snippy

Tags: snp variant-calling phylogeny core-genome snippy bactopia-tool

Rapid haplotype variant calling and core genome alignment.

This Bactopia Tool uses Snippy to find SNPs between a reference genome and a set of reads, perform core genome alignment, and generate phylogenetic trees. It includes optional recombination detection with Gubbins and phylogenetic tree construction with IQ-Tree.

Usage

Bactopia CLI:

bactopia --wf snippy \
  --bactopia /path/to/your/bactopia/results

Nextflow:

nextflow run bactopia/bactopia/workflows/bactopia-tools/snippy/main.nf \
  --bactopia /path/to/your/bactopia/results

Outputs

Expected Output Files

<BACTOPIA_DIR>
├── <SAMPLE_NAME>
│   └── tools
│       └── snippy-<TIMESTAMP>
│           └── GCF_000292685
│               ├── <SAMPLE_NAME>.aligned.fa.gz
│               ├── <SAMPLE_NAME>.annotated.vcf.gz
│               ├── <SAMPLE_NAME>.bam
│               ├── <SAMPLE_NAME>.bam.bai
│               ├── <SAMPLE_NAME>.bed.gz
│               ├── <SAMPLE_NAME>.consensus.fa.gz
│               ├── <SAMPLE_NAME>.consensus.subs.fa.gz
│               ├── <SAMPLE_NAME>.consensus.subs.masked.fa.gz
│               ├── <SAMPLE_NAME>.coverage.txt.gz
│               ├── <SAMPLE_NAME>.csv.gz
│               ├── <SAMPLE_NAME>.filt.vcf.gz
│               ├── <SAMPLE_NAME>.gff.gz
│               ├── <SAMPLE_NAME>.html
│               ├── <SAMPLE_NAME>.raw.vcf.gz
│               ├── <SAMPLE_NAME>.subs.vcf.gz
│               ├── <SAMPLE_NAME>.tab
│               ├── <SAMPLE_NAME>.txt
│               ├── <SAMPLE_NAME>.vcf.gz
│               └── logs
│                   ├── <SAMPLE_NAME>.log
│                   ├── nf.command.{begin,err,log,out,run,sh,trace}
│                   └── versions.yml
├── ERR6005894
│   └── tools
│       └── snippy-<TIMESTAMP>
│           └── GCF_000292685
│               ├── ERR6005894.aligned.fa.gz
│               ├── ERR6005894.annotated.vcf.gz
│               ├── ERR6005894.bam
│               ├── ERR6005894.bam.bai
│               ├── ERR6005894.bed.gz
│               ├── ERR6005894.consensus.fa.gz
│               ├── ERR6005894.consensus.subs.fa.gz
│               ├── ERR6005894.consensus.subs.masked.fa.gz
│               ├── ERR6005894.coverage.txt.gz
│               ├── ERR6005894.csv.gz
│               ├── ERR6005894.filt.vcf.gz
│               ├── ERR6005894.gff.gz
│               ├── ERR6005894.html
│               ├── ERR6005894.raw.vcf.gz
│               ├── ERR6005894.subs.vcf.gz
│               ├── ERR6005894.tab
│               ├── ERR6005894.txt
│               ├── ERR6005894.vcf.gz
│               └── logs
│                   ├── ERR6005894.log
│                   ├── nf.command.{begin,err,log,out,run,sh,trace}
│                   └── versions.yml
├── ERR6005894SE
│   └── tools
│       └── snippy-<TIMESTAMP>
│           └── GCF_000292685
│               ├── ERR6005894SE.aligned.fa.gz
│               ├── ERR6005894SE.annotated.vcf.gz
│               ├── ERR6005894SE.bam
│               ├── ERR6005894SE.bam.bai
│               ├── ERR6005894SE.bed.gz
│               ├── ERR6005894SE.consensus.fa.gz
│               ├── ERR6005894SE.consensus.subs.fa.gz
│               ├── ERR6005894SE.consensus.subs.masked.fa.gz
│               ├── ERR6005894SE.coverage.txt.gz
│               ├── ERR6005894SE.csv.gz
│               ├── ERR6005894SE.filt.vcf.gz
│               ├── ERR6005894SE.gff.gz
│               ├── ERR6005894SE.html
│               ├── ERR6005894SE.raw.vcf.gz
│               ├── ERR6005894SE.subs.vcf.gz
│               ├── ERR6005894SE.tab
│               ├── ERR6005894SE.txt
│               ├── ERR6005894SE.vcf.gz
│               └── logs
│                   ├── ERR6005894SE.log
│                   ├── nf.command.{begin,err,log,out,run,sh,trace}
│                   └── versions.yml
├── SRR2838702
│   └── tools
│       └── snippy-<TIMESTAMP>
│           └── GCF_000292685
│               ├── SRR2838702.aligned.fa.gz
│               ├── SRR2838702.annotated.vcf.gz
│               ├── SRR2838702.bam
│               ├── SRR2838702.bam.bai
│               ├── SRR2838702.bed.gz
│               ├── SRR2838702.consensus.fa.gz
│               ├── SRR2838702.consensus.subs.fa.gz
│               ├── SRR2838702.consensus.subs.masked.fa.gz
│               ├── SRR2838702.coverage.txt.gz
│               ├── SRR2838702.csv.gz
│               ├── SRR2838702.filt.vcf.gz
│               ├── SRR2838702.gff.gz
│               ├── SRR2838702.html
│               ├── SRR2838702.raw.vcf.gz
│               ├── SRR2838702.subs.vcf.gz
│               ├── SRR2838702.tab
│               ├── SRR2838702.txt
│               ├── SRR2838702.vcf.gz
│               └── logs
│                   ├── SRR2838702.log
│                   ├── nf.command.{begin,err,log,out,run,sh,trace}
│                   └── versions.yml
