Hello, I have four haploid fungi strains of the same species that I have conducted short-read whole genome sequencing on. I want to devise a way to differentiate the strains through creating PCR primers.

So far, I have run the Manta to call insertion and deletion structural variants larger than 50-bp. I was able to design a set of primers targeting a 125 bp deletion and another set targeting a 120 bp insertion and this combination should show different length targets for each of the four strains. However, when I ran my PCR I only got the same bands for all of them, nothing was differentiated between them.

I felt that running only one caller was perhaps not as accurate as I anticipated, I then ran a different variant caller, SVIM-asm, and used SURVIVOR to compare the two callers and I got only a few matches between them and I couldn't find a good way to design PCR primers using fewer variant sites.

Is there a better method to differentiate four closely related strains in-house using PCR without sending them off for sequencing? Is there another approach I should be doing to call structural variants for short-reads? Thank you for your time.