How can I quickly test many primers against my sequence?

When there are a lot of primers, the testing process can take a long time, especially when degenerate primers are being used or if you’re testing primers as pairs. Testing as pairs can be especially slow with a lot of primers because Geneious has to test every possible combination.  This can turn a task that should take seconds into one that will take hours, so turn off the option to test as pairs if you don’t need it.

If you want to do a quick screen to identify primers that match your sequence before running the actual primer testing tool, the best option is the Map to Reference tool.  This is a short read assembler which can handle mismatches and aligns the whole short read against the reference.  To use this for testing primers, select the sequences you want to test the primers against as the reference sequences (to do multiple sequences they need to be combined into a sequence list) and then select the primer sequences as the reads then do a medium setting assembly. This will map all the primers that can match onto the references while retaining the regions that don’t match. Note that it will reverse complement reads that match the other strand so you need to reference the primer annotation to see the primer sequence. If you want to check that primers don’t match in multiple locations, be sure to switch to ‘Custom Sensitivity’ and turn on the ‘Map multiple best matches’ option in the ‘More Options’ section.

BLAST is not a good option for testing primers for reasons described under “Can I BLAST primers or short sequences” in the BLAST section.

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