├── SRR2838702SE
│   └── tools
│       └── snippy-<TIMESTAMP>
│           └── GCF_000292685
│               ├── SRR2838702SE.aligned.fa.gz
│               ├── SRR2838702SE.annotated.vcf.gz
│               ├── SRR2838702SE.bam
│               ├── SRR2838702SE.bam.bai
│               ├── SRR2838702SE.bed.gz
│               ├── SRR2838702SE.consensus.fa.gz
│               ├── SRR2838702SE.consensus.subs.fa.gz
│               ├── SRR2838702SE.consensus.subs.masked.fa.gz
│               ├── SRR2838702SE.coverage.txt.gz
│               ├── SRR2838702SE.csv.gz
│               ├── SRR2838702SE.filt.vcf.gz
│               ├── SRR2838702SE.gff.gz
│               ├── SRR2838702SE.html
│               ├── SRR2838702SE.raw.vcf.gz
│               ├── SRR2838702SE.subs.vcf.gz
│               ├── SRR2838702SE.tab
│               ├── SRR2838702SE.txt
│               ├── SRR2838702SE.vcf.gz
│               └── logs
│                   ├── SRR2838702SE.log
│                   ├── nf.command.{begin,err,log,out,run,sh,trace}
│                   └── versions.yml
├── SRR2838702SE_2
│   └── tools
│       └── snippy-<TIMESTAMP>
│           └── GCF_000292685
│               ├── SRR2838702SE_2.aligned.fa.gz
│               ├── SRR2838702SE_2.annotated.vcf.gz
│               ├── SRR2838702SE_2.bam
│               ├── SRR2838702SE_2.bam.bai
│               ├── SRR2838702SE_2.bed.gz
│               ├── SRR2838702SE_2.consensus.fa.gz
│               ├── SRR2838702SE_2.consensus.subs.fa.gz
│               ├── SRR2838702SE_2.consensus.subs.masked.fa.gz
│               ├── SRR2838702SE_2.coverage.txt.gz
│               ├── SRR2838702SE_2.csv.gz
│               ├── SRR2838702SE_2.filt.vcf.gz
│               ├── SRR2838702SE_2.gff.gz
│               ├── SRR2838702SE_2.html
│               ├── SRR2838702SE_2.raw.vcf.gz
│               ├── SRR2838702SE_2.subs.vcf.gz
│               ├── SRR2838702SE_2.tab
│               ├── SRR2838702SE_2.txt
│               ├── SRR2838702SE_2.vcf.gz
│               └── logs
│                   ├── SRR2838702SE_2.log
│                   ├── nf.command.{begin,err,log,out,run,sh,trace}
│                   └── versions.yml
├── SRR2838702_2
│   └── tools
│       └── snippy-<TIMESTAMP>
│           └── GCF_000292685
│               ├── SRR2838702_2.aligned.fa.gz
│               ├── SRR2838702_2.annotated.vcf.gz
│               ├── SRR2838702_2.bam
│               ├── SRR2838702_2.bam.bai
│               ├── SRR2838702_2.bed.gz
│               ├── SRR2838702_2.consensus.fa.gz
│               ├── SRR2838702_2.consensus.subs.fa.gz
│               ├── SRR2838702_2.consensus.subs.masked.fa.gz
│               ├── SRR2838702_2.coverage.txt.gz
│               ├── SRR2838702_2.csv.gz
│               ├── SRR2838702_2.filt.vcf.gz
│               ├── SRR2838702_2.gff.gz
│               ├── SRR2838702_2.html
│               ├── SRR2838702_2.raw.vcf.gz
│               ├── SRR2838702_2.subs.vcf.gz
│               ├── SRR2838702_2.tab
│               ├── SRR2838702_2.txt
│               ├── SRR2838702_2.vcf.gz
│               └── logs
│                   ├── SRR2838702_2.log
│                   ├── nf.command.{begin,err,log,out,run,sh,trace}
│                   └── versions.yml
└── bactopia-runs
    └── snippy-<TIMESTAMP>
        ├── GCF_000292685.samples.txt
        ├── core-snp-clean.full.aln.gz
        ├── core-snp.distance.tsv
        ├── core-snp.full.aln.gz
        ├── core-snp.masked.aln.gz
        ├── core-snp.masked.distance.tsv
        ├── gubbins
        │   ├── core-snp.branch_base_reconstruction.embl.gz
        │   ├── core-snp.filtered_polymorphic_sites.fasta.gz
        │   ├── core-snp.filtered_polymorphic_sites.phylip
        │   ├── core-snp.final_tree.tre
        │   ├── core-snp.node_labelled.final_tree.tre
        │   ├── core-snp.per_branch_statistics.csv
        │   ├── core-snp.recombination_predictions.embl.gz
        │   ├── core-snp.recombination_predictions.gff.gz
        │   ├── core-snp.summary_of_snp_distribution.vcf.gz
        │   └── logs
        │       ├── core-snp.log
        │       ├── nf.command.{begin,err,log,out,run,sh,trace}
        │       └── versions.yml
        ├── nf-reports
        │   ├── snippy-dag.dot
        │   ├── snippy-report.html
        │   └── snippy-timeline.html
        ├── snippy-core
        │   ├── core-snp.aln.gz
        │   ├── core-snp.tab.gz
        │   ├── core-snp.txt
        │   ├── core-snp.vcf.gz
        │   └── logs
        │       ├── nf.command.{begin,err,log,out,run,sh,trace}
        │       └── versions.yml
        ├── snpdists
        │   └── logs
        │       ├── nf.command.{begin,err,log,out,run,sh,trace}
        │       └── versions.yml
        └── snpdists-masked
            └── logs
                ├── nf.command.{begin,err,log,out,run,sh,trace}
                └── versions.yml

