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.
About
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.