primalscheme is a tool for designing primer panels for multiplex PCR. It uses a greedy algorithm to find primers for tiling amplicon generation for multiple reference genomes. It works best on viral isolates of known lineages e.g. outbreak strains.
- Download complete genomes from GenBank and concatenate into a single FASTA file.
cat "sequence (1).fasta" "sequence (2).fasta" > input.fasta
- primalscheme does not currently support inputs with ambiguity codes.
- Align all sequences using Clustal Omega.
- The genomes should be complete and roughly the same length.
- Primers are only designed within the limits of the alignment.
- The first reference will be used for the coordinate system in the output scheme.
- Check the 'Result Summary' tab then the 'Percent Identity Matrix' link to check the identity matrix of all sequences.
- Check that the maximum sequence divergence is not more than 5%.
- If your sequences are more divergent, it may be necessary to separate them into multiple independent schemes; the 'Phylogenetic Tree' tab may be useful for this.
- Remove sequences with 99-100% identity to other genomes in the file as these will bias the primer selection
- Run primalscheme on your FASTA file.
- Optionally, set amplicon length as required by your sequencing technology.
We strongly recommend using the PCR conditions outlined in the Nature Protocols paper.
Command Line Interface
If you prefer a CLI, primalscheme is available as a python package. See our GitHub repo for further details.
If you use primalscheme please cite:
Quick J et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc. 2017 Jun;12(6):1261-1276.