bactopia

Tags: bacteria assembly annotation amr mlst genomics pipeline named-workflow

Comprehensive bacterial analysis pipeline for complete genomic characterization.

This workflow performs end-to-end analysis including quality control, assembly, annotation, antimicrobial resistance detection, MLST typing, and optional pathogen-specific analysis through Merlin. It processes raw sequencing reads and produces a complete genomic characterization suitable for downstream analysis.

Pipeline Overview

Looking at the workflow overview above, it might not look like much is happening, but I can assure you that a lot is going on. The workflow is broken down into 8 steps, which are:

1. Gather - Collect all the data in one place
2. QC - Quality control of the data
3. Assembler - Assemble the reads into contigs
4. Annotator - Annotate the contigs
5. Sketcher - Create a sketch of the contigs, and query databases
6. Sequence Typing - Determine the sequence type of the contigs
7. Antibiotic Resistance - Determine the antibiotic resistance of the contigs and proteins
8. Merlin - Automatically run species-specific tools based on distance

If you are looking for a guide to get started quickly, please check out the Beginner's Guide.

Step 1 - Gather

The main purpose of the gather step is to get all the samples into a single place. This includes downloading samples from ENA/SRA or NCBI Assembly. The tools used are:

Tool	Description
art	For simulating error-free reads for an input assembly
fastq-dl	Downloading FASTQ files from ENA/SRA
ncbi-genome-download	Downloading FASTA files from NCBI Assembly

This gather step also does basic QC checks to help prevent downstream failures.

Failed Quality Checks

Filename	Description
-gzip-error.txt	Sample failed Gzip checks and excluded from further analysis
-low-basepair-proportion-error.txt	Sample failed basepair proportion checks and excluded from further analysis
-low-read-count-error.txt	Sample failed read count checks and excluded from further analysis
-low-sequence-depth-error.txt	Sample failed sequenced basepair checks and excluded from further analysis

Poor samples are excluded to prevent downstream failures

Samples that fail any of the QC checks will be excluded from further analysis. Those samples will generate a *-error.txt file with the error message. Excluding these samples prevents downstream failures that cause the whole workflow to fail.

Example Error: Input FASTQ(s) failed Gzip checks

If input FASTQ(s) fail to pass Gzip test, the sample will be excluded from further analysis.

Example Text from <SAMPLE_NAME>-gzip-error.txt <SAMPLE_NAME> FASTQs failed Gzip tests. Please check the input FASTQs. Further analysis is discontinued.

Example Error: Input FASTQs have disproportionate number of reads

If input FASTQ(s) for a sample have disproportionately different number of reads between the two pairs, the sample will be excluded from further analysis. You can adjust this minimum read count using the --min_proportion parameter.

Example Text from <SAMPLE_NAME>-low-basepair-proportion-error.txt <SAMPLE_NAME> FASTQs failed to meet the minimum shared basepairs. They shared Y basepairs, with R1 having A bp and R2 having B bp. Further analysis is discontinued.

Example Error: Input FASTQ(s) has too few reads

If input FASTQ(s) for a sample have less than the minimum required reads, the sample will be excluded from further analysis. You can adjust this minimum read count using the --min_reads parameter.

Example Text from <SAMPLE_NAME>-low-read-count-error.txt <SAMPLE_NAME> FASTQ(s) contain X total reads. This does not exceed the required minimum Y read count. Further analysis is discontinued.

Example Error: Input FASTQ(s) has too little sequenced basepairs

If input FASTQ(s) for a sample fails to meet the minimum number of sequenced basepairs, the sample will be excluded from further analysis. You can adjust this minimum read count using the --min_basepairs parameter.

Example Text from <SAMPLE_NAME>-low-sequence-depth-error.txt <SAMPLE_NAME> FASTQ(s) contain X total basepairs. This does not exceed the required minimum Y bp. Further analysis is discontinued.

