Hello,

I am designing short nucleotide probes and need to check them for off-target hybridisation against the transcriptome of my organism. My probes are only 25 nucleotides long, and I would like to remove all the probes which have off-target hybridisation sites with less than five mismatches. What parameters in my blast query can I best alter to balance speed, noise and sensitivity to obtain results which include such low identity? I have tried to decrease the word count to 4 for example, but I fear that a lot of noise is included in the results as a consequence.

Thank you very much!