primalscheme3 3.2.1

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

Currently the best way to use is to use poetry to handle dependencies.

git clone https://github.com/ChrisgKent/primalscheme3
cd primalscheme3
poetry install
poetry build

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.