Page Comparison

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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)

CEPH is an international genetic research center that provides a resource of immortalized cell cultures used to map genetic markers. The whole pedigree was sequenced to 50x depth on a HiSeq 2000 Illumina system, which is considered a platinum standard, where platinum refers to the quality and completeness of the resulting assembly, such as providing full chromosome scaffolds with phasing and haplotypes resolved across the entire genome.

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This figure depicts the pedigree of the family sequenced for this study, where the ID for each sample is defined by adding the prefix NA128 to each numbered individual, so that 77 = NA12877 and 78 = NA12878, corresponding to the VCF tracks available in this track set. The dark orange individuals indicate sequences used in the analysis methods, whereas the blue represent the founder generations (grandparents), which were also sequenced and used in validation steps. The genomes of the parent-child trio on the top right side, 91-92-78, were also sequenced during Phase I of the 1000 Genomes Project.

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

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:

Pipeline requirements

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 (see below)

STEP1: Create the metadata file (samplesheet.csv):

Change to List FASTQ files in the data folder directory of the family trio:

Code Block
cd $HOME/workshopls -l /work/training/sarek/data/WES/trio pwd

Copy the python script “create_samplesheet_nf-core_sarek.py" to the working (run2) folder

Code Block
cp $HOME/work/trainingworkshop/sarek/scripts/create_samplesheet_nf-core_sarek.py $HOME/workshop/sarek/data//run2_trio cd $HOME/workshop/sarek/run2_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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Code Block
python create_samplesheet_nf-core_sarek.py --dir $HOME/work/workshoptraining/sarek/data/WES/trio \ --read1_extension R1.fastq.gz \ --read2_extension R2.fastq.gz \ --out samplesheet.csv

Alternatively copy the samplesheet.csv file:

Code Block
cp /work/training/sarek/data/WES/trio/samplesheet.csv .

Check the newly created samplesheet.csv file:

Code Block
ls -l cat samplesheet.cvscsv

patient,sample,lane,fastq_1,fastq_2

SRR14724455,NA12892a,L001,/sarek/data/WES/trio/SRR14724455_NA12892a_L001_R1.fastq.gz,/sarek/data/WES/trio/SRR14724455_NA12892a_L001_R2.fastq.gz

SRR14724456,NA12891a,L001,/sarek/data/WES/trio/SRR14724456_NA12891a_L001_R1.fastq.gz,/sarek/data/WES/trio/SRR14724456_NA12891a_L001_R2.fastq.gz

SRR14724463,NA12878a,L001,/sarek/data/WES/trio/SRR14724463_NA12878a_L001_R1.fastq.gz,/sarek/data/WES/trio/SRR14724463_NA12878a_L001_R2.fastq.gz

SRR14724474,NA12892b,L001,/sarek/data/WES/trio/SRR14724474_NA12892b_L001_R1.fastq.gz,/sarek/data/WES/trio/SRR14724474_NA12892b_L001_R2.fastq.gz

SRR14724475,NA12891b,L001,/sarek/data/WES/trio/SRR14724475_NA12891b_L001_R1.fastq.gz,/sarek/data/WES/trio/SRR14724475_NA12891b_L001_R2.fastq.gz

SRR14724483,NA12878b,L001,/sarek/data/WES/trio/SRR14724483_NA12878b_L001_R1.fastq.gz,/sarek/data/WES/trio/SRR14724483_NA12878b_L001_R2.fastq.gz

Copy the PBS Pro script for STEP 2: 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 directoryLine 2: copy the launch_nf-core_sarek_trio.pbs submission script to the working directory
Line 32: move to the working directory

View the content of the launch_nf-core_RNAseqsarek_QCtrio.pbs script:

Code Block
cat launch_nf-core_RNAseqsarek_QCtrio.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”
Pipeline steps:
- mapping
- markduplicates
- prepare_recalibration
- recalibrate
- variant_calling
- annotation
  - See more information on the input requirements for each step https://nf-co.re/sarek/3.3.2/docs/usage

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:

Code Block
qsub launch_nf-core_sarek_trio.pbs

Monitoring the Run

Code Block
qjobs

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

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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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Code Block
\\hpc-fs\work\training\rnaseqsarek

Mac

Code Block
smb://hpc-fs/work/training/rnaseqsarek

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:

Code Block

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_20-14-17.html
│   ├── execution_timeline_2024-05-11_20-14-17.html
│   ├── execution_trace_2024-05-11_20-14-17.txt
│   ├── params_2024-05-12_00-00-57.json
│   ├── pipeline_dag_2024-05-11_20-14-17.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

Versions Compared

Old Version 3

New Version Current

Key

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

Public data

Pipeline requirements

STEP1: Create the metadata file (samplesheet.csv):

Submitting the job

Monitoring the Run

Once the pipeline has finished running - Assess the

results as follows:

Page Comparison

Versions Compared

Old Version 3

New Version Current

Key

<span class="diff-html-changed" data-a11y-before="Start of changed content" data-a11y-after="End of changed content" id="changed-diff-0">[data-colorid=</span>

Public data

Pipeline requirements

STEP1: Create the metadata file (samplesheet.csv):

Submitting the job

Monitoring the Run

Once the pipeline has finished running - Assess the

results as follows: