A mapping-based pipeline for creating a phylogeny from bacterial whole genome sequences

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Introduction

nf-core/bactmap is a bioinformatics best-practice analysis pipeline for mapping short reads from bacterial WGS to a reference sequence, creating filtered VCF files, making pseudogenomes based on high quality positions in the VCF files and optionally creating a phylogeny from an alignment of the pseudogenomes.

The pipeline is built using Nextflow , a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses Docker/Singularity containers making installation trivial and results highly reproducible. The Nextflow DSL2 implementation of this pipeline uses one container per process which makes it much easier to maintain and update software dependencies. Where possible, these processes have been submitted to and installed from nf-core/modules in order to make them available to all nf-core pipelines, and to everyone within the Nextflow community!

On release, automated continuous integration tests run the pipeline on a full-sized dataset on the AWS cloud infrastructure. This ensures that the pipeline runs on AWS, has sensible resource allocation defaults set to run on real-world datasets, and permits the persistent storage of results to benchmark between pipeline releases and other analysis sources. The results obtained from the full-sized test can be viewed on the nf-core website .

Pipeline summary

Pipeline summary schematic

The pipeline is composed of the following steps:

  1. Index reference fasta file ( BWA index )

  2. Trim reads for quality and adapter sequence (Optional) ( fastp )

  3. Estimate genome size ( mash sketch )

  4. Downsample fastq files (Optional) ( Rasusa )

  5. Variant calling

    1. Read mapping ( BWA mem )

    2. Sort and index alignments ( SAMtools )

    3. Call and filter variants ( BCFtools )

    4. Convert filtered bcf to pseudogenome fasta ( vcf2pseudogenome.py )

  6. Create alignment from pseudogenome by concatenating fasta files having first checked that the sample sequences are high quality( calculate_fraction_of_non_GATC_bases.py )

  7. Remove recombination (Optional) ( Gubbins )

  8. Extract variant sites from alignment ( SNP-sites )

  9. Construct phylogenetic tree (Optional)

    1. Fast/less accurate

    2. Slow/more accurate, maximum likelihood

Quick Start

  1. Install Nextflow ( >=21.04.0 )

  2. Install any of Docker , Singularity , Podman , Shifter or Charliecloud for full pipeline reproducibility (please only use Conda as a last resort; see docs )

  3. Download the pipeline and test it on a minimal dataset with a single command: 

    nextflow run nf-core/bactmap -profile test,<docker/singularity/podman/shifter/charliecloud/conda/institute>

    • Please check nf-core/configs to see if a custom config file to run nf-core pipelines already exists for your Institute. If so, you can simply use -profile <institute> in your command. This will enable either docker or singularity and set the appropriate execution settings for your local compute environment.

    • If you are using singularity then the pipeline will auto-detect this and attempt to download the Singularity images directly as opposed to performing a conversion from Docker images. If you are persistently observing issues downloading Singularity images directly due to timeout or network issues then please use the --singularity_pull_docker_container parameter to pull and convert the Docker image instead. Alternatively, it is highly recommended to use the nf-core download command to pre-download all of the required containers before running the pipeline and to set the NXF_SINGULARITY_CACHEDIR or singularity.cacheDir Nextflow options to be able to store and re-use the images from a central location for future pipeline runs.

    • If you are using conda , it is highly recommended to use the NXF_CONDA_CACHEDIR or conda.cacheDir settings to store the environments in a central location for future pipeline runs.

  4. Start running your own analysis! 

    nextflow run nf-core/bactmap -profile <docker/singularity/podman/conda/institute> --input samplesheet.csv --reference chromosome.fasta

Documentation

The nf-core/bactmap pipeline comes with documentation about the pipeline usage , parameters and output .

Credits

nf-core/bactmap was originally written by Anthony Underwood , Andries van Tonder and Thanh Le Viet .

We thank the following people for their extensive assistance in the development of this pipeline:

Anthony Underwood's time working on the project was funded by the National Institute for Health Research (NIHR) Global Health Research Unit for the Surveillance of Antimicrobial Resistance (Grant Reference Number 16/136/111) NIHR funded

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines .

For further information or help, don't hesitate to get in touch on the Slack #bactmap channel (you can join with this invite ).

Citations

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x .

