Preprint comming soon
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.
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
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 panelrepair-mode: Repairs a primer scheme via adding more...scheme-create: Creates a tiling overlap scheme for each...scheme-replace: Replaces a primerpair in a bedfilevisualise-bedfile: Visualise the bedfilevisualise-primer-mismatches: Visualise mismatches between primers and...
Shows all the primer-primer interactions within a bedfile
Usage:
$ primalscheme3 interactions [OPTIONS] BEDFILEArguments:
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.
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.
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.
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.
Replaces a primerpair in a bedfile
Usage:
$ primalscheme3 scheme-replace [OPTIONS] PRIMERNAME PRIMERBED MSAArguments:
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.
Visualise the bedfile
Usage:
$ primalscheme3 visualise-bedfile [OPTIONS] BEDFILE REF_PATHArguments:
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.
Visualise mismatches between primers and the input genomes
Usage:
$ primalscheme3 visualise-primer-mismatches [OPTIONS] MSA BEDFILEArguments:
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.