Step 2 - QC

The qc module uses a variety of tools to perform quality control on Illumina and Oxford Nanopore reads. The tools used are:

Tool	Technology	Description
bbtools	Illumina	A suite of tools for manipulating reads
fastp	Illumina	A tool designed to provide fast all-in-one preprocessing for FastQ files
fastqc	Illumina	A quality control tool for high throughput sequence data
fastq_scan	Nanopore	A tool for quickly scanning FASTQ files
lighter	Illumina	A tool for correcting sequencing errors in Illumina reads
NanoPlot	Nanopore	A tool for plotting long read sequencing data
nanoq	Nanopore	A tool for calculating quality metrics for Oxford Nanopore reads
porechop	Nanopore	A tool for removing adapters from Oxford Nanopore reads
rasusa	Nanopore	Randomly subsample sequencing reads to a specified coverage

Similar to the gather step, the qc step will also stop samples that fail to meet basic QC checks from continuing downstream.

Failed Quality Checks

Filename	Description
.error-fastq.gz	A gzipped FASTQ file of reads that failed QC
-low-read-count-error.txt	Sample failed read count checks and excluded from further analysis
-low-sequence-coverage-error.txt	Sample failed sequenced coverage checks and excluded from further analysis
-low-sequence-depth-error.txt	Sample failed sequenced basepair checks and excluded from further analysis

Poor samples are excluded to prevent downstream failures

Samples that fail any of the QC checks will be excluded from further analysis. Those samples will generate a *-error.txt file with the error message. Excluding these samples prevents downstream failures that cause the whole workflow to fail.

Example Error: After QC, too few reads remain

If after cleaning reads, a sample has less than the minimum required reads, the sample will be excluded from further analysis. You can adjust this minimum read count using the --min_reads parameter.

Example Text from <SAMPLE_NAME>-low-read-count-error.txt <SAMPLE_NAME> FASTQ(s) contain X total reads. This does not exceed the required minimum Y read count. Further analysis is discontinued.

Example Error: After QC, too little sequence coverage remains

If after cleaning reads, a sample has failed to meet the minimum sequence coverage required, the sample will be excluded from further analysis. You can adjust this minimum read count using the --min_coverage parameter.

Note: This check is only performed when a genome size is available.

Example Text from <SAMPLE_NAME>-low-sequence-coverage-error.txt After QC, <SAMPLE_NAME> FASTQ(s) contain X total basepairs. This does not exceed the required minimum Y bp (Zx coverage). Further analysis is discontinued.

Example Error: After QC, too little sequenced basepairs remain

If after cleaning reads, a sample has failed to meet the minimum number of sequenced basepairs, the sample will be excluded from further analysis. You can adjust this minimum read count using the --min_basepairs parameter.

Example Text from <SAMPLE_NAME>-low-sequence-depth-error.txt <SAMPLE_NAME> FASTQ(s) contain X total basepairs. This does not exceed the required minimum Y bp. Further analysis is discontinued.

Step 3 - Assembler

The assembler module uses a variety of assembly tools to create an assembly of Illumina and Oxford Nanopore reads. The tools used are:

Tool	Description
Dragonflye	Assembly of Oxford Nanopore reads, as well as hybrid assembly with short-read polishing
Shovill	Assembly of Illumina paired-end reads
Shovill-SE	Assembly of Illumina single-end reads
Unicycler	Hybrid assembly, using short-reads first then long-reads

Summary statistics for each assembly are generated using assembly-scan.

Prefer --short_polish over --hybrid with recent ONT sequencing

Using Unicycler (--hybrid) to create a hybrid assembly works great when you have low-coverage noisy long-reads. However, if you are using recent ONT sequencing, you likely have high-coverage and using the --short_polish method is going to yield better results (and be faster!) than --hybrid.

Failed Quality Checks

Filename	Description
-assembly-error.txt	Sample failed assembly checks and excluded from further analysis

Poor samples are excluded to prevent downstream failures

Samples that fail any of the QC checks will be excluded from further analysis. Those samples will generate a *-error.txt file with the error message. Excluding these samples prevents downstream failures that cause the whole workflow to fail.

Example Error: Assembled Successfully, but 0 Contigs

If a sample assembles successfully, but 0 contigs are formed, the sample will be excluded from further analysis.

