Degenerate primers from an alignment

1. 01One primer designed across an ALIGNMENT rather than a single template
2. 02The maximum-coverage degenerate oligo at every column, under a degeneracy ceiling you set as a real constraint rather than a filter after the fact
3. 03Five searches, including an exact branch-and-bound that refuses rather than guessing
4. 04Coverage, distinct-sequence count and entropy reported per column: how many members of the family each candidate binds, per site
5. 05Column eligibility controlled: how much gapping a column may carry and still be designed on
6. 06A melting-temperature RANGE over every molecule in the pool, not a single number for a mixture
7. 07Amplifying pairs inside a product-size window, with the reverse oligo as ordered
8. 08Marks on the canvas through both coordinate hops, and every designed site drawn on the alignment

A faithful port of DegePrime (Hugerth et al. 2014), the published maximum-coverage degenerate primer method, validated row for row against the real program over thousands of oracle rows, plus an exact solver the original has no equivalent of, so the heuristics are graded against the true optimum rather than against each other; melting temperatures come from the same golden-validated engine the other primer tools use. The panel runs on @ports/enzymes, @ports/oligo-structure, @ports/primer.

Everything runs client-side. Files are parsed in the browser and projects are saved on this machine, so an unpublished sequence is never uploaded.

Why does the input have to be an alignment?

Because the question is which positions the family varies at. The alignment is what says which columns are conserved and which are not, so a wrong alignment produces a confidently wrong primer.

What does degeneracy actually cost?

The concentration you order is split across every variant the synthesis makes. At degeneracy 4 each molecule is a quarter of the nominal concentration; at 64, under 2%. Past a point the correct primer for your template is too dilute to prime while the wrong ones are numerous enough to mis-prime, which is why the ceiling is the most consequential control on the panel.

Why was my degeneracy ceiling lowered?

A ceiling has to be a product of 1, 2, 3 and 4, because those are the only counts a single position can contribute. Ask for 10 and the search uses 9. The panel says so when it happens rather than quietly using a different number.

Can I use it for barcoding?

That is one of the cases it is for. Barcoding primers have to work across a whole clade, which is the same problem as amplifying a gene from a species whose sequence you do not have, using its relatives.

My family has diverged too far and every oligo is fully degenerate. What now?

That is the point where nucleotide-level design stops working, because silent third-base changes accumulate fastest. Align the proteins instead and use CODEHOP, which designs a small degenerate core over a conserved amino-acid motif with a non-degenerate clamp in front of it.