Variant Calling

File	Description
*.vcf	Variant calls in VCF format
*.bam	Alignment file
*.txt	Snippy summary report

Core Genome Alignment

File	Description
core.full.aln	Full core genome alignment
core.snps.aln	Core SNP alignment

Recombination Analysis

note

Only created if recombination analysis is enabled

File	Description
*.filtered.aln	Alignment with recombination regions removed
*.gff	Recombination predictions

Phylogeny

note

Only created if phylogeny analysis is enabled

File	Description
*.treefile	Phylogenetic tree in Newick format

Merged Results

File	Description
snippy.tsv	Merged summary of Snippy analyses

Audit Trail

Below are files that can assist you in understanding which parameters and program versions were used.

Logs

Each process that is executed will have a folder named logs. In this folder are helpful files for you to review if the need ever arises.

Extension	Description
.begin	An empty file used to designate the process started
.err	Contains STDERR outputs from the process
.log	Contains both STDERR and STDOUT outputs from the process
.out	Contains STDOUT outputs from the process
.run	The script Nextflow uses to stage/unstage files and queue processes based on given profile
.sh	The script executed by bash for the process
.trace	The Nextflow trace report for the process
versions.yml	A YAML formatted file with program versions

Nextflow Reports

These Nextflow reports provide great a great summary of your run. These can be used to optimize resource usage and estimate expected costs if using cloud platforms.

