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Exercise 2: Run nf-core/sarek using a family trio data (HapMap; Genome in a Bottle)

Public data

Family ID: 1463
Family information: family lineage from Utah of four grandparents, two parents, and 11 children (17 family members)
Genomics consortia: Genome in a Bottle, 1000 Genomes Project, International HapMap Project, Centre d'Etude du Polymorphisme Humain (CEPH)

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Sample ID

Description

Biological sample source

NA12878 (Daughter)

Mother; donor subject has a single bp (G-to-A) transition at nucleotide 681 in exon 5 of the CYP2C19 gene (CYP2C19*2) which creates an aberrant splice site. The change altered the reading frame of the mRNA starting with amino acid 215 and produced a premature stop codon 20 amino acids downstream, resulting in a truncated, nonfunctional protein. Because of the aberrant splice site, a 40-bp deletion occurred at the beginning of exon 5 (from bp 643 to bp 682), resulting in deletion of amino acids 215 to 227. The truncated protein had 234 amino acids and would be catalytically inactive because it lacked the heme-binding region.

https://catalog.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=NA12878&Product=DNA

NA12891 (Father)

Maternal Grandfather; donor subject is homozygous for a single bp (G-to-A) transition at nucleotide 681 in exon 5 of the CYP2C19 gene (CYP2C19*2) which creates an aberrant splice site. The change altered the reading frame of the mRNA starting with amino acid 215 and produced a premature stop codon 20 amino acids downstream, resulting in a truncated, nonfunctional protein. Because of the aberrant splice site, a 40-bp deletion occurred at the beginning of exon 5 (from bp 643 to bp 682), resulting in deletion of amino acids 215 to 227. The truncated protein had 234 amino acids and would be catalytically inactive because it lacked the heme-binding region.

https://catalog.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=NA12891&Product=DNA

NA12892 (Mother)

Maternal Grandmother

https://catalog.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=NA12892&Product=DNA

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Location of raw data:

Code Block
/work/training/sarek/data/WES/trio

Code Block

├── trio
│   ├── samplesheet.csv
│   ├── SRR14724455_NA12892a_L001_R1.fastq.gz
│   ├── SRR14724455_NA12892a_L001_R2.fastq.gz
│   ├── SRR14724456_NA12891a_L001_R1.fastq.gz
│   ├── SRR14724456_NA12891a_L001_R2.fastq.gz
│   ├── SRR14724463_NA12878a_L001_R1.fastq.gz
│   ├── SRR14724463_NA12878a_L001_R2.fastq.gz
│   ├── SRR14724474_NA12892b_L001_R1.fastq.gz
│   ├── SRR14724474_NA12892b_L001_R2.fastq.gz
│   ├── SRR14724475_NA12891b_L001_R1.fastq.gz
│   ├── SRR14724475_NA12891b_L001_R2.fastq.gz
│   ├── SRR14724483_NA12878b_L001_R1.fastq.gz
│   └── SRR14724483_NA12878b_L001_R2.fastq.gz

Where:

The pipeline requires preparing at least 2 files:

Metadata file (samplesheet.csv) thatspecifies the following information:

Code Block
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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Code Block
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:

Code Block

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:

Code Block
cat launch_nf-core_RNAseq_QC.pbs

#!/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

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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Versions Compared

Old Version 5

New Version 6

Key

Exercise 2: Run nf-core/sarek using a family trio data (HapMap; Genome in a Bottle)

Public data

Create the metadata file (samplesheet.csv):

Submitting the job

Once the pipeline has finished running - Assess the results as follows: