primer-finder

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/ ──┐                                                   ┌──► 1. kmers shared by all inclusion genomes (KMC)
             ├──► kmers ──► subtract ──► assemble ──► map ──► blast
exclusion/ ──┘              (KMC)       (SKESA or   (minimap2)  (every genome)
                                         SPAdes)
                                                                 └──► final_kmers.fasta: the candidate regions,
                                                                      with the specific bases in lower case

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/

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 install from the source code instead, see Installation.

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. 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/v1.0.0.tar.gz | tar -xz --strip-components=1 primer-finder-1.0.0/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 (see CITATION.cff) together with the programs it runs: KMC, SKESA or SPAdes, minimap2 and BLAST.

License

MIT