Filename	Description
snippy-dag.dot	The Nextflow DAG visualization
snippy-report.html	The Nextflow Execution Report
snippy-timeline.html	The Nextflow Timeline Report
snippy-trace.txt	The Nextflow Trace report

Parameters

Required Parameters

Define where the pipeline should find input data and save output data.

Parameter	Type	Default	Description
--bactopia	string		The path to bactopia results to use as inputs

NCBI Genome Download Parameters

Parameter	Type	Default	Description
--species	string		Name of the species to download assemblies
--accession	string		An NCBI Assembly accession to be downloaded
--accessions	string		An file of NCBI Assembly accessions (one per line) to be downloaded
--format	string	fasta	Comma separated list of formats to download
--section	string	refseq	NCBI section to download
--assembly_level	string	complete	Comma separated list of assembly levels to download
--kingdom	string	bacteria	Comma separated list of formats to download
--limit	string		Limit the number of assemblies to download
--keep_downloads	boolean	false	Save downloaded files into the bactopia-runs folder

Snippy Run Parameters

Parameter	Type	Default	Description
--reference	string		Reference genome in GenBank format
--snippy_mapqual	integer	60	Minimum read mapping quality to consider
--snippy_basequal	integer	13	Minimum base quality to consider
--snippy_mincov	integer	10	Minimum site depth to for calling alleles
--snippy_minfrac	integer	0	Minimum proportion for variant evidence (0=AUTO)
--snippy_minqual	integer	100	Minimum QUALITY in VCF column 6
--snippy_maxsoft	integer	10	Maximum soft clipping to allow
--snippy_bwaopt	string		Extra BWA MEM options, eg. -x pacbio
--snippy_fbopt	string		Extra Freebayes options, eg. --theta 1E-6 --read-snp-limit 2
--snippy_remove_bam	boolean	false	Delete BAM files after variant calling
--snippy_opts	string		Extra options in quotes for Snippy

Snippy-Core Parameters

Parameter	Type	Default	Description
--snippy_core_maxhap	integer	100	Largest haplotype to decompose
--snippy_core_mask	string		BED file of sites to mask
--snippy_core_mask_char	string	X	Masking character
--snippy_core_opts	string		Extra options in quotes for snippy-core

SNP-Dists Parameters

Parameter	Type	Default	Description
--snpdists_a	boolean	false	Count all differences not just [AGTC]
--snpdists_b	boolean	false	Keep top left corner cell
--snpdists_csv	boolean	false	Output CSV instead of TSV
--snpdists_k	boolean	false	Keep case, don't uppercase all letters

Gubbins Parameters

Parameter	Type	Default	Description
--gubbins_iterations	integer	5	Maximum number of iterations
--gubbins_min_snps	integer	3	Min SNPs to identify a recombination block
--gubbins_min_window_size	integer	100	Minimum window size
--gubbins_max_window_size	integer	10000	Maximum window size
--gubbins_filter_percentage	number	25.0	Filter out taxa with more than this percentage of gaps
--gubbins_remove_identical_sequences	boolean	false	Remove identical sequences
--gubbins_opts	string		Extra Gubbins options in quotes
--skip_recombination	boolean	false	Skip Gubbins execution in subworkflows

IQ-TREE Parameters

Parameter	Type	Default	Description
--iqtree_model	string	HKY	Substitution model name
--iqtree_bb	integer	1000	Ultrafast bootstrap replicates
--iqtree_alrt	integer	1000	SH-like approximate likelihood ratio test replicates
--iqtree_asr	boolean	false	Ancestral state reconstruction by empirical Bayes
--iqtree_opts	string		Extra IQ-TREE options in quotes.
--skip_phylogeny	boolean	false	Skip IQ-TREE execution in subworkflows

Filtering Parameters

Use these parameters to specify which samples to include or exclude.

Parameter	Type	Default	Description
--include	string		A text file containing sample names (one per line) to include from the analysis
--exclude	string		A text file containing sample names (one per line) to exclude from the analysis

Optional Parameters

These optional parameters can be useful in certain settings.

Parameter	Type	Default	Description
--outdir	string	bactopia	Base directory to write results to
--skip_compression	boolean	false	Output files will not be compressed
--datasets	string		The path to cache datasets to
--keep_all_files	boolean	false	Keeps all analysis files created

Max Job Request Parameters

Set the top limit for requested resources for any single job.