Example Text from <SAMPLE_NAME>-assembly-error.txt <SAMPLE_NAME> assembled successfully, but 0 contigs were formed. Please investigate <SAMPLE_NAME> to determine a cause (e.g. metagenomic, contaminants, etc...) for this outcome. Further assembly-based analysis of <SAMPLE_NAME> will be discontinued.

Example Error: Assembled successfully, but poor assembly size

If your sample assembles successfully, but the assembly size is less than the minimum allowed genome size, the sample will be excluded from further analysis. You can adjust this minimum size using the --min_genome_size parameter.

Example Text from <SAMPLE_NAME>-assembly-error.txt <SAMPLE_NAME> assembled size (000 bp) is less than the minimum allowed genome size (000 bp). If this is unexpected, please investigate <SAMPLE_NAME> to determine a cause (e.g. metagenomic, contaminants, etc...) for the poor assembly. Otherwise, adjust the --min_genome_size parameter to fit your need. Further assembly based analysis of <SAMPLE_NAME> will be discontinued.

Step 4 - Annotator

The annotator step uses either Prokka (default) or Bakta (via --use_bakta) to annotate assembled contigs with functional information including genes, proteins, rRNA, tRNA, and other genomic features.

Step 5 - Sketcher

The sketcher module uses Mash and Sourmash to create sketches and query RefSeq and GTDB.

Step 6 - Sequence Typing

The mlst step uses mlst to scan assemblies against PubMLST typing schemes and determine the sequence type.

Step 7 - Antibiotic Resistance

The amrfinderplus step uses AMRFinder+ to identify antimicrobial resistance genes and point mutations from both assembled contigs and annotated protein sequences.

Step 8 - Merlin

The merlin step automatically selects and runs species-specific typing tools based on Mash distance results from the sketcher step. Enable with --ask_merlin. See the Pathogen-Specific Analysis output section below for the full list of supported organisms and tools.

Usage

Bactopia CLI:

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

Nextflow:

nextflow run bactopia/bactopia \
  --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-se.log
│   │   │   │   └── versions.yml
│   │   │   └── supplemental
│   │   │       ├── illumina.txt
│   │   │       └── shovill.corrections
│   │   ├── gather
│   │   │   ├── <SAMPLE_NAME>-meta.tsv
│   │   │   └── logs
│   │   │       ├── nf.command.{begin,err,log,out,run,sh,trace}
│   │   │       └── versions.yml
│   │   ├── qc
│   │   │   ├── <SAMPLE_NAME>_SE.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>_SE-final.json
│   │   │       ├── <SAMPLE_NAME>_SE-final_fastqc.html
│   │   │       ├── <SAMPLE_NAME>_SE-final_fastqc.zip
│   │   │       ├── <SAMPLE_NAME>_SE-original.json
│   │   │       ├── <SAMPLE_NAME>_SE-original_fastqc.html
│   │   │       └── <SAMPLE_NAME>_SE-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
│       ├── 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
└── bactopia-runs
    └── bactopia-<TIMESTAMP>
        ├── merged-results
        │   ├── amrfinderplus.tsv
        │   ├── assembly-scan.tsv
        │   ├── logs
        │   │   ├── 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
        │   ├── meta.tsv
        │   └── mlst.tsv
        └── nf-reports
            ├── bactopia-dag.dot
            ├── bactopia-report.html
            └── bactopia-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)
supplemental/*_original.json	Quality metrics for original reads
supplemental/*_final.json	Quality metrics for final reads

Assembly

File	Description
*.fasta	Assembled genome sequences
assembly-stats.tsv	Assembly quality metrics
merged-assembly-stats.tsv	Consolidated assembly statistics

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)
*.ffn.gz	Feature nucleotide sequences (compressed)
annotation.tsv	Annotation summary tables
blastdb.*	BLAST database created from annotation

Typing

File	Description
mlst.tsv	MLST sequence type results
merged-mlst.tsv	Consolidated MLST results

Antimicrobial Resistance

File	Description
amrfinderplus.tsv	AMR gene detection results
amrfinderplus.mutation.tsv	AMR point mutation results
merged-amrfinderplus.tsv	Consolidated AMR 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

