primerforge 1.1.1

software to identify primers that can be used to distinguish genomes

primerForge

software to identify primers that can be used to distinguish genomes

Installation

pip installation

pip install primerforge

conda installation

conda install -c bioconda -c conda-forge primerforge

Manual installation

[!NOTE] This might take up to ten minutes.

git clone https://github.com/dr-joe-wirth/primerForge.git
conda env create -f primerForge/environment.yml
conda activate primerforge

Docker Installation

A Docker image for the latest release is available at DockerHub

Checking installation

If primerForge is installed correctly, then the following command should execute without errors:

primerForge --check_install

If you installed manually, you may need to use the following command instead

python primerForge.py --check_install

Usage

usage:
    primerForge [-ioaubfpgtrdnkvh]

required arguments:
    -i, --ingroup        [file] ingroup filename or a file pattern inside double-quotes (eg."*.gbff")

optional arguments: 
    -o, --out            [file] output filename for primer pair data (default: results.tsv)
    -a, --analysis       [file] output basename for primer analysis data (default: distribution)
    -u, --outgroup       [file(s)] outgroup filename or a file pattern inside double-quotes (eg."*.gbff")
    -b, --bad_sizes      [int,int] a range of PCR product lengths that the outgroup cannot produce (default: same as '--pcr_prod')
    -f, --format         [str] file format of the ingroup and outgroup genbank|fasta (default: genbank)
    -p, --primer_len     [int(s)] a single primer length or a range specified as 'min,max' (default: 16,20)
    -g, --gc_range       [float,float] a min and max percent GC specified as a comma separated list (default: 40.0,60.0)
    -t, --tm_range       [float,float] a min and max melting temp (Tm) specified as a comma separated list (default: 55.0,68.0)
    -r, --pcr_prod       [int(s)] a single PCR product length or a range specified as 'min,max' (default: 120,2400)
    -d, --tm_diff        [float] the maximum allowable Tm difference between a pair of primers (default: 5.0)
    -n, --num_threads    [int] the number of threads for parallel processing (default: 1)
    -k, --keep           keep intermediate files (default: False)
    -v, --version        print the version
    -h, --help           print this message
    --check_install      check installation
    --debug              run in debug mode (default: False)

Workflow

flowchart TB
    ingroup[/"ingroup genomes"/]
    ingroup --> A

    %% get unique kmers
    subgraph A["for each genome"]
        uniqKmer["get unique kmers"]
    end

    %% get shared kmers
    sharedKmers(["shared kmers"])
    uniqKmer -- intersection --> sharedKmers

    %% get candidate kmers
    subgraph B["for each genome"]
        subgraph B0["for each kmer start position"]
            subgraph B1["pick one kmer"]
                GC{"GC in
                 range?"}
                Tm{"Tm in
                range?"}
                homo{"repeats
                ≤ 3bp?"}
                hair{"no hairpins?"}
                dime{"no homo-
                dimers?"}
                GC-->Tm-->homo-->hair-->dime
            end
        end
    end

    %% connections up to candidate kmers
    sharedKmers --> B
    dump1[/"dump to file"/]
    sharedKmers --> dump1
    candidates(["unique, shared kmers; one per start position"])
    dime --> candidates

    %% get primer pairs
    subgraph C["for one genome"]
        bin1["bin overlapping kmers (64bp max)"]
        bin2["get bin pairs"]
        candPair(["candidate primer pairs"])
        sharePair(["shared primer pairs"])

        %% evaluate one kmer pair
        subgraph C0["for each bin pair"]
            size{"is PCR
            size ok?"}
            subgraph C4["for each primer pair"]
                prime{"is 3' end
                G or C?"}
                temp{"is Tm
                difference ok?"}
                hetero{"no hetero-
                dimers?"}
            end
            size --> C4
        end


        %% get shared primer pairs
        subgraph C2["for each candidate primer pair"]
            subgraph C3["for each other genome"]
                pcr{"PCR size ok?"}
            end
        end

        bin1 --> bin2
        bin2 --> C0
        prime --> temp --> hetero --> candPair
        candPair --> C2
        pcr --> sharePair
    end

    allSharePair(["all shared primer pairs"])
    dump2[/"dump to file"/]
    dump3[/"dump to file"/]

    candidates --> dump2
    candidates --> C
    sharePair --> allSharePair
    allSharePair --> dump3

    %% outgroup removal
    outgroup[/"outgroup genomes"/]

    allSharePair --> D0
    outgroup --> D
    subgraph D["for each outgroup genome"]
        subgraph D0["for each primer pair"]
            ogsize{"PCR size outside
            disallowed range?"}
        end
    end
    
    allPairs(["all suitable primer pairs"])
    ogsize --> allPairs

    %% one pair per bin pair
    subgraph E["for each bin pair"]
        keep["keep only one primer
        pair per bin pair"]
    end

    allPairs --> E

    final(["final set of pairs"])
    keep --> final

    write[/"write pairs to file"/]
    plots[/"make plots"/]

    final --> write
    final --> plots