singularity & snakemake workflow to detect mutations with several tools to detect mutations

public public 1yr ago Version: v0.1.0 0 bookmarks
View Workflow
Report
detectmutations
detectmutations
View Workflow

detect Mutations

Sylvain Schmitt April 20, 2021

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

Installation

  • [x] Python ≥3.5

  • [x] Snakemake ≥5.24.1

  • [x] Golang ≥1.15.2

  • [x] Singularity ≥3.7.3

  • [x] 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

gatk_idx

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

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

gatk_genotypegvcfs

strelka2

  • Tools: Strelka2

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

varscan

  • Tools: VarScan

  • Singularity: docker://alexcoppe/varscan

varscan2vcf

somaticsniper

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

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

Code Snippets

13
14
shell:
    "bedtools subtract -header -a {input[0]} -b {input[1]} > {output}"
SnakeMake BEDTools From line 13 of rules/bedtools_substract.smk 
13
14
shell:
    "bwa index {input}"
SnakeMake BWA From line 13 of rules/bwa_index.smk 
21
22
23
shell:
    "bwa mem -M -R '{params.rg_mutated}' -t {threads} {input[0]} {input[1]} {input[2]} > {output[0]} ; "
    "bwa mem -M -R '{params.rg_base}' -t {threads} {input[0]} {input[3]} {input[4]} > {output[1]} ; "
SnakeMake BWA From line 21 of rules/bwa_mem.smk 
12
13
shell:
    "cp {input} {output}"
SnakeMake From line 12 of rules/cp_vcfs.smk 
13
14
script:
    "../scripts/evaluate_call.R"
SnakeMake From line 13 of rules/evaluate_call.smk 
16
17
shell:
    "fastqc -t {threads} -q {input} --outdir=results/{wildcards.library}/"
SnakeMake FastQC From line 16 of rules/fastqc.smk 
16
17
18
19
shell:
    "freebayes -f {input[0]} --pooled-continuous --pooled-discrete --genotype-qualities --report-genotype-likelihood-max"
    " --allele-balance-priors-off --min-alternate-fraction 0.03 --min-repeat-entropy 1 --min-alternate-count 2"
    " {input[2]} {input[1]} > {output}"
SnakeMake FreeBayes From line 16 of rules/freebayes.smk 
12
13
shell:
    "gatk CreateSequenceDictionary R={input} O={output}"
SnakeMake gatk From line 12 of rules/gatk_dict.smk 
15
16
shell:
    "gatk GenotypeGVCFs -R {input[0]} -V {input[1]} -O {output}"
SnakeMake gatk From line 15 of rules/gatk_genotypegvcfs.smk 
16
17
shell:
    "gatk HaplotypeCaller -R {input[0]} -I {input[1]} -O {output} -ERC GVCF"
SnakeMake gatk From line 16 of rules/gatk_haplotypecaller.smk 
12
13
shell:
    "gatk IndexFeatureFile -I {input}"
SnakeMake gatk From line 12 of rules/gatk_idx.smk 
16
17
18
shell:
    "gatk MarkDuplicates --java-options \"-Xmx16G -Xms1G -Djava.io.tmpdir=tmp\" I={input[0]} O={output[0]} M={output[2]} ; "
    "gatk MarkDuplicates --java-options \"-Xmx16G -Xms1G -Djava.io.tmpdir=tmp\" I={input[1]} O={output[1]} M={output[3]}"
SnakeMake gatk From line 16 of rules/gatk_markduplicates.smk 
18
19
20
shell:
    "gatk Mutect2 -R {input[0]} -I {input[1]} -tumor {wildcards.lib}_REP{wildcards.REP}_mutated  -I {input[2]} -normal {wildcards.lib}_REP{wildcards.REP}_base "
    " --panel-of-normals {input[3]} -dont-use-soft-clipped-bases true -O {output}"
SnakeMake gatk From line 18 of rules/gatk_mutect2.smk 
16
17
18
19
shell:
    "multiqc results/{wildcards.library}/ -o results/{wildcards.library} ; "
    "rm -r results/{wildcards.library}/qualimap_mutated ; "
