Parameters for primer blasting

Hi. May I please have whatever parameter changes are necessary to BLAST short (ie primer) sequences?

Hi @corn26 :wave:

a few key points can help when using BLAST to check for specificity of PCR primers:

  1. in the advanced parameters box at the bottom, use -task blastn-short as this decreases the word-size and thus increases the sensitivity
  2. 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 false into the advanced parameters box.
  3. 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.
  4. 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 :thinking:,


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

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