staphopia

Tags: staphylococcus-aureus assembly annotation amr mlst spa-typing agr-typing sccmec named-workflow

Comprehensive analysis pipeline for Staphylococcus aureus isolates.

This workflow performs complete bacterial analysis including quality control, assembly, annotation, antimicrobial resistance detection, MLST typing, and Staphylococcus-specific analysis using Spatyper, AgrVATE, and SCCmecFinder. It processes raw sequencing reads and produces a comprehensive genomic characterization for S. aureus isolates.

Usage

staphopia CLI:

staphopia \
  --input samples.csv \
  --outdir results/

Nextflow:

nextflow run bactopia/bactopia/workflows/staphopia/main.nf \
  --input samples.csv \
  --outdir results/

Outputs

Expected Output Files

<BACTOPIA_DIR>
├── <SAMPLE_NAME>
│   ├── main
│   │   ├── annotator
│   │   │   └── prokka
│   │   │       ├── <SAMPLE_NAME>-blastdb.tar.gz
│   │   │       ├── <SAMPLE_NAME>.faa.gz
│   │   │       ├── <SAMPLE_NAME>.ffn.gz
│   │   │       ├── <SAMPLE_NAME>.fna.gz
│   │   │       ├── <SAMPLE_NAME>.fsa.gz
│   │   │       ├── <SAMPLE_NAME>.gbk.gz
│   │   │       ├── <SAMPLE_NAME>.gff.gz
│   │   │       ├── <SAMPLE_NAME>.sqn.gz
│   │   │       ├── <SAMPLE_NAME>.tbl.gz
│   │   │       ├── <SAMPLE_NAME>.tsv
│   │   │       ├── <SAMPLE_NAME>.txt
│   │   │       └── logs
│   │   │           ├── <SAMPLE_NAME>.err
│   │   │           ├── <SAMPLE_NAME>.log
│   │   │           ├── nf.command.{begin,err,log,out,run,sh,trace}
│   │   │           └── versions.yml
│   │   ├── assembler
│   │   │   ├── <SAMPLE_NAME>.fna.gz
│   │   │   ├── <SAMPLE_NAME>.tsv
│   │   │   ├── logs
│   │   │   │   ├── nf.command.{begin,err,log,out,run,sh,trace}
│   │   │   │   ├── shovill.log
│   │   │   │   └── versions.yml
│   │   │   └── supplemental
│   │   │       ├── flash.hist
│   │   │       ├── flash.histogram
│   │   │       └── shovill.corrections
│   │   ├── gather
│   │   │   ├── <SAMPLE_NAME>-meta.tsv
│   │   │   └── logs
│   │   │       ├── nf.command.{begin,err,log,out,run,sh,trace}
│   │   │       └── versions.yml
│   │   ├── qc
│   │   │   ├── <SAMPLE_NAME>_R1.fastq.gz
│   │   │   ├── <SAMPLE_NAME>_R2.fastq.gz
│   │   │   ├── logs
│   │   │   │   ├── <SAMPLE_NAME>-fastp.log
│   │   │   │   ├── nf.command.{begin,err,log,out,run,sh,trace}
│   │   │   │   └── versions.yml
│   │   │   └── supplemental
│   │   │       ├── <SAMPLE_NAME>.fastp.html
│   │   │       ├── <SAMPLE_NAME>.fastp.json
│   │   │       ├── <SAMPLE_NAME>_R1-final.json
│   │   │       ├── <SAMPLE_NAME>_R1-final_fastqc.html
│   │   │       ├── <SAMPLE_NAME>_R1-final_fastqc.zip
│   │   │       ├── <SAMPLE_NAME>_R1-original.json
│   │   │       ├── <SAMPLE_NAME>_R1-original_fastqc.html
│   │   │       ├── <SAMPLE_NAME>_R1-original_fastqc.zip
│   │   │       ├── <SAMPLE_NAME>_R2-final.json
│   │   │       ├── <SAMPLE_NAME>_R2-final_fastqc.html
│   │   │       ├── <SAMPLE_NAME>_R2-final_fastqc.zip
│   │   │       ├── <SAMPLE_NAME>_R2-original.json
│   │   │       ├── <SAMPLE_NAME>_R2-original_fastqc.html
│   │   │       └── <SAMPLE_NAME>_R2-original_fastqc.zip
│   │   └── sketcher
│   │       ├── <SAMPLE_NAME>-k21.msh
│   │       ├── <SAMPLE_NAME>-k31.msh
│   │       ├── <SAMPLE_NAME>-mash-refseq88-k21.txt
│   │       ├── <SAMPLE_NAME>-sourmash-gtdb-rs207-k31.txt
│   │       ├── <SAMPLE_NAME>.sig