    "rm -r results/{wildcards.library}/qualimap_base ; "
SnakeMake MultiQC From line 16 of rules/multiqc.smk 
21
22
shell:
    "muse.py -f {input[0]} --tumor-bam {input[1]} --normal-bam {input[2]} -O {output} -n {threads} -w results/{wildcards.lib}_REP{wildcards.REP}/muse/"
SnakeMake From line 21 of rules/muse.smk 
15
16
17
18
19
shell:
    "qualimap bamqc -bam {input[0]} --paint-chromosome-limits -nt {threads} -skip-duplicated --skip-dup-mode " 
    "0 -outdir results/{wildcards.library}/qualimap_mutated -outformat HTML ; "
    "qualimap bamqc -bam {input[1]} --paint-chromosome-limits -nt {threads} -skip-duplicated --skip-dup-mode " 
    "0 -outdir results/{wildcards.library}/qualimap_base -outformat HTML"
SnakeMake QualiMap From line 15 of rules/qualimap.smk 
12
13
shell:
    "samtools faidx {input}"
SnakeMake SAMtools From line 12 of rules/samtools_faidx.smk 
12
13
shell:
    "samtools index {input[0]} ; samtools index {input[1]}"
SnakeMake SAMtools From line 12 of rules/samtools_index_md.smk 
12
13
shell:
    "samtools index {input[0]} ; samtools index {input[1]}"
SnakeMake SAMtools From line 12 of rules/samtools_index.smk 
16
17
18
shell:
    "samtools mpileup -f {input[0]} {input[1]} > {output[0]} ; "
    "samtools mpileup -f {input[0]} {input[2]} > {output[1]}"
SnakeMake SAMtools From line 16 of rules/samtools_mpileup.smk 
12
13
14
shell:
    "samtools sort --threads {threads} {input[0]} > {output[0]} ; "
    "samtools sort --threads {threads} {input[1]} > {output[1]}"
SnakeMake SAMtools From line 12 of rules/samtools_sort.smk 
12
13
14
shell:
    "samtools stats --threads {threads} {input[0]} > {output[0]} ; "
    "samtools stats --threads {threads} {input[1]} > {output[1]}"
SnakeMake SAMtools From line 12 of rules/samtools_stats.smk 
16
17
shell:
    "/opt/somatic-sniper/build/bin/bam-somaticsniper -q 25 -Q 15 -s 0.0001 -F vcf -f {input[0]} {input[1]}  {input[2]} {output}"
SnakeMake From line 16 of rules/somaticsniper.smk 
19
20
21
22
23
24
25
26
27
shell:
    "configureStrelkaSomaticWorkflow.py "
    "--normalBam {input[2]} "
    "--tumorBam {input[1]} "
    "--referenceFasta {input[0]} "
    "--runDir results/{wildcards.lib}_{wildcards.REP}/strelka2/ ; "
    "results/{wildcards.lib}_{wildcards.REP}/strelka2/runWorkflow.py -m local -j {threads} ; "
    "zcat results/{wildcards.lib}_{wildcards.REP}/strelka2/results/variants/somatic.snvs.vcf.gz > {output} ; "
    "rm -r results/{wildcards.lib}_{wildcards.REP}/strelka2"
SnakeMake From line 19 of rules/strelka2.smk 
17
18
19
20
21
shell:
    "trimmomatic PE -threads {threads} {input[0]} {input[1]} {output[0]} {output[4]} {output[1]} {output[5]} "
    "ILLUMINACLIP:TruSeq3-PE.fa:2:30:10:2:keepBothReads SLIDINGWINDOW:4:15 2> {output[8]} ; "
    "trimmomatic PE -threads {threads} {input[2]} {input[3]} {output[2]} {output[6]} {output[3]} {output[7]} "
    "ILLUMINACLIP:TruSeq3-PE.fa:2:30:10:2:keepBothReads SLIDINGWINDOW:4:15 2> {output[9]}"
SnakeMake Trimmomatic From line 17 of rules/trimmomatic.smk 
15
16
script:
    "../scripts/varscan2vcf.R"
SnakeMake From line 15 of rules/varscan2vcf.smk 
12
13
shell:
    "/.run somatic {input[1]} {input[0]} results/{wildcards.library}/varscan/{wildcards.library}"
SnakeMake From line 12 of rules/varscan.smk 
 1
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
callfile <- snakemake@input[[1]]
mutationsfile <- snakemake@input[[2]]
statsfile <-  snakemake@output[[1]] 