Pathogen-Specific Analysis

note

Only created if --ask_merlin is enabled

File	Description
merlin/clermontyping/*	E. coli phylogroup typing
merlin/ectyper/*	Enterotoxigenic E. coli typing
merlin/shigatyper/*	Shigella serotype prediction
merlin/shigapass/*	Shigella passive surveillance
merlin/shigeifinder/*	Shigella and EIEC detection
merlin/stecfinder/*	STEC detection and typing
merlin/emmtyper/*	S. pyogenes emm typing
merlin/hicap/*	H. influenzae capsular typing
merlin/hpsuissero/*	H. parasuis serotyping
merlin/kleborate/*	Klebsiella species typing
merlin/staphtyper/*	S. aureus spa typing
merlin/agrvate/*	S. aureus agr typing
merlin/sccmec/*	S. aureus SCCmec typing

Merged Results

note

Run-level aggregated results from all samples

File	Description
samplesheet.tsv	Sample metadata and quality metrics

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
bactopia-dag.dot	The Nextflow DAG visualization
bactopia-report.html	The Nextflow Execution Report
bactopia-timeline.html	The Nextflow Timeline Report
bactopia-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

AMRFinder+ Parameters

Parameter	Type	Default	Description
--amrfinderplus_ident_min	number	-1	Minimum proportion of identical amino acids in alignment for hit (0..1)
--amrfinderplus_coverage_min	number	0.5	Minimum coverage of the reference protein (0..1)
--amrfinderplus_organism	string		Taxonomy group to run additional screens against
--amrfinderplus_translation_table	integer	11	NCBI genetic code for translated BLAST
--amrfinderplus_noplus	boolean	false	Disable running AMRFinder+ with the --plus option
--amrfinderplus_report_common	boolean	false	Report proteins common to a taxonomy group
--amrfinderplus_report_all_equal	boolean	false	Report all equally-scoring BLAST and HMM matches
--amrfinderplus_opts	string		Extra AMRFinder+ options in quotes.
--amrfinderplus_db	string		A custom AMRFinder+ database to use, either a tarball or a folder

csvtk concat Parameters

Parameter	Type	Default	Description
--csvtk_concat_opts	string		Extra csvtk concat options in quotes

Assembler Parameters

Parameter	Type	Default	Description
--shovill_assembler	string	skesa	Assembler to be used by Shovill (choices: skesa, megahit, spades, velvet)
--dragonflye_assembler	string	flye	Assembler to be used by Dragonflye (choices: flye, miniasm, raven)
--use_unicycler	boolean		Use unicycler for paired end assembly
--min_contig_len	integer	500	Minimum contig length <0=AUTO>
--min_contig_cov	integer	2	Minimum contig coverage <0=AUTO>
--contig_namefmt	string		Format of contig FASTA IDs in 'printf' style
--shovill_opts	string		Extra assembler options in quotes for Shovill
--shovill_kmers	string		K-mers to use <blank=AUTO>
--dragonflye_opts	string		Extra assembler options in quotes for Dragonflye
--trim	boolean		Enable adaptor trimming
--no_stitch	boolean		Disable read stitching for paired-end reads
--no_corr	boolean		Disable post-assembly correction
--unicycler_mode	string	normal	Bridging mode used by Unicycler (choices: conservative, normal, bold)
--min_component_size	integer	1000	Graph dead ends smaller than this size (bp) will be removed from the final graph
--min_dead_end_size	integer	1000	Graph dead ends smaller than this size (bp) will be removed from the final graph
--nanohq	boolean	false	For Flye, use '--nano-hq' instead of --nano-raw
--medaka_model	string		The model to use for Medaka polishing
--medaka_rounds	integer	0	The number of Medaka polishing rounds to conduct
--racon_rounds	integer	1	The number of Racon polishing rounds to conduct
--no_polish	boolean		Skip the assembly polishing step
--no_miniasm	boolean		Skip miniasm+Racon bridging
--no_rotate	boolean		Do not rotate completed replicons to start at a standard gene
--reassemble	boolean	false	If reads were simulated, they will be used to create a new assembly.
--polypolish_rounds	integer	1	Number of polishing rounds to conduct with Polypolish for short read polishing
--pilon_rounds	integer	0	Number of polishing rounds to conduct with Pilon for short read polishing