│   │       └── logs
│   │           ├── nf.command.{begin,err,log,out,run,sh,trace}
│   │           └── versions.yml
│   └── tools
│       ├── agrvate
│       │   ├── <SAMPLE_NAME>.tsv
│       │   ├── logs
│       │   │   ├── nf.command.{begin,err,log,out,run,sh,trace}
│       │   │   └── versions.yml
│       │   └── supplemental
│       │       ├── <SAMPLE_NAME>-agr_gp.tab
│       │       ├── <SAMPLE_NAME>-blastn_log.txt
│       │       ├── <SAMPLE_NAME>-hmm-log.txt
│       │       ├── <SAMPLE_NAME>-hmm.tab
│       │       └── <SAMPLE_NAME>.fna-error-report.tab
│       ├── amrfinderplus
│       │   ├── <SAMPLE_NAME>.tsv
│       │   └── logs
│       │       ├── nf.command.{begin,err,log,out,run,sh,trace}
│       │       └── versions.yml
│       ├── mlst
│       │   ├── <SAMPLE_NAME>.tsv
│       │   └── logs
│       │       ├── nf.command.{begin,err,log,out,run,sh,trace}
│       │       └── versions.yml
│       ├── sccmec
│       │   ├── <SAMPLE_NAME>.regions.blastn.tsv
│       │   ├── <SAMPLE_NAME>.regions.details.tsv
│       │   ├── <SAMPLE_NAME>.targets.blastn.tsv
│       │   ├── <SAMPLE_NAME>.targets.details.tsv
│       │   ├── <SAMPLE_NAME>.tsv
│       │   └── logs
│       │       ├── nf.command.{begin,err,log,out,run,sh,trace}
│       │       └── versions.yml
│       └── spatyper
│           ├── <SAMPLE_NAME>.tsv
│           └── logs
│               ├── nf.command.{begin,err,log,out,run,sh,trace}
│               └── versions.yml
└── bactopia-runs
    └── staphopia-<TIMESTAMP>
        ├── merged-results
        │   ├── agrvate.tsv
        │   ├── amrfinderplus.tsv
        │   ├── assembly-scan.tsv
        │   ├── logs
        │   │   ├── agrvate-concat
        │   │   │   ├── nf.command.{begin,err,log,out,run,sh,trace}
        │   │   │   └── versions.yml
        │   │   ├── amrfinderplus-concat
        │   │   │   ├── nf.command.{begin,err,log,out,run,sh,trace}
        │   │   │   └── versions.yml
        │   │   ├── assembly-scan-concat
        │   │   │   ├── nf.command.{begin,err,log,out,run,sh,trace}
        │   │   │   └── versions.yml
        │   │   ├── meta-concat
        │   │   │   ├── nf.command.{begin,err,log,out,run,sh,trace}
        │   │   │   └── versions.yml
        │   │   ├── mlst-concat
        │   │   │   ├── nf.command.{begin,err,log,out,run,sh,trace}
        │   │   │   └── versions.yml
        │   │   ├── sccmec-concat
        │   │   │   ├── nf.command.{begin,err,log,out,run,sh,trace}
        │   │   │   └── versions.yml
        │   │   └── spatyper-concat
        │   │       ├── nf.command.{begin,err,log,out,run,sh,trace}
        │   │       └── versions.yml
        │   ├── meta.tsv
        │   ├── mlst.tsv
        │   ├── sccmec.tsv
        │   └── spatyper.tsv
        └── nf-reports
            ├── staphopia-dag.dot
            ├── staphopia-report.html
            └── staphopia-timeline.html

Quality Control

File	Description
`supplemental/_fastqc.`	FastQC quality control reports for raw and cleaned reads
`supplemental/-NanoPlot.`	NanoPlot reports for Nanopore reads
`supplemental/.fastp.`	Fastp quality reports (when applicable)

Assembly

File	Description
`*.fna`	Assembled genome sequences in FASTA format
`assembly-stats.tsv`	Assembly quality metrics per sample

Annotation

note

Output format depends on chosen annotation tool (Bakta or Prokka)