library(tidyverse)
library(vcfR)

mutations <- read_tsv(mutationsfile, col_types = cols(
  CHROM = col_character(),
  POS = col_double(),
  REF = col_character(),
  TYPE = col_character(),
  ALT = col_character()
)) %>% 
  mutate(True = 1) %>%
  filter(REF != "N")
call <- try(read.vcfR(callfile, verbose = F)@fix, silent = T)
if(!inherits(call, "try-error")){
  call <- as.data.frame(call) %>% 
    mutate_at(c("CHROM", "REF", "ALT"), as.character) %>% 
    mutate(POS = as.numeric(as.character(POS))) %>% 
    mutate(Called = 1) %>% 
    mutate(REF = substr(REF, 1, 1), ALT = substr(ALT, 1, 1))
} else {
  call <- data.frame(CHROM = character(), POS = double(), REF = character(), ALT = character(), Called = integer())
}
stats <- full_join(mutations, call, by = c("CHROM", "POS", "REF", "ALT")) %>% 
  mutate_at(c("True", "Called"), funs(ifelse(is.na(.), 0, .))) %>% 
  mutate(Confusion = recode(paste0(True, Called), "01" = "FP", "10" = "FN", "11" = "TP")) %>% 
  group_by(Confusion) %>% 
  summarise(N = n()) %>% 
  reshape2::dcast(mutationsfile + callfile ~ Confusion, value.var = "N")
if(!("FP" %in% names(stats)))
  stats$FP <- 0
if(!("FN" %in% names(stats)))
  stats$FN <- 0
if(!("TP" %in% names(stats)))
  stats$TP <- 0
stats <- mutate(stats, Precision = round(TP/(TP+FP), 2), Recall = round(TP/(TP+FN), 2)) %>% 
  mutate(callfile = callfile) %>% 
  mutate(mutationsfile = mutationsfile)
write_tsv(stats, statsfile)
R tidyverse vcfR From line 1 of scripts/evaluate_call.R 
 2
 3
 4
 5
 6
 7
 8
 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
varscanfile <- snakemake@input[[1]]
snpfile <- snakemake@input[[2]]
# output
vcffile <-  snakemake@output[[1]] 

# manual
# varscanfile <- "results/N100_R2_AF0.1_NR7000/varscan/N100_R2_AF0.1_NR7000.snp"
# snpfile <- file.path("results/reference/", yaml::read_yaml("config/config.dev.yml")$snps)
# vcffile <-  "results/N100_R2_AF0.1_NR7000/varscan/N100_R2_AF0.1_NR7000.vcf"

library(tidyverse)
library(Biostrings)

cat("##fileformat=VCFv4.1\n##fileDate=20210521\n##source=varscan\n#CHROM	POS	ID	REF	ALT\n",
    file = vcffile)

snps <-   read_tsv(snpfile, skip = 11) %>% 
  dplyr::rename(CHROM = `#CHROM`) %>% 
  select(CHROM, POS, ID, REF, ALT) %>% 
  mutate(ID = ".") %>% 
  as_tibble() %>% 
  mutate_all(as.character) %>% 
  mutate_at("POS", as.numeric)

read_tsv(varscanfile) %>% 
  mutate(ID = ".", CHROM	= chrom, POS = position, REF =ref,	ALT	= var) %>% 
  dplyr::select(CHROM,	POS,	ID,	REF,	ALT) %>% 
  anti_join(snps) %>% 
  write_tsv(file = vcffile, append = T, col_names = F)
R tidyverse From line 2 of scripts/varscan2vcf.R
ShowHide 19 more snippets with no or duplicated tags.