Parameter	Type	Default	Description
--max_retry	integer	3	Maximum times to retry a process before allowing it to fail.
--max_cpus	integer	4	Maximum number of CPUs that can be requested for any single job.
--max_memory	string	128.GB	Maximum amount of memory that can be requested for any single job.
--max_time	string	240.h	Maximum amount of time that can be requested for any single job.
--max_downloads	integer	3	Maximum number of samples to download at a time

Nextflow Configuration Parameters

Parameters to fine-tune your Nextflow setup.

Parameter	Type	Default	Description
--nfconfig	string		A Nextflow compatible config file for custom profiles, loaded last and will overwrite existing variables if set.
--publish_dir_mode	string	copy	Method used to save pipeline results to output directory. (choices: symlink, rellink, link, copy, copyNoFollow, move)
--infodir	string	${params.outdir}/pipeline_info	Directory to keep pipeline Nextflow logs and reports.
--force	boolean	false	Nextflow will overwrite existing output files.
--cleanup_workdir	boolean	false	After Bactopia is successfully executed, the work directory will be deleted.

Institutional config options

Parameters used to describe centralized config profiles. These should not be edited.

Parameter	Type	Default	Description
--custom_config_version	string	master	Git commit id for Institutional configs.
--custom_config_base	string	https://raw.githubusercontent.com/nf-core/configs/master	Base directory for Institutional configs.
--config_profile_name	string		Institutional config name.
--config_profile_description	string		Institutional config description.
--config_profile_contact	string		Institutional config contact information.
--config_profile_url	string		Institutional config URL link.

Nextflow Profile Parameters

Parameters to fine-tune your Nextflow setup.

Parameter	Type	Default	Description
--condadir	string		Directory to Nextflow should use for Conda environments
--registry	string	quay.io	Registry to pull Docker containers from.
--datasets_cache	string	<HOME>/.bactopia/datasets	Directory where downloaded datasets should be stored.
--singularity_cache	string		Directory where remote Singularity images are stored.
--singularity_pull_docker_container	boolean		Instead of directly downloading Singularity images for use with Singularity, force the workflow to pull and convert Docker containers instead.
--force_rebuild	boolean	false	Force overwrite of existing pre-built environments.
--queue	string	general,high-memory	Comma-separated name of the queue(s) to be used by a job scheduler (e.g. AWS Batch or SLURM)
--cluster_opts	string		Additional options to pass to the executor. (e.g. SLURM: '--account=my_acct_name'
--container_opts	string		Additional options to pass to Apptainer, Docker, or Singularity. (e.g. Singularity: '-D pwd'
--disable_scratch	boolean	false	All intermediate files created on worker nodes of will be transferred to the head node.

Helpful Parameters

Uncommonly used parameters that might be useful.

Parameter	Type	Default	Description
--monochrome_logs	boolean		Do not use coloured log outputs.
--nfdir	boolean		Print directory Nextflow has pulled Bactopia to
--sleep_time	integer	5	The amount of time (seconds) Nextflow will wait after setting up datasets before execution.
--validate_params	boolean	true	Boolean whether to validate parameters against the schema at runtime
--help	boolean		Display help text.
--wf	string	bactopia	Specify which workflow or Bactopia Tool to execute
--list_wfs	boolean		List the available workflows and Bactopia Tools to use with '--wf'
--show_hidden_params	boolean		Show all params when using --help
--help_all	boolean		An alias for --help --show_hidden_params
--version	boolean		Display version text.

Composition

This workflow uses the following subworkflows:

• gubbins - Detect and filter recombination regions in bacterial alignments.
• iqtree - Construct maximum likelihood phylogenetic trees from alignments.
• ncbigenomedownload - Download bacterial genomes from NCBI's RefSeq database.
• snippy_core - Generate core-genome SNP alignment from per-sample Snippy outputs.
• snippy_run - Call variants against a reference genome using Snippy.

Citations

If you use this in your analysis, please cite the following.

• Bactopia
  Petit III RA, Read TD Bactopia - a flexible pipeline for complete analysis of bacterial genomes. mSystems 5 (2020)

• Snippy
  Seemann T Snippy: fast bacterial variant calling from NGS reads (GitHub)

• Gubbins
  Croucher NJ, Page AJ, Connor TR, Delaney AJ, Keane JA, Bentley SD, Parkhill J, Harris SR Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Research 43(3), e15. (2015)

• IQ-TREE
  Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Mol. Biol. Evol. 32:268-274 (2015)

Source

View source on GitHub