Code Snippets

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"""
touch low_quality_pseudogenomes.tsv
touch aligned_pseudogenomes.fas
for pseudogenome in ${pseudogenomes}
do
    fraction_non_GATC_bases=\$(calculate_fraction_of_non_GATC_bases.py -f \$pseudogenome | tr -d '\\n')
    if awk 'BEGIN { exit !(\$fraction_non_GATC_bases < ${params.non_GATC_threshold}) }'; then
        cat \$pseudogenome >> aligned_pseudogenomes.fas
    else
        echo "\$pseudogenome\t\$fraction_non_GATC_bases" >> low_quality_pseudogenomes.tsv
    fi
done
reference2single_sequence.py -r ${reference} -o final_reference.fas
cat final_reference.fas >> aligned_pseudogenomes.fas

NUM_ALIGNMENT_GENOMES=\$(grep -c ">" aligned_pseudogenomes.fas)
"""
NextFlow From line 30 of local/alignpseudogenomes.nf 
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"""
echo $workflow.manifest.version > pipeline.version.txt
echo $workflow.nextflow.version > nextflow.version.txt
scrape_software_versions.py &> software_versions_mqc.yaml
"""
NextFlow From line 27 of local/get_software_versions.nf 
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"""
multiqc -f $options.args $custom_config .
multiqc --version | sed -e "s/multiqc, version //g" > ${software}.version.txt
"""
NextFlow MultiQC From line 39 of local/multiqc.nf 
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"""
check_samplesheet.py $samplesheet samplesheet.valid.csv
"""
NextFlow From line 26 of local/samplesheet_check.nf 
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"""
vcf2pseudogenome.py  -r ${reference} -b ${vcf} -o ${meta.id}.fas
"""
NextFlow From line 30 of local/vcf2pseudogenome.nf 
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"""
bcftools filter \\
    --output ${prefix}.vcf.gz \\
    $options.args \\
    $vcf

echo \$(bcftools --version 2>&1) | sed 's/^.*bcftools //; s/ .*\$//' > ${software}.version.txt
"""
NextFlow BCFtools From line 31 of filter/main.nf 
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"""
echo "${meta.id}" > sample_name.list
bcftools mpileup \\
    --fasta-ref $fasta \\
    $options.args \\
    $bam \\
    | bcftools call --output-type v $options.args2 \\
    | bcftools reheader --samples sample_name.list \\
    | bcftools view --output-file ${prefix}.vcf.gz --output-type z $options.args3
tabix -p vcf -f ${prefix}.vcf.gz
bcftools stats ${prefix}.vcf.gz > ${prefix}.bcftools_stats.txt
echo \$(bcftools --version 2>&1) | sed 's/^.*bcftools //; s/ .*\$//' > ${software}.version.txt
"""
NextFlow BCFtools tabix From line 34 of mpileup/main.nf 
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"""
mkdir index
bwa index $options.args -p index/${fasta.baseName} $fasta 
echo \$(bwa 2>&1) | sed 's/^.*Version: //; s/Contact:.*\$//' > ${software}.version.txt
"""
NextFlow BWA From line 30 of index/main.nf 
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"""
INDEX=`find -L ./ -name "*.amb" | sed 's/.amb//'`

bwa mem \\
    $options.args \\
    $read_group \\
    -t $task.cpus \\
    \$INDEX \\
    $reads \\
    | samtools view $options.args2 -@ $task.cpus -bhS -o ${prefix}.bam -

echo \$(bwa 2>&1) | sed 's/^.*Version: //; s/Contact:.*\$//' > ${software}.version.txt
"""
NextFlow SAMtools BWA From line 33 of mem/main.nf 
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"""
[ ! -f  ${prefix}.fastq.gz ] && ln -s $reads ${prefix}.fastq.gz
fastp \\
    --in1 ${prefix}.fastq.gz \\
    --out1 ${prefix}.trim.fastq.gz \\
    --thread $task.cpus \\
    --json ${prefix}.fastp.json \\
    --html ${prefix}.fastp.html \\
    $fail_fastq \\
    $options.args \\
    2> ${prefix}.fastp.log
echo \$(fastp --version 2>&1) | sed -e "s/fastp //g" > ${software}.version.txt
"""
NextFlow fastp From line 39 of fastp/main.nf 
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"""
[ ! -f  ${prefix}_1.fastq.gz ] && ln -s ${reads[0]} ${prefix}_1.fastq.gz
[ ! -f  ${prefix}_2.fastq.gz ] && ln -s ${reads[1]} ${prefix}_2.fastq.gz
fastp \\
    --in1 ${prefix}_1.fastq.gz \\
    --in2 ${prefix}_2.fastq.gz \\
    --out1 ${prefix}_1.trim.fastq.gz \\
    --out2 ${prefix}_2.trim.fastq.gz \\
    --json ${prefix}.fastp.json \\
    --html ${prefix}.fastp.html \\
    $fail_fastq \\
    --thread $task.cpus \\
    --detect_adapter_for_pe \\
    $options.args \\
    2> ${prefix}.fastp.log

