primalscheme3 3.3.0

Generation of highly multiplexed primer schemes

Primalscheme3

This is a command-line interface tool that generates a primer scheme from a Multiple Sequence Alignment (MSA) file, utilising degenerate primers to handle variation in the genomes.

Installation

Via pip!

pip install primalscheme3

From source

git clone https://github.com/artic-network/primalscheme3
cd primalscheme3
uv sync
uv run primalscheme3

PrimalScheme3

Usage:

$ primalscheme3 [OPTIONS] COMMAND [ARGS]...

Options:

• --version
• --install-completion: Install completion for the current shell.
• --show-completion: Show completion for the current shell, to copy it or customize the installation.
• --help: Show this message and exit.

Commands:

• interactions: Shows all the primer-primer interactions...
• panel-create: Creates a primer panel
• repair-mode: Repairs a primer scheme via adding more...
• scheme-create: Creates a tiling overlap scheme for each...
• scheme-replace: Replaces a primerpair in a bedfile
• visualise-bedfile: Visualise the bedfile
• visualise-primer-mismatches: Visualise mismatches between primers and...

primalscheme3 interactions

Shows all the primer-primer interactions within a bedfile

Usage:

$ primalscheme3 interactions [OPTIONS] BEDFILE

Arguments:

• BEDFILE: Path to the bedfile [required]

Options:

• --threshold FLOAT: Only show interactions more severe (Lower score) than this value [default: -26.0]
• --help: Show this message and exit.

primalscheme3 panel-create

Creates a primer panel

Usage:

$ primalscheme3 panel-create [OPTIONS]

Options:

• --msa PATH: Paths to the MSA files [required]
• --output PATH: The output directory [required]
• --region-bedfile FILE: Path to the bedfile containing the wanted regions
• --input-bedfile FILE: Path to a primer.bedfile containing the pre-calculated primers
• --mode [entropy|region-only|equal]: Select what run mode [default: region-only]
• --amplicon-size INTEGER: The size of an amplicon [default: 400]
• --n-pools INTEGER RANGE: Number of pools to use [default: 2; x>=1]
• --dimer-score FLOAT: Threshold for dimer interaction [default: -26.0]
• --min-base-freq FLOAT RANGE: Min freq to be included,[0<=x<=1] [default: 0.0; 0.0<=x<=1.0]
• --mapping [first|consensus]: How should the primers in the bedfile be mapped [default: first]
• --max-amplicons INTEGER RANGE: Max number of amplicons to create [x>=1]
• --max-amplicons-msa INTEGER RANGE: Max number of amplicons for each MSA [x>=1]
• --max-amplicons-region-group INTEGER RANGE: Max number of amplicons for each region [x>=1]
• --force / --no-force: Override the output directory [default: no-force]
• --high-gc / --no-high-gc: Use high GC primers [default: no-high-gc]
• --offline-plots / --no-offline-plots: Includes 3Mb of dependencies into the plots, so they can be viewed offline [default: offline-plots]
• --use-matchdb / --no-use-matchdb: Create and use a mispriming database [default: use-matchdb]
• --help: Show this message and exit.

primalscheme3 repair-mode

Repairs a primer scheme via adding more primers to account for new mutations

Usage:

$ primalscheme3 repair-mode [OPTIONS]

Options:

• --bedfile PATH: Path to the bedfile [required]
• --msa PATH: An MSA, with the reference.fasta, aligned to any new genomes with mutations [required]
• --config PATH: Path to the config.json [required]
• --output PATH: The output directory [required]
• --force / --no-force: Override the output directory [default: no-force]
• --help: Show this message and exit.

primalscheme3 scheme-create

Creates a tiling overlap scheme for each MSA file

Usage:

$ primalscheme3 scheme-create [OPTIONS]

Options:

• --msa PATH: The MSA to design against. To use multiple MSAs, use multiple --msa flags. (--msa 1.fasta --msa 2.fasta) [required]
• --output PATH: The output directory [required]
• --amplicon-size INTEGER: The size of an amplicon. Min / max size are ± 10 percent [100<=x<=2000] [default: 400]
• --bedfile PATH: An existing bedfile to add primers to
• --min-overlap INTEGER RANGE: min amount of overlap between primers [default: 10; x>=0]
• --n-pools INTEGER RANGE: Number of pools to use [default: 2; x>=1]
• --dimer-score FLOAT: Threshold for dimer interaction [default: -26.0]
• --min-base-freq FLOAT RANGE: Min freq to be included,[0<=x<=1] [default: 0.0; 0.0<=x<=1.0]
• --mapping [first|consensus]: How should the primers in the bedfile be mapped [default: first]
• --circular / --no-circular: Should a circular amplicon be added [default: no-circular]
• --backtrack / --no-backtrack: Should the algorithm backtrack [default: no-backtrack]
• --ignore-n / --no-ignore-n: Should N in the input genomes be ignored [default: no-ignore-n]
• --force / --no-force: Override the output directory [default: no-force]
• --input-bedfile PATH: Path to a primer.bedfile containing the pre-calculated primers
• --high-gc / --no-high-gc: Use high GC primers [default: no-high-gc]
• --offline-plots / --no-offline-plots: Includes 3Mb of dependencies into the plots, so they can be viewed offline [default: offline-plots]
• --use-matchdb / --no-use-matchdb: Create and use a mispriming database [default: use-matchdb]
• --help: Show this message and exit.

primalscheme3 scheme-replace

Replaces a primerpair in a bedfile

Usage:

$ primalscheme3 scheme-replace [OPTIONS] PRIMERNAME PRIMERBED MSA

Arguments:

• PRIMERNAME: The name of the primer to replace [required]
• PRIMERBED: The bedfile containing the primer to replace [required]
• MSA: The msa used to create the original primer scheme [required]

Options:

• --amplicon-size INTEGER: The size of an amplicon. Use single value for ± 10 percent [100<=x<=2000] [required]
• --config PATH: The config.json used to create the original primer scheme [required]
• --help: Show this message and exit.

primalscheme3 visualise-bedfile

Visualise the bedfile

Usage:

$ primalscheme3 visualise-bedfile [OPTIONS] BEDFILE REF_PATH

Arguments:

• BEDFILE: The bedfile containing the primers [required]
• REF_PATH: The bedfile containing the primers [required]

Options:

• --ref-id TEXT: The reference genome ID [required]
• --output FILE: Output location of the plot [default: bedfile.html]
• --help: Show this message and exit.

primalscheme3 visualise-primer-mismatches

Visualise mismatches between primers and the input genomes

Usage:

$ primalscheme3 visualise-primer-mismatches [OPTIONS] MSA BEDFILE

Arguments:

• MSA: The MSA used to design the scheme [required]
• BEDFILE: The bedfile containing the primers [required]

Options:

• --output FILE: Output location of the plot [default: primer.html]
• --include-seqs / --no-include-seqs: Reduces plot filesize, by excluding primer sequences [default: include-seqs]
• --offline-plots / --no-offline-plots: Includes 3Mb of dependencies into the plots, so they can be viewed offline [default: offline-plots]
• --help: Show this message and exit.