Gather Parameters

Parameter	Type	Default	Description
--skip_fastq_check	boolean		Skip minimum requirement checks for input FASTQs
--min_basepairs	integer	2241820	The minimum amount of basepairs required to continue downstream analyses.
--min_reads	integer	7472	The minimum amount of reads required to continue downstream analyses.
--min_coverage	integer	10	The minimum amount of coverage required to continue downstream analyses.
--min_proportion	number	0.5	The minimum proportion of basepairs for paired-end reads to continue downstream analyses.
--min_genome_size	integer	100000	The minimum estimated genome size allowed for the input sequence to continue downstream analyses.
--max_genome_size	integer	18040666	The maximum estimated genome size allowed for the input sequence to continue downstream analyses.
--attempts	integer	3	Maximum times to attempt downloads
--use_ena	boolean		Download FASTQs from ENA
--no_cache	boolean		Skip caching the assembly summary file from ncbi-genome-download

Sketcher Parameters

Parameter	Type	Default	Description
--sketch_size	integer	10000	Sketch size. Each sketch will have at most this many non-redundant min-hashes.
--sourmash_scale	integer	10000	Choose number of hashes as 1 in FRACTION of input k-mers
--no_winner_take_all	boolean		Disable winner-takes-all strategy for identity estimates
--screen_i	number	0.8	Minimum identity to report.

MLST Parameters

Parameter	Type	Default	Description
--mlst_scheme	string		Don't autodetect, force this scheme on all inputs
--mlst_minid	integer	95	Minimum DNA percent identity of full allele to consider 'similar'
--mlst_mincov	integer	10	Minimum DNA percent coverage to report partial allele at all
--mlst_minscore	integer	50	Minimum score out of 100 to match a scheme
--mlst_nopath	boolean	false	Strip filename paths from FILE column
--mlst_db	string		A custom MLST database to use, either a tarball or a directory

QC Parameters

Parameter	Type	Default	Description
--use_bbmap	boolean		Illumina reads will be QC'd using BBMap
--use_porechop	boolean	false	Use Porechop to remove adapters from ONT reads
--skip_qc	boolean		The QC step will be skipped and it will be assumed the inputs sequences have already been QCed.
--skip_qc_plots	boolean		QC Plot creation by FastQC or Nanoplot will be skipped
--skip_error_correction	boolean		FLASH error correction of reads will be skipped.
--adapters	string		A FASTA file containing adapters to remove
--adapter_k	integer	23	Kmer length used for finding adapters.
--phix	string		phiX174 reference genome to remove
--phix_k	integer	31	Kmer length used for finding phiX174.
--ktrim	string	r	Trim reads to remove bases matching reference kmers (choices: f, r, l)
--mink	integer	11	Look for shorter kmers at read tips down to this length, when k-trimming or masking.
--hdist	integer	1	Maximum Hamming distance for ref kmers (subs only)
--tpe	string	t	When kmer right-trimming, trim both reads to the minimum length of either (choices: f, t)
--tbo	string	t	Trim adapters based on where paired reads overlap (choices: f, t)
--qtrim	string	rl	Trim read ends to remove bases with quality below trimq. (choices: rl, f, r, l, w)
--trimq	integer	6	Regions with average quality BELOW this will be trimmed if qtrim is set to something other than f
--maq	integer	10	Reads with average quality (after trimming) below this will be discarded
--minlength	integer	35	Reads shorter than this after trimming will be discarded
--ftm	integer	5	If positive, right-trim length to be equal to zero, modulo this number
--tossjunk	string	t	Discard reads with invalid characters as bases (choices: f, t)
--ain	string	f	When detecting pair names, allow identical names (choices: f, t)
--qout	string	33	PHRED offset to use for output FASTQs (choices: 33, 64)
--maxcor	integer	1	Max number of corrections within a 20bp window
--sampleseed	integer	42	Set to a positive number to use as the random number generator seed for sampling
--ont_minlength	integer	1000	ONT Reads shorter than this will be discarded
--ont_minqual	integer	0	Minimum average read quality filter of ONT reads
--porechop_opts	string		Extra Porechop options in quotes
--nanoplot_opts	string		Extra NanoPlot options in quotes
--bbduk_opts	string		Extra BBDuk options in quotes
--fastp_opts	string		Extra fastp options in quotes

