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How to call variants for bacterial genomes

This recipe demonstrates a bacterial variant caller with minimap2 and bcftools

1 result • updated 4.5 years ago by Istvan Albert

This recipe demonstrates a bacterial variant caller with minimap2 and bcftools

This recipe contains the code presented by Torsten Seemann in the blog post titled A Unix one-liner to call bacterial variants

The code from the above website has been generalized a bit with additional utility.

This recipe provides code that:

Downloads a reference genome (default value: NZ_CP008918 Pasteurella multocida strain ATCC 43137, complete genome)
Downloads a sequencing run from SRA (default value: SRR4124989)
Generates statistics on the sequencing data
Runs minimap2 to align the sequences and create a SAM output
Runs bcftools mpileup to generate the genotype likelihoods of each base
Runs bcftools call to filter for multiallelic variants only
Runs bcftools norm to normalize each variant to a standard form
Runs bcftools filter to remove variants with low quality (QUAL) or low coverage (DP)

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# Stop on errors.
set -uexo pipefail

# The reference genome.
ACC=NZ_CP008918

# Select sequencing run.
SRR=SRR4124989

# The directory that stores the reference sequence.
mkdir -p ref

# The file that stores the reference genome.
REF=ref/${ACC}.fa

# Download the reference accession number.
efetch -db nuccore -id $ACC -format fasta > $REF

# The directory that stores the sequencing reads.
mkdir -p reads

# Download the sequencing data.
fastq-dump --origfmt  --split-files -O reads $SRR

# Optional step. Generate a report on the data size
echo "*** Sequence statistics"
seqkit stat reads/${SRR}_?.fastq
echo "*** "
#
# The one-liner code comes from:
#
# https://thegenomefactory.blogspot.com/2018/10/a-unix-one-liner-to-call-bacterial.html
#

# The number of CPUs to use.
CPUS=4

# The names for the read pairs.
R1=reads/${SRR}_1.fastq
R2=reads/${SRR}_2.fastq

# Unix trick: A single command line that is too long may be broken
# into several lines as long as each ends with a \ symbol.
#
# Call the variants.
minimap2 -a -x sr -t $CPUS $REF $R1 $R2 \
 | samtools sort -l 0 --threads $CPUS \
 | bcftools mpileup -Ou -B --min-MQ 60 -f $REF - \
 | bcftools call -Ou -v -m - \
 | bcftools norm -Ou -f $REF -d all - \
 | bcftools filter -Ov -e 'QUAL<40 || DP<10 || GT!="1/1"' \
 > variants.vcf

# Generate a statistics on variants.
bcftools stats variants.vcf > variants-stats.txt

# Plot the variant statistics.
plot-vcfstats -P -p plots variants-stats.txt

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[acc]
label = "Genome Accession Number"
display = "TEXTBOX"
value = "NZ_CP008918"
regex = "^\\w{1,20}$"
help = "An NCBI accession number"

[srr]
label = "SRA Run Number"
display = "TEXTBOX"
value = "SRR4124989"
help = "An SRR Run number"

[settings]
id = 85
recipe_uid = "recipe-bacterial"
uid = "recipe-bacterial"
name = "How to call variants for bacterial genomes"
template = "How_to_call_variants_for_bacterial_genomes_bio-data-analysis_85.sh"
image = "How_to_call_variants_for_bacterial_genomes_bio-data-analysis_85.png"
project_uid = "bio-data-analysis"
url = "http://localhost8000"
help = "This recipe demonstrates a bacterial variant caller with minimap2 and bcftools\n\nThis recipe contains the code presented by Torsten Seemann in the blog post titled [A Unix one-liner to call bacterial variants][code]\n\nThe code from the above website has been generalized a bit with additional utility.\n\nThis recipe provides code that:\n\n1. Downloads a reference genome (default value: [NZ_CP008918][NZ_CP008918] *Pasteurella multocida strain ATCC 43137, complete genome*)\n1. Downloads a sequencing run from SRA (default value: [SRR4124989][SRR4124989])\n1. Generates statistics on the sequencing data\n1. Runs `minimap2` to align the sequences and create a SAM output\n1. Runs `bcftools mpileup` to generate the genotype likelihoods of each base\n1. Runs `bcftools call` to filter for multiallelic variants only\n1. Runs `bcftools norm` to normalize each variant to a standard form\n1. Runs `bcftools filter` to remove variants with low quality (QUAL) or low coverage (DP)\n\n[NZ_CP008918]: https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP008918.1\n[SRR4124989]: https://www.ncbi.nlm.nih.gov/sra/?term=SRR4124989\n\n[code]: https://thegenomefactory.blogspot.com/2018/10/a-unix-one-liner-to-call-bacterial.html"

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This recipe demonstrates a bacterial variant caller with minimap2 and bcftools

This recipe contains the code presented by Torsten Seemann in the blog post titled [A Unix one-liner to call bacterial variants][code]

The code from the above website has been generalized a bit with additional utility.

This recipe provides code that:

1. Downloads a reference genome (default value: [NZ_CP008918][NZ_CP008918] *Pasteurella multocida strain ATCC 43137, complete genome*)
1. Downloads a sequencing run from SRA (default value: [SRR4124989][SRR4124989])
1. Generates statistics on the sequencing data
1. Runs `minimap2` to align the sequences and create a SAM output
1. Runs `bcftools mpileup` to generate the genotype likelihoods of each base
1. Runs `bcftools call` to filter for multiallelic variants only
1. Runs `bcftools norm` to normalize each variant to a standard form
1. Runs `bcftools filter` to remove variants with low quality (QUAL) or low coverage (DP)

[NZ_CP008918]: https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP008918.1
[SRR4124989]: https://www.ncbi.nlm.nih.gov/sra/?term=SRR4124989

[code]: https://thegenomefactory.blogspot.com/2018/10/a-unix-one-liner-to-call-bacterial.html

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