primer-finder 1.8.0

primer-finder finds the sequences that tell one group of genomes from another, so that a selective (q)PCR assay can be designed on them. Give it two folders of assembled genomes — the ones the assay should amplify (inclusion) and the ones it must not (exclusion) — and it reports the regions that every inclusion genome carries, that no exclusion genome carries, and whose differences are close enough together to sit in one primer or probe.

inclusion/ ──┐                                                        ┌─► final_kmers.fasta
             ├─► kmers ─► subtract ─► assemble ─► map ─────► blast ───┤   the candidate regions
exclusion/ ──┘   (KMC)     (KMC)     (SKESA,     (minimap2)  (every   │
                                      SPAdes or              genome)  └─► primer-finder design
                                      unitig)
                                                                          Primer3 + in silico PCR
                                                                          ─► assays.tsv

Quick start

conda create -n primer-finder -c conda-forge -c bioconda primer-finder
conda activate primer-finder

primer-finder -i inclusion/ -e exclusion/ -o results/

The bioconda package is awaiting review (bioconda-recipes#70034). Until it is merged, use pip install primer-finder in an environment that already holds KMC, SKESA or SPAdes, minimap2 and BLAST (-a unitig needs no assembler), or install from the source code with environment.yml, which brings them.

inclusion/ and exclusion/ hold one assembled genome per file (.fasta, .fna, .fa, gzipped or not; subfolders and symbolic links are followed). The answer is results/final_kmers.fasta: one record per candidate region, the specific bases in lower case and their positions in the header, the most promising first. results/run_info.json records the parameters, the genomes and the version of every program used.

To turn those regions into assays, with Primer3 and a check of what would actually make each one selective:

primer-finder design results/ -o assays/ --insilico-pcr /path/to/insilicoPCR-linux-x64

--insilico-pcr is optional and points at insilicoPCR, a separate program: given it, the designed assays are amplified in silico against both groups and each one is marked with whether it amplifies every inclusion genome and no exclusion genome.

Every oligo proposed satisfies Primer3's length, melting temperature and GC limits and folds or dimerises — with itself and with its partner — below 47 °C; and unless the request pinned a 3' end on a differing base, each primer carries a G or a C at that end. All of it is predicted for an ordinary TaqMan reaction whose salt, dNTP and oligo concentrations are options. So an oligo outside those limits is never proposed. Whether one inside them works is a question for the bench: two of the four published Brucella assays in the validation suite are over the hairpin limit and are validated on 427 strains. See Designing assays.

To install from the source code instead, see Installation.

Given the genomes of five Xylella fastidiosa subspecies, it reports the regions that four independently published subspecies-specific qPCR assays were designed on, three of them in the top five candidates — see Validation.

primer-finder only keeps perfect matches: a kmer must be in all the inclusion genomes with no mismatch, and in none of the exclusion genomes (-p/--min-inclusion relaxes the first half when some of the inclusion genomes are incomplete). It is therefore very sensitive to the quality of the assemblies and to how the genomes were assigned to the two groups. Curate the input genomes; genome_comparator helps with that.

To check an installation, run the bundled example (simulated genomes with a known answer, a few seconds). It is in the repository, not in the conda package:

curl -sL https://github.com/duceppemo/primer-finder/archive/refs/tags/v1.8.0.tar.gz | tar -xz --strip-components=1 primer-finder-1.8.0/example
bash example/run_example.sh

Ordering the assays

Once an assay has been designed from a candidate region and ordered, primer-finder idt turns the IDT order sheet into a fasta file of oligos, one record per primer and probe:

primer-finder idt order.xlsx assays.fasta my_target

Documentation

Everything else is in the wiki, whose sources are maintained in docs/wiki:

Citation

If primer-finder helped your work, please cite it — doi:10.5281/zenodo.23226411, which always resolves to the latest version (see CITATION.cff) — together with the programs it runs: KMC, SKESA or SPAdes, minimap2, BLAST and, for the design step, Primer3 and insilicoPCR.

License

MIT