blast nucleotide short sequence high sensitivity

Question

[ireen.vandolderen]

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!

[GenoMax]

NCBI provides primer-BLAST for this purpose: https://www.ncbi.nlm.nih.gov/tools/primer-blast/ You can find the settings they use that you can use locally.

Answer 1

[SequenceServer]

Hiya,

-word_size 4 is great. You can do a few extra things, tweaking match rewards and gap penalties.

We (makers of SequenceServer Blast analysis software) have written a small overview of how to tweak BLAST for primer checking.

Take care

[ireen.vandolderen]

Very helpful, thank you so much!