detect Mutations

Sylvain Schmitt April 20, 2021

• Installation
• Usage
  • Get data
  • Locally
  • HPC
• Workflow
  • Reference
  • Reads
  • Alignments
  • Detection
  • Mutations
  • Quality check
• Results

singularity & snakemake workflow to detect mutations with several alignment and mutation detection tools.

Installation

• Python ≥3.5
• Snakemake ≥5.24.1
• Golang ≥1.15.2
• Singularity ≥3.7.3
• This workflow

# Python
sudo apt-get install python3.5
# Snakemake
sudo apt install snakemake`
# Golang
export VERSION=1.15.8 OS=linux ARCH=amd64  # change this as you need
wget -O /tmp/go${VERSION}.${OS}-${ARCH}.tar.gz https://dl.google.com/go/go${VERSION}.${OS}-${ARCH}.tar.gz && \
sudo tar -C /usr/local -xzf /tmp/go${VERSION}.${OS}-${ARCH}.tar.gz
echo 'export GOPATH=${HOME}/go' >> ~/.bashrc && \
echo 'export PATH=/usr/local/go/bin:${PATH}:${GOPATH}/bin' >> ~/.bashrc && \
source ~/.bashrc
# Singularity
mkdir -p ${GOPATH}/src/github.com/sylabs && \
  cd ${GOPATH}/src/github.com/sylabs && \
  git clone https://github.com/sylabs/singularity.git && \
  cd singularity
git checkout v3.7.3
cd ${GOPATH}/src/github.com/sylabs/singularity && \
  ./mconfig && \
  cd ./builddir && \
  make && \
  sudo make install
# detect Mutations
git clone git@github.com:sylvainschmitt/detectMutations.git
cd detectMutations

Usage

Get data

Generate data using the generate Mutations workflow.

git clone git@github.com:sylvainschmitt/generateMutations.git
cd ../generateMutations
snakemake --use-singularity --cores 4
cd ../detectMutations
bash scripts/get_data.sh

Locally

snakemake -np # dry run
snakemake --dag | dot -Tsvg > dag/dag.svg # dag
snakemake --use-singularity --cores 4 # run
snakemake --use-singularity --cores 1 --verbose # debug
snakemake --report report.html # report

HPC

module purge ; module load bioinfo/snakemake-5.25.0 # for test on node
snakemake -np # dry run
sbatch job.sh ; watch 'squeue -u sschmitt' # run
less detMut.*.err # snakemake outputs, use MAJ+F
less detMut.*.out # snakemake outputs, use MAJ+F
snakemake --dag | dot -Tsvg > dag/dag.svg # dag
module purge ; module load bioinfo/snakemake-5.8.1 ; module load system/Python-3.6.3 # for report
snakemake --report report.html # report
module purge ; module load system/R-3.6.2 ; R # to build results

Workflow

Reference

Index reference and SNPs for software to work with.

bwa_index

• Tools: BWA index
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/bwa/bwa:latest

samtools_faidx

• Tools: samtools faidx
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/samtools/samtools:latest

gatk_dict

• Tools: gatk CreateSequenceDictionary
• Singularity: docker://broadinstitute/gatk:4.2.6.1

gatk_idx

• Tools: gatk IndexFeatureFile
• Singularity: docker://broadinstitute/gatk:4.2.6.1

Reads

CReport quality and trim.

fastqc

• Tools: fastQC
• Singularity: docker://biocontainers/fastqc:v0.11.9_cv8

trimmomatic

• Tools: Trimmomatic
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/trimmomatic/trimmomatic:latest

Alignments

Align reads against reference, mark duplicated, and report alignment quality.

bwa_mem

• Tools: BWA mem
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/bwa/bwa:latest

samtools_sort

• Tools: Samtools sort
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/samtools/samtools:latest

samtools_index

• Tools: Samtools index
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/samtools/samtools:latest

gatk_markduplicates

• Tools: gatk MarkDuplicates
• Singularity: docker://broadinstitute/gatk:4.2.6.1

samtools_index_md

• Tools: Samtools index
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/samtools/samtools:latest

samtools_mpileup

• Tools: Samtools mpileup
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/samtools/samtools:latest

samtools_stats

• Tools: Samtools stats
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/samtools/samtools:latest

qualimap

• Tools: QualiMap
• Singularity: docker://pegi3s/qualimap:2.2.1

Detection

Detect mutations.

gatk_mutect2

• Tools: gatk Mutect2
• Singularity: docker://broadinstitute/gatk:4.2.6.1

freebayes

• Tools: freebayes
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/freebayes/freebayes:latest

gatk_haplotypecaller

• Tools: gatk HaplotypeCaller
• Singularity: docker://broadinstitute/gatk:4.2.6.1

gatk_genotypegvcfs

• Tools: gatk GenotypeGVCFs
• Singularity: docker://broadinstitute/gatk:4.2.6.1

strelka2

• Tools: Strelka2
• Singularity: docker://quay.io/wtsicgp/strelka2-manta

varscan

• Tools: VarScan
• Singularity: docker://alexcoppe/varscan

varscan2vcf

• Script: varscan2vcf.R
• Singularity: https://github.com/sylvainschmitt/singularity-template/releases/download/0.0.1/sylvainschmitt-singularity-tidyverse-Biostrings.latest.sif

somaticsniper

• Tools: Somatic Sniper
• Singularity: docker://lethalfang/somaticsniper:1.0.5.0

muse

• Tools: MuSe
• Singularity: docker://opengenomics/muse:v0.1.1

Mutations

cp_vcfs

• Tools: cp

bedtools_substract

• Tools: bedtools substract
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/bedtools/bedtools:latest

Quality check

Combined quality information from QualiMap, Picard, Samtools, Trimmomatic, and FastQC (see previous steps) and assess calls performance.

multiqc

• Tools: MultiQC
• Singularity: oras://registry.forgemia.inra.fr/gafl/singularity/multiqc/multiqc:latest

evaluate_call

• Script: evaluate_call.R
• Singularity: https://github.com/sylvainschmitt/singularity-template/releases/download/0.0.1/sylvainschmitt-singularity-tidyverse-Biostrings.latest.sif

Results

module load system/singularity-3.7.3
singularity pull https://github.com/sylvainschmitt/singularity-r-bioinfo/releases/download/0.0.3/sylvainschmitt-singularity-r-bioinfo.latest.sif
singularity shell sylvainschmitt-singularity-r-bioinfo.latest.sif
library(tidyverse)
lapply(list.files("results/stats", full=T), read_tsv) %>% 
  bind_rows() %>% 
  write_tsv("stats.tsv")
quit()
exit