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
`--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_bwaopt`	string		Extra BWA MEM options, eg. -x pacbio
`--snippy_fbopt`	string		Extra Freebayes options, eg. --theta 1E-6 --read-snp-limit 2
`--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]

Gubbins Parameters

Parameter	Type	Default	Description
`--gubbins_iterations`	integer	`5`	Maximum number of iterations
`--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
`--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

Nextflow Profile Parameters

Parameters to fine-tune your Nextflow setup.

Parameter	Type	Default	Description
`--datasets_cache`	string	`<HOME>/.bactopia/datasets`	Directory where downloaded datasets should be stored.

Helpful Parameters

Uncommonly used parameters that might be useful.

Parameter	Type	Default	Description
`--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'
`--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

Usage​

Outputs​

Expected Output Files​

Variant Calling​

Core Genome Alignment​

Recombination Analysis​

Phylogeny​

Merged Results​

Audit Trail​

Logs​

Nextflow Reports​

Parameters​

Required Parameters​

NCBI Genome Download Parameters​

Snippy Run Parameters​

Snippy-Core Parameters​

SNP-Dists Parameters​

Gubbins Parameters​

IQ-TREE Parameters​

Composition​

Citations​

Source​