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:
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List of sequence IDs from P.generosa genes FastA (GitHub Issue comment from Steven)
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P.generosa genes FastA (OSF repo)
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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):
RESULTS
Output folder:
The number of matches identified in the P.generosa genes FastA file for each primer set are in the table below. Note the following:
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Primer sets selected/tested were the first primer set generated by Primer3.
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There was a 20% mismatch allowed when checking specificity.
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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.
| SeqID | PrimerName | Matches |
|---|---|---|
| PGEN_.00g025890-vv0.74.a | TIF3s12 | 2 |
| PGEN_.00g070040-vv0.74.a | APLP | 2 |
| PGEN_.00g188130-vv0.74.a | FEN1 | 2 |
| PGEN_.00g194630-vv0.74.a | ECHD3 | 2 |
| PGEN_.00g338640-vv0.74.a | NSF | 2 |
| PGEN_.00g288180-vv0.74.a | TIF3s4a | 4 |
| PGEN_.00g245080-vv0.74.a | TIF3s10 | 8 |
| PGEN_.00g132030-vv0.74.a | TIF3s8-1 | 10 |
| PGEN_.00g079690-vv0.74.a | TIF3s7 | 14 |
| PGEN_.00g088260-vv0.74.a | NFIP1 | 36 |
| PGEN_.00g224740-vv0.74.a | GLYG | 46 |
| PGEN_.00g280110-vv0.74.a | SPTN1 | 496 |
| PGEN_.00g082590-vv0.74.a | TIF3s5 | 742 |
| PGEN_.00g287540-vv0.74.a | RPL5 | 2570 |
| PGEN_.00g132040-vv0.74.a | TIF3s8-2 | 7800 |
| PGEN_.00g114060-vv0.74.a | GSK3B | 8596 |
| PGEN_.00g000750-vv0.74.a | TIF3s6b | 15512 |