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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  • Avatar
    Britt Henke

    How do I upload my primers? What format do they need to be in?

  • Avatar
    Moreland Gibbs

    Hi Britt,

    You can import a collection of primers from a tab (.tsv) or comma (.csv) delimited file. You can create this file type manually with a text editor, or in something like Excel.

    At it's simplest the file would contain two columns.

    Name sequence
    A cgatctaggtactacg
    B cgatcgatgcatcggt

    Simply drag the delimited file into the Geneious sources panel. You will asked to confirm format, and then you will get a panel that will allow you assign columns to metadata types. Make sure you set the data type to "Primer" and check the option to "Determine Characteristics". The sequences will then import as individual primer files.

    Edited by Moreland Gibbs
  • Avatar
    Britt Henke


    Thank you for this helpful answer. I have successfully imported my primers. I imported two .csv files. Each with the reverse, forward, and probe. But I'm still not getting the results I want. Do I need to reverse complement the reverse primer and save it that way before running the search? What does this mean, "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."

    Can I talk to someone on the phone?