We generally talk about degenerate nucleotide positions in synthesized oligo (like primers) as if they are this automatic and even thing, but like everything else, things can be a lot more complicated. We recently ordered some primers with six degenerate positions (so NNNNNN) from ThermoFisher, and they were predominantly filled with T’s, while the same oligo ordered from IDT was a lot more balanced in terms of nucleotide composition. While this was done at really small (Sanger) scale and not quantitative, this reminded me of nucleotide biases I observed in libraries created in the Fowler lab using Inverse PCR (where the degenerate sequence is not hybridizing with a partner strand during initial hybridization, so no degenerate sequence should have a competitive edge over another, at least in terms of hybridization). These were all made with IDT primers. I dug up that slide, and have posted it below:

So in this analysis, you can see that there is a ubiquitous albeit somewhat variable G bias across the libraries. This often resulted in the presence of more glycines than expected. Overall, I don’t think this is ALL that bad; certainly still WAY better than the rampant (like, 86% T) bias with the ThermoFisher primer (When I emailed them about this, they told me “Unfortunately, we do know exactly what the issue is. This is an inherent instrument issue with how automated degeneracies are produced on the existing synthesizer. The only way to fix it from a customer perspective is to order using special instructions to “hand mix” the degeneracy in an optimal ratio to produce as close to a 25% balance as possible.”). So while I’ll still buy regular cloning primers from Thermo (b/c they are the cheapest for me), all degenerate primers will have to go through IDT (or maybe eventually EuroFins, as the Roth lab has noted good luck with them).

5/15/21 update: OK, got some Illumina sequencing back to actually quantitate this, and the ThermoFisher degenerate primer definitely suffered from too many T’s, with ~ 49% of all nucleotides being T’s. Actually, the IDT hand mixed did worse than IDT machine mixed, which had the closest to the target values. Interesting.