File	Description
`*.gff.gz`	Genome annotation in GFF3 format (compressed)
`*.gbk.gz`	Genome annotation in GenBank format (compressed)
`*.faa.gz`	Protein sequences (compressed)
`*.fna.gz`	Nucleotide sequences from annotation (compressed)
`annotation.tsv`	Annotation summary tables

Typing

File	Description
`mlst.tsv`	MLST sequence type results
`agrvate-*`	Agr locus typing results
`spatyper-*`	spa typing results
`sccmec-*`	SCCmec typing results (targets, regions, details)

Antimicrobial Resistance

File	Description
`amrfinderplus.tsv`	AMR gene detection results
`amrfinderplus.mutation.tsv`	AMR point mutation results

Comparative Analysis

File	Description
`*-k21.msh`	Mash sketch files (k=21)
`*-k31.msh`	Mash sketch files (k=31)
`-mash-refseq88-.txt`	Mash screening results against RefSeq
`*.sig`	Sourmash signatures
`sourmash-*.txt`	Sourmash classification results

Merged Results

note

Run-level aggregated results from all samples

File	Description
`merged-assembly-stats.tsv`	Consolidated assembly statistics
`merged-mlst.tsv`	Consolidated MLST results
`staphtyper.tsv`	Consolidated Staphylococcus typing summary

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

Parameters

Required Parameters

The following parameters are how you will provide either local or remote samples to be processed by Bactopia.

Parameter	Type	Default	Description
`--samples`	string		A FOFN (via bactopia prepare) with sample names and paths to FASTQ/FASTAs to process

`--r1`	string		First set of compressed (gzip) Illumina paired-end FASTQ reads (requires --r2 and --sample)
`--r2`	string		Second set of compressed (gzip) Illumina paired-end FASTQ reads (requires --r1 and --sample)
`--se`	string		Compressed (gzip) Illumina single-end FASTQ reads (requires --sample)
`--ont`	string		Compressed (gzip) Oxford Nanopore FASTQ reads (requires --sample)
`--hybrid`	boolean	`false`	Create hybrid assembly using Unicycler. (requires --r1, --r2, --ont and --sample)
`--short_polish`	boolean	`false`	Create hybrid assembly from long-read assembly and short read polishing. (requires --r1, --r2, --ont and --sample)
`--sample`	string		Sample name to use for the input sequences

`--accessions`	string		A file containing ENA/SRA Experiment accessions or NCBI Assembly accessions to processed
`--accession`	string		Sample name to use for the input sequences

`--assembly`	string		A assembled genome in compressed FASTA format. (requires --sample)
`--check_samples`	boolean	`false`	Validate the input FOFN provided by --samples

Dataset Parameters

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

Parameter	Type	Default	Description
`--species`	string		Name of species for species-specific dataset to use
`--ask_merlin`	boolean		Ask Merlin to execute species specific Bactopia tools based on Mash distances
`--coverage`	integer	`100`	Reduce samples to a given coverage, requires a genome size
`--genome_size`	integer	`0`	Expected genome size (bp) for all samples, required for read error correction and read subsampling
`--use_bakta`	boolean		Use Bakta for annotation, instead of Prokka

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:

amrfinderplus - Find antimicrobial resistance genes and point mutations.
bactopia_assembler - Assemble bacterial genomes using automated assembler selection.
bactopia_datasets - Download and provide pre-compiled datasets required by Bactopia.
bactopia_gather - Search, validate, gather, and standardize input samples.
bactopia_qc - Perform comprehensive quality control on sequencing reads.
bactopia_sketcher - Create genomic sketches and perform rapid taxonomic classification.
bakta - Rapid bacterial genome annotation.
mlst - Determine multilocus sequence types (MLST) from bacterial assemblies.
prokka - Annotate bacterial genomes with functional information.
staphtyper - Determine the agr, spa and SCCmec types for Staphylococcus aureus genomes.

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)
Staphopia
Petit III RA, Read TD Staphylococcus aureus viewed from the perspective of 40,000+ genomes. PeerJ 6, e5261 (2018)

Source

View source on GitHub

Usage​

Outputs​

Expected Output Files​

Quality Control​

Assembly​

Annotation​

Typing​

Antimicrobial Resistance​

Comparative Analysis​

Merged Results​

Audit Trail​

Logs​

Nextflow Reports​

Parameters​

Required Parameters​

Composition​

Citations​

Source​