf-core/sarek variant calling pipeline.
...
Conda installation: https://docs.conda.io/projects/conda/en/latest/user-guide/install/linux.html
Code Block wget https://repo.anaconda.com/miniconda/Miniconda3-py37_22.11.1-1-Linux-x86_64.sh bash Miniconda3-py37_22.11.1-1-Linux-x86_64.shnextflow installation: Nextflow
...
To install several tools, prepare a file called environment.yml (example below). Tip: use a text editor (i.e., vim, nano, or other) to copy and paste the code below into the file.
...
Move to the folder where all the BAM files are present and prepare the following script (i.e.,launch_BAM2FASTQ.pbs):
| Code Block |
|---|
#!/bin/bash -l
#PBS -N BAM2FASTQ
#PBS -l walltime=24:00:00
#PBS -l mem=8gb
#PBS -l ncpus=4
cd $PBS_O_WORKDIR
#activate the conda environment with the necessary tools
conda activate liver
#Sort reads in BAM file by indentifier-name (-n) using 4 CPUs (-@ 4). Note 'prefix' for sorted file noted after $i (input BAM file)
for i in `ls --color=never *.bam`
do
echo $i
samtools sort -@ 4 -n $i ${i%%.bam}_sorted
done
#Extract paired end reads in FASTQ format
for file in `ls --color=never *sorted.bam`
do
echo $file
bedtools bamtofastq -i $file -fq ${file%%.bam}_R1.fastq -fq2 ${file%%.bam}_R2.fastq
#compress FASTQ files to run using the sarek pipeline
gzip -c -9 ${file%%.bam}_R1.fastq > ${file%%.bam}_R1.fastq.gz
gzip -c -9 ${file%%.bam}_R1.fastq > ${file%%.bam}_R2.fastq.gz
done |
...
To run Sarek 3 files are required:
launch.pbs → details on how to run the workflow
~/.nextflow/config → specify how to run the workflow in the HPC
samplesheet.csv → provides information on the samples and data to be used (i.e., FASTQ, BAM or CRAM)
...
PHASE I - preprocessing
Below is an example of a launch_phase1.pbs file for mapping onto the selected genome:
...
| Code Block |
|---|
singularity {
cacheDir = '$HOME/NXF_SINGULARITY_CACHEDIR'
autoMounts = true
}
conda {
cacheDir = '$HOME/NXF_CONDA_CACHEDIR'
}
singularity {
enabled = true
autoMounts = true
}
process {
executor = 'pbspro'
beforeScript = {
"""
source $HOME/.bashrc
source $HOME/.profile
"""
}
scratch = false
cleanup = false
} |
Example of a samplesheet.csv file:
| Code Block |
|---|
patient,sample,lane,fastq_1,fastq_2 healthy_11,1,1,/path/to/data/1.Healthy/Healthy_Combined_11_sorted_R1.fastq.gz,/path/to/data/1.Healthy/Healthy_Combined_11_sorted_R2.fastq.gz |
...
Prepare/edit the following launch_phase2.pbs script:
| Code Block |
|---|
#!/bin/bash -l
#PBS -N sarek_II
#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
#run the sarek pipeline
nextflow run nf-core/sarek \
-r 3.1.1 \
-profile singularity \
--genome GATK.GRCh38 \
--step variant_calling \
--tools haplotypecaller \
--wes \
-resume |
...
Prepare/edit the following launch_phase3.pbs script:
| Code Block |
|---|
#!/bin/bash -l
#PBS -N sarek_III
#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
#run the sarek pipeline
nextflow run nf-core/sarek \
-r 3.1.1 \
-profile singularity \
--genome GATK.GRCh38 \
--step annotate \
--tools vep,snpeff \
--wes \
-resume |
...