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primer-finder finds the sequences that tell one group of assembled genomes from another, so that a selective (q)PCR assay can be designed on them.
Give it two folders of genomes — the ones the assay should amplify (inclusion) and the ones it must not (exclusion) — and it:
- counts the kmers shared by every inclusion genome and the kmers of any exclusion genome (KMC), and subtracts the second set from the first;
- assembles the inclusion-specific kmers into contigs (SKESA, or SPAdes), so that there are fewer and longer sequences to examine;
- maps the contigs to one exclusion genome (minimap2) and keeps those whose differences — mismatches, insertions, deletions — are close enough together to fit in one primer or probe;
- checks every candidate against all the genomes with blast: it must be present in every inclusion genome, and its differences must hold up against every exclusion genome, not just the one it was mapped to.
primer-finder design then runs Primer3 on those regions and checks what would make each assay selective,
writing the primer files insilicoPCR reads.
The answer of find is final_kmers.fasta: one record per candidate region, with the specific bases in
lower case and their positions in the header, the most promising first. Design the assay on those bases — ideally with the
variable positions in the primers, at their 3' end, rather than in the probe.
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.
| Page | Contents |
|---|---|
| Installation | conda, bioconda, from source, checking the installation |
| Usage | inputs, every option, choosing the two groups, performance |
| Designing assays | Primer3 on the regions, how assays are scored, in silico PCR |
| Methods | what each step does, the filtering rules, limits |
| Outputs | every file and field |
| Example | the simulated dataset and its expected result |
| Validation | finding four published qPCR assays in public genomes |
| FAQ | troubleshooting, "no contig passed" |
| Development | tests, continuous integration, releases |