Does a mismatch mean the assay has failed?

No. Where it sits is what matters. A mismatch in the last few 3′ bases is the one a polymerase has to extend through; one further away is usually carried by an oligo whose melting temperature is above the annealing temperature. The panel counts the two apart and does not model the reaction.

What does the rate actually describe?

The genomes you gave it. A corpus drawn from one country, one month or one lineage answers that narrower question, and the interval beside the rate says how firmly the corpus supports it.

My probe has an R or a Y in it. Is that counted as a mismatch?

No. A designer who wrote R meant both bases, so a position the code covers is never a mismatch. That is what makes a pan-species probe readable here.

Is anything uploaded?

No. Every calculation runs in the browser on the records in your project.

Analyze

Assay decay: does a published assay still match current genomes, and is the mismatch at the 3′ end

Take an assay somebody published years ago and ask whether its primers and probes still match the sequences circulating now. Each oligo's best binding site is found in every genome of a corpus, the mismatching positions are reported from the oligo's own 5′ end, and the ones inside the 3′ window a polymerase must extend through are counted apart from the ones a primer usually carries. The rate comes with an exact interval, so a reading over forty genomes cannot be mistaken for one over four hundred.

Open Assay decay: does a published assay still match current genomes, and is the mismatch at the 3′ end Open the Assay decay sample

Twelve published SARS-CoV-2 RT-PCR assays against 40 genomes deposited in 2026, to see which of their primers and probes still match and where the mismatch sits

What it does

01Each primer and probe of a published assay scored against every genome in a corpus, on either strand, by the site