Bakta Download Parameters

Parameter	Type	Default	Description
--bakta_db	string		Tarball or path to the Bakta database
--bakta_db_type	string	full	Which Bakta DB to download 'full' (~30GB) or 'light' (~2GB) (choices: full, light)
--bakta_save_as_tarball	boolean	false	Save the Bakta database as a tarball
--download_bakta	boolean	false	Download the Bakta database to the path given by --bakta_db

Bakta Parameters

Parameter	Type	Default	Description
--bakta_proteins	string		FASTA file of trusted proteins to first annotate from
--bakta_prodigal_tf	string		Training file to use for Prodigal
--bakta_replicons	string		Replicon information table (tsv/csv)
--bakta_min_contig_length	integer	1	Minimum contig size to annotate
--bakta_keep_contig_headers	boolean	false	Keep original contig headers
--bakta_compliant	boolean	false	Force Genbank/ENA/DDJB compliance
--bakta_skip_trna	boolean	false	Skip tRNA detection & annotation
--bakta_skip_tmrna	boolean	false	Skip tmRNA detection & annotation
--bakta_skip_rrna	boolean	false	Skip rRNA detection & annotation
--bakta_skip_ncrna	boolean	false	Skip ncRNA detection & annotation
--bakta_skip_ncrna_region	boolean	false	Skip ncRNA region detection & annotation
--bakta_skip_crispr	boolean	false	Skip CRISPR array detection & annotation
--bakta_skip_cds	boolean	false	Skip CDS detection & annotation
--bakta_skip_sorf	boolean	false	Skip sORF detection & annotation
--bakta_skip_gap	boolean	false	Skip gap detection & annotation
--bakta_skip_ori	boolean	false	Skip oriC/oriT detection & annotation
--bakta_opts	string		Extra Bakta options in quotes. Example: '--gram +'

Prokka Parameters

Parameter	Type	Default	Description
--prokka_proteins	string	${projectDir}/data/proteins.faa	FASTA file of trusted proteins to first annotate from
--prokka_prodigal_tf	string		Training file to use for Prodigal
--prokka_compliant	boolean	false	Force Genbank/ENA/DDJB compliance
--prokka_centre	string	Bactopia	Sequencing centre ID
--prokka_coverage	integer	80	Minimum coverage on query protein
--prokka_evalue	string	1e-09	Similarity e-value cut-off
--prokka_opts	string		Extra Prokka options in quotes.
--prokka_debug	boolean	false	Enable debug mode for Prokka

mashdist Parameters

Parameter	Type	Default	Description
--mash_sketch	string		The reference sequence as a Mash Sketch (.msh file)
--mash_seed	integer	42	Seed to provide to the hash function
--mash_table	boolean	false	Table output (fields will be blank if they do not meet the p-value threshold)
--mash_m	integer	1	Minimum copies of each k-mer required to pass noise filter for reads
--mash_w	number	0.01	Probability threshold for warning about low k-mer size.
--mash_max_p	number	1.0	Maximum p-value to report.
--mash_max_dist	number	1.0	Maximum distance to report.
--merlin_dist	number	0.1	Maximum distance to report when using Merlin .
--full_merlin	boolean	false	Go full Merlin and run all species-specific tools, no matter the Mash distance
--mash_use_fastqs	boolean	false	Query with FASTQs instead of the assemblies

ClermonTyping Parameters

Parameter	Type	Default	Description
--clermontyping_threshold	integer	0	Do not use contigs under this size

