Primer Design and In-Silico Testing - Geoduck Reproduction Primers

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:

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:

  • 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 2 only 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