Shelly asked that I re-run the primer design pipeline that Kaitlyn had previously run to design a set of reproduction-related qPCR primers. Unfortunately, Kaitlyn’s Jupyter Notebook wasn’t backed up and she accidentally deleted it, I believe, so there’s no real record of how she designed the primers. However, I do know that she was unable to run the EMBOSS primersearch tool, which will check your primers against a set of sequences for any other matches. This is useful for confirming specificity.
In an attempt to replicate what Katilyn previously did, I used the following:
List of sequence IDs from P.generosa genes FastA (GitHub Issue comment from Steven)
P.generosa genes FastA (OSF repo)
Abbreviated gene names used to name primers (Google Sheet)
Primers were designed using Primer3.
Primers were checked for specificity, allowing a 20 percent mismatch, using the EMBOSS primersearch program.
This was all documented and run in a Jupyter Notebook (GitHub):
The number of matches identified in the P.generosa genes FastA file for each primer set are in the table below. Note the following:
Primer sets selected/tested were the first primer set generated by Primer3.
There was a 20% mismatch allowed when checking specificity.
Counts listed in table should be divided by 2 (this is explained in the Jupyter Notebook). Thus, an entry with a value of
2only has a single match in the entire P.generosa genes FastA file.