I seek advice in the form a bioinformatics strategy to design primers to distinguish 10 sequences from one another.
These 10 sequences are genic sequences, and across which their respective exonic sequences are highly conserved, and so is the number of exons. Their non-exonic sequences, as expected, are not always as highly conserved.
The Multiple Sequence Alignment of these 10 genes that I have generated factor in the rules of splicing at intron-exon junctions.
My goal is to design primers that anneal to a common conserved region across these 10 sequences. But the amplicon that is generated across these should be different enough in size or sequence or both, so that I can figure out which sequence was the PCR template from among 10 possible ones. Better yet if the primers can be used for template discrimination during both qPCR (with gDNA template) and q RT-PCR (with mRNA template).
Since I have ~ 3000 such alignments, I want and need to automate this, and not squint looking at them onscreen! I could follow a purely MSA parsing approach, but I wonder if newer tools used for completely different purposes (e.g. genome mappers etc) can be co-opted for my task. Thanks, folks!