So I'm working on a problem which involves aligning PCR primers to a reference and then assessing the likelihood of those primers generating a successful amplification based on the number/location of mismatches. My issue lies in the fact that one of the PCR primer sets has a stretch of 5 inosines (included to span a highly variable region in the target). Essentially, the two problems I have are:
1) What do I do with these inosine residues when reverse-complementing the reverse primer sequence before aligning them to the reference?
2) How should I treat the inosines when deciding what is/isn't a mismatch between primer and target?
I was thinking I could substitute the inosines to either N or another degenerate nucleotide code to superficially get around the issue as I also have the issue of my alignment tool (EMBOSS-Water) not recognising inosine?
Is there a more biologically accurate solution?