ECTyper Parameters

Parameter	Type	Default	Description
--ectyper_opid	integer	90	Percent identity required for an O antigen allele match
--ectyper_opcov	integer	90	Minimum percent coverage required for an O antigen allele match
--ectyper_hpid	integer	95	Percent identity required for an H antigen allele match
--ectyper_hpcov	integer	50	Minimum percent coverage required for an H antigen allele match
--ectyper_verify	boolean	false	Enable E. coli species verification
--ectyper_print_alleles	boolean	false	Prints the allele sequences if enabled as the final column

emmtyper Parameters

Parameter	Type	Default	Description
--emmtyper_wf	string	blast	Workflow for emmtyper to use. (choices: blast, pcr)
--emmtyper_blastdb	string		Path to custom EMM BLAST DB.
--emmtyper_cluster_distance	integer	500	Distance between cluster of matches to consider as different clusters
--emmtyper_percid	integer	95	Minimal percent identity of sequence
--emmtyper_culling_limit	integer	5	Total hits to return in a position
--emmtyper_mismatch	integer	5	Threshold for number of mismatch to allow in BLAST hit
--emmtyper_align_diff	integer	5	Threshold for difference between alignment length and subject length in BLAST
--emmtyper_gap	integer	2	Threshold gap to allow in BLAST hit
--emmtyper_min_perfect	integer	15	Minimum size of perfect match at 3 primer end
--emmtyper_min_good	integer	15	Minimum size where there must be 2 matches for each mismatch
--emmtyper_max_size	integer	2000	Maximum size of PCR product

hicap Parameters

Parameter	Type	Default	Description
--hicap_database_dir	string		Directory containing locus database
--hicap_model_fp	string		Path to prodigal model
--hicap_full_sequence	boolean	false	Write the full input sequence out to the genbank file rather than just the region surrounding and including the locus
--hicap_debug	boolean	false	hicap will print debug messages
--hicap_gene_coverage	number	0.8	Minimum percentage coverage to consider a single gene complete
--hicap_gene_identity	number	0.7	Minimum percentage identity to consider a single gene complete
--hicap_broken_gene_length	integer	60	Minimum length to consider a broken gene
--hicap_broken_gene_identity	number	0.8	Minimum percentage identity to consider a broken gene

Mykrobe Parameters

Parameter	Type	Default	Description
--mykrobe_species	string		Species panel to use (choices: sonnei, staph, tb, typhi)
--mykrobe_kmer	integer	21	K-mer length
--mykrobe_min_depth	integer	1	Minimum depth
--mykrobe_model	string	kmer_count	Genotype model used. (choices: kmer_count, median_depth)
--mykrobe_report_all_calls	boolean	false	Report all calls
--mykrobe_opts	string		Extra Mykrobe options in quotes

GenoTyphi Parameters

Parameter	Type	Default	Description
--genotyphi_kmer	integer	21	K-mer length
--genotyphi_min_depth	integer	1	Minimum depth
--genotyphi_model	string	kmer_count	Genotype model used. (choices: kmer_count, median_depth)
--genotyphi_report_all_calls	boolean	false	Report all calls
--genotyphi_mykrobe_opts	string		Extra Mykrobe options in quotes

Kleborate Parameters

Parameter	Type	Default	Description
--kleborate_preset	string	kpsc	Preset module to use for Kleborate (choices: kpsc, kosc, escherichia)
--kleborate_opts	string		Extra options in quotes for Kleborate

legsta Parameters

Parameter	Type	Default	Description
--legsta_noheader	boolean	false	Don't print header row

LisSero Parameters

Parameter	Type	Default	Description
--lissero_min_id	number	95.0	Minimum percent identity to accept a match
--lissero_min_cov	number	95.0	Minimum coverage of the gene to accept a match

ngmaster Parameters

Parameter	Type	Default	Description
--ngmaster_csv	boolean	false	output comma-separated format (CSV) rather than tab-separated

pasty Parameters

Parameter	Type	Default	Description
--pasty_min_pident	integer	95	Minimum percent identity to count a hit
--pasty_min_coverage	integer	95	Minimum percent coverage to count a hit

pbptyper Parameters

Parameter	Type	Default	Description
--pbptyper_min_pident	integer	95	Minimum percent identity to count a hit
--pbptyper_min_coverage	integer	95	Minimum percent coverage to count a hit

SeqSero2 Parameters

Parameter	Type	Default	Description
--seqsero2_run_mode	string	k	Workflow to run. 'a' allele mode, or 'k' k-mer mode (choices: a, k)
--seqsero2_input_type	string	assembly	Input format to analyze. 'assembly' or 'fastq' (choices: assembly, fastq)
--seqsero2_bwa_mode	string	mem	Algorithms for bwa mapping for allele mode (choices: mem, sam)

