I'm writing a tool to automate Sanger sequencing primer design for a production lab that uses a universal-tail chemistry Sanger sequencing to verify NGS results. Essentially, the template DNA is amplification using primers with universal 5' tails (M13F/R), and then the "BigDye" reaction is performed with an M13F and M13R primers in separate wells.
I'm able to design logical primers for each region in a request using Primer3 (v2.5.0), but then I'd like to calculate folding thermodynamics for the M13-appended primers to determine the best primer partner for the 5' M13 appendages.
In other words, in the last step, I would like to know if I should use:
M13F-forward_primer + M13R-reverse_primer
M13R-forward_primer + M13F-reverse_primer
The problem is that Primer3 won't fold an oligo > 36 nt, since the algorithm for Tm is diminishingly accurate for longer oligos. However, I believe thermodynamic predictions are valuable, even if only relatively, i.e. comparing one 5' appendage to another 5' appendage on the same allele-specific sequence, so I'd like the most painless answer, even if it isn't accurate in absolute terms.
I'm aware that UNAFold will do this, but it seems like extreme overkill for what I need to do.
For lack of a better solution, I've implemented a workaround wherein I pair each allele-specific primer with either M13F or M13R and submit the pair as a
check_primer task. In other words, the left primer is one of the allele-specific primers that primer3 designed, and the right primer is either M13F, or M13R. I then choose the M13 appendage orientation based on the
PRIMER_PAIR_0_COMPL_ANY_TH output values for the two possible orientations.
I would like to know: Is there a better solution? Primer3 docs say that the Tm calculation (and not necessarily folding thermodynamic calculations) is inaccurate for 37+ mers. Is there a way to force Primer3 to fold a sequence > 36 nt? If not, is there a simple tool for folding >36-mers?
Follow-Up: The workaround described in my OP was inadequate and UNAFold would cost $9k (which would take months for my employer to approve - don't get me started), so I tried @MichaelG's solution below. It turned out to work quite well. I'm computing the maximum Tm value for all fragments from every oligo, and that number agrees quite well with the browser-based UNAFold output.