echo \$(fastp --version 2>&1) | sed -e "s/fastp //g" > ${software}.version.txt
"""
NextFlow fastp From line 54 of fastp/main.nf 
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"""
fasttree \\
    $options.args \\
    -log fasttree_phylogeny.tre.log \\
    -nt $alignment \\
    > fasttree_phylogeny.tre

echo \$(fasttree -help 2>&1) | head -1  | sed 's/^FastTree \\([0-9\\.]*\\) .*\$/\\1/' > ${software}.version.txt
"""
NextFlow fasttree From line 30 of fasttree/main.nf 
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"""
run_gubbins.py \\
    --threads $task.cpus \\
    $options.args \\
    $alignment
echo \$(run_gubbins.py --version 2>&1) > ${software}.version.txt
"""
NextFlow From line 37 of gubbins/main.nf 
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"""
iqtree \\
    $fconst_args \\
    $options.args \\
    -s $alignment \\
    -nt AUTO \\
    -ntmax $task.cpus \\
    -mem $memory \\

echo \$(iqtree -version 2>&1) | sed 's/^IQ-TREE multicore version \\([0-9\\.]*\\) .*\$/\\1/' > ${software}.version.txt
"""
NextFlow IQ-TREE From line 32 of iqtree/main.nf 
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"""
mash \\
    sketch \\
    $options.args \\
    -p $task.cpus \\
    -o ${prefix} \\
    -r $reads \\
    2> ${prefix}.mash_stats
echo \$(mash --version 2>&1) > ${software}.version.txt
"""
NextFlow Mash From line 30 of sketch/main.nf 
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"""
python \\
    -c 'from Bio import SeqIO; SeqIO.convert("$alignment", "fasta", "alignment.sth", "stockholm")'

rapidnj \\
    alignment.sth \\
    $options.args \\
    -i sth \\
    -c $task.cpus \\
    -x rapidnj_phylogeny.tre

# Doesn't appear to be a way of getting the version number
echo 2.3.2 > ${software}.version.txt
"""
NextFlow RapidNJ From line 31 of rapidnj/main.nf 
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"""
rasusa \\
    $options.args \\
    --coverage $depth_cutoff \\
    --genome-size $genome_size \\
    --input $reads \\
    $output
echo \$(rasusa --version 2>&1) | sed -e "s/rasusa //g" > ${software}.version.txt
"""
NextFlow rasusa From line 33 of rasusa/main.nf 
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"""
raxml-ng \\
    $options.args \\
    --msa $alignment \\
    --threads $task.cpus \\
    --prefix output

echo \$(raxml-ng --version 2>&1) | sed 's/^.*RAxML-NG v. //; s/released.*\$//' > ${software}.version.txt
"""
NextFlow raxml-ng From line 31 of raxmlng/main.nf 
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"""
samtools flagstat $bam > ${bam}.flagstat
echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//' > ${software}.version.txt
"""
NextFlow SAMtools From line 30 of flagstat/main.nf 
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"""
samtools idxstats $bam > ${bam}.idxstats
echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//' > ${software}.version.txt
"""
NextFlow SAMtools From line 30 of idxstats/main.nf 
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"""
samtools index $bam
echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//' > ${software}.version.txt
"""
NextFlow SAMtools From line 30 of index/main.nf 
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"""
samtools sort $options.args -@ $task.cpus -o ${prefix}.bam -T $prefix $bam
echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//' > ${software}.version.txt
"""
NextFlow SAMtools From line 31 of sort/main.nf 
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"""
samtools stats $bam > ${bam}.stats
echo \$(samtools --version 2>&1) | sed 's/^.*samtools //; s/Using.*\$//' > ${software}.version.txt
"""
NextFlow SAMtools From line 30 of stats/main.nf 
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"""
snp-sites \\
    $alignment \\
    > filtered_alignment.fas

echo \$(snp-sites -C $alignment) > constant.sites.txt

CONSTANT_SITES=\$(cat constant.sites.txt)

echo \$(snp-sites -V 2>&1) | sed 's/snp-sites //' > ${software}.version.txt
"""
NextFlow From line 31 of snpsites/main.nf
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Created: 1yr ago
Updated: 1yr ago
Maitainers: public
URL: https://nf-co.re/bactmap
Name: bactmap
Version: 1.0.0
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Copyright: Public Domain
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