Login to post a comment if you would like to share your experience with this workflow.

Do you know this workflow well? If so, you can request seller status , and start supporting this workflow.

Free

Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://github.com/sylvainschmitt/detectMutations
Name: detectmutations
Version: v0.1.0
Badge:
workflow icon

Insert copied code into your website to add a link to this workflow.

Downloaded: 0
Copyright: Public Domain
License: GNU General Public License v3.0
  • Future updates

Related Workflows

psychip_snakemake
Show Details View Workflow
ENCODE pipeline for histone marks developed for the psychENCODE project
public

public

psychip pipeline is an improved version of the ENCODE pipeline for histone marks developed for the psychENCODE project. The o...
raw sequence reads Alignment Sequence alignment report macs2 ucsc-bedclip bedGraphToBigWig BEDTools BWA Picard SAMtools Snakemake
Free
74
ncov_2
Show Details View Workflow
Near-real time tracking of SARS-CoV-2 in Connecticut
public

public

Repository containing scripts to perform near-real time tracking of SARS-CoV-2 in Connecticut using genomic data. This pipeli...
JSON nextclade Augur Biopython FOCUS Pandas Snakemake bs4 epiweeks geopy matplotlib numpy pycountry pycountry-convert uszipcode
Free
28
cellranger-snakemake-gke
Show Details View Workflow
snakemake workflow to run cellranger on a given bucket using gke.
public

public

A Snakemake workflow for running cellranger on a given bucket using Google Kubernetes Engine. The usage of this workflow ...
macs2 ucsc-bedclip bedGraphToBigWig BEDTools BWA Picard SAMtools Snakemake
Free
74
atlas
Show Details View Workflow
ATLAS - Three commands to start analyzing your metagenome data
public

public

Metagenome-atlas is a easy-to-use metagenomic pipeline based on snakemake. It handles all steps from QC, Assembly, Binning, t...
raw sequence reads Genome assembly Annotation track checkm2 gunc prodigal snakemake-wrapper-utils MEGAHIT Atlas BBMap Biopython BioRuby Bwa-mem2 cd-hit CheckM DAS Diamond eggNOG-mapper v2 MetaBAT 2 Minimap2 MMseqs MultiQC Pandas Picard pyfastx SAMtools SemiBin Snakemake SPAdes SqueezeMeta TADpole VAMB CONCOCT ete3 gtdbtk h5py networkx numpy plotly psutil utils metagenomics
Free
175
rna-seq-star-deseq2
Show Details View Workflow
RNA-seq workflow using STAR and DESeq2
public

public

This workflow performs a differential gene expression analysis with STAR and Deseq2. The usage of this workflow is described ...
raw sequence reads Gene expression profiling ID list snakemake-wrapper-utils biomaRt PCAtools BiocParallel BWA Cutadapt DESeq2 GFFutils MultiQC Pandas SAMtools Snakemake STAR cli stringr tidyverse RNA-Seq
Free
41
dna-seq-gatk-variant-calling
Show Details View Workflow
This Snakemake pipeline implements the GATK best-practices workflow
public

public

This Snakemake pipeline implements the GATK best-practices workflow for calling small germline variants. The usage of thi...
VCF raw sequence reads Variant calling genetic variants gatk rust-bio-tools snakemake-wrapper-utils tabix BCFtools BWA FastQC MultiQC Pandas Picard SAMtools Snakemake Trimmomatic Variant Effect Predictor (VEP) common matplotlib numpy seaborn DNA
Free
57