- Download complete genomes from GenBank and concatenate into a single FASTA file.
cat "sequence (1).fasta" "sequence (2).fasta" > input.fasta
- 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
- 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.