Hi. May I please have whatever parameter changes are necessary to BLAST short (ie primer) sequences?
a few key points can help when using BLAST to check for specificity of PCR primers:
- in the advanced parameters box at the bottom, use
-task blastn-shortas this decreases the word-size and thus increases the sensitivity
- also remove low complexity filter and low confidence filter. This is because part of your primer may sometimes hit a repetitive or ambiguous sequence. For this, put
-dust no -soft_masking falseinto the advanced parameters box.
- reduce the scope of your search (e.g., search against only your organism of interest, rather than a multi-genome databases). This is because greater database size decreases e-value strength.
- Check the coordinates of the hits. If you have many, it can be helpful to download the table of all hits into excel. Make sure you also check query coordinates - blast doesn’t necessarily align from the first to the last nucleotide (i.e., the hit may focus on the middle part of the sequence that does match perfectly).
Hope this helps ,
P.S.: for primer search, some people also modify the way BLAST calculates scores… because a mismatch can severely reduce annealing. So they might add
-penalty -3 -reward 1. Same thing for gaps (insertion/deletions), you might add
-gapopen 5 -gapextend 2.
P.P.S.: So if you put all of everything together, use the following BLASTN options for checking PCR primers:
-task blastn-short -dust no -soft_masking false -penalty -3 -reward 1 -gapopen 5 -gapextend 2