SeroBA Parameters

Parameter	Type	Default	Description
--seroba_noclean	boolean	false	Do not clean up intermediate files
--seroba_coverage	integer	20	Threshold for k-mer coverage of the reference sequence

SISTR Parameters

Parameter	Type	Default	Description
--sistr_full_cgmlst	boolean	false	Use the full set of cgMLST alleles which can include highly similar alleles

AgrVATE Parameters

Parameter	Type	Default	Description
--agrvate_typing_only	boolean	false	agr typing only. Skips agr operon extraction and frameshift detection

spaTyper Parameters

Parameter	Type	Default	Description
--spatyper_repeats	string		List of spa repeats
--spatyper_repeat_order	string		List spa types and order of repeats
--spatyper_do_enrich	boolean	false	Do PCR product enrichment

sccmec Parameters

Parameter	Type	Default	Description
--sccmec_min_targets_pident	integer	90	Minimum percent identity to count a target hit
--sccmec_min_targets_coverage	integer	80	Minimum percent coverage to count a target hit
--sccmec_min_regions_pident	integer	85	Minimum percent identity to count a region hit
--sccmec_min_regions_coverage	integer	93	Minimum percent coverage to count a region hit

STECFinder Parameters

Parameter	Type	Default	Description
--stecfinder_use_reads	boolean	false	Paired-end Illumina reads will be used instead of assemblies
--stecfinder_hits	boolean	false	Show detailed gene search results
--stecfinder_cutoff	number	10.0	Minimum read coverage for gene to be called
--stecfinder_length	number	50.0	Percentage of gene length needed for positive call
--stecfinder_ipah_length	number	10.0	Percentage of ipaH gene length needed for positive gene call
--stecfinder_ipah_depth	number	1.0	Minimum depth for positive ipaH gene call (requires --stecfinder_use_reads)
--stecfinder_stx_length	number	10.0	Percentage of stx gene length needed for positive gene call
--stecfinder_stx_depth	number	1.0	Minimum depth for positive stx gene call (requires --stecfinder_use_reads)
--stecfinder_o_length	number	60.0	Percentage of wz_ gene length needed for positive call
--stecfinder_o_depth	number	1.0	Minimum depth for positive qz_ gene call (requires --stecfinder_use_reads)
--stecfinder_h_length	number	60.0	Percentage of fliC gene length needed for positive call
--stecfinder_h_depth	number	1.0	Minimum depth for positive fliC gene call (requires --stecfinder_use_reads)

TB-Profiler Profile Parameters

Parameter	Type	Default	Description
--tbprofiler_call_whole_genome	boolean	false	Call whole genome
--tbprofiler_mapper	string	bwa	Mapping tool to use. If you are using nanopore data it will default to minimap2 (choices: bwa, minimap2, bowtie2, bwa-mem2)
--tbprofiler_caller	string	freebayes	Variant calling tool to use (choices: bcftools, gatk, freebayes)
--tbprofiler_calling_params	string		Extra variant caller options in quotes
--tbprofiler_suspect	boolean	false	Use the suspect suite of tools to add ML predictions
--tbprofiler_no_flagstat	boolean	false	Don't collect flagstats
--tbprofiler_no_delly	boolean	false	Don't run delly
--tbprofiler_opts	string		Extra options in quotes for TBProfiler

TB-Profiler Collate Parameters

Parameter	Type	Default	Description
--tbprofiler_itol	boolean	false	Generate itol config files
--tbprofiler_full	boolean	false	Output mutations in main result file
--tbprofiler_all_variants	boolean	false	Output all variants in variant matrix
--tbprofiler_mark_missing	boolean	false	An asterisk will be used to mark predictions which are affected by missing data at a drug resistance position

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
--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:

• 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.
• merlin - MinER assisted species-specific bactopia tool seLectIoN.
• mlst - Determine multilocus sequence types (MLST) from bacterial assemblies.
• prokka - Annotate bacterial genomes with functional information.

Source

View source on GitHub