Hello, I'm trying to design specific primers and probes (for quantitative real time PCR) to detect diffent fungi species from a complex soil sample (environmental sample). Right now the main difficulty is to detect a non-conserved sequence to amplify only the species I want and not other species from the same genus.
I'm using tools such as MEGA 7.0, Clustal Omega and Blast to align ITS sequences of several fungi from the same genus in order to detect a non-conserved region to use as a target. Then I manually select the region and generate primers (with Primer3Plus) but they are always lacking specificity (after checking Blast primer design tool i get as result other species plus the one I need).
Is there a tool/software (preference for free ones) that can help me with this problem? One with an option to detect species-specific sequences in an alignment?
Or, can I be aproaching this problem the wrong way?
Thank you in advance for your support
BBMap has a tool called KmerCountExact that will allow you to gather and list kmers, and specify count cutoffs. You can use this to perform set intersections operations. So, for example, you could get a set of all kmers in the fungi of interest, and then subtract kmers in all other known fungal genomes. The resulting set of kmers would be specific to the fungi of interest and you could choose your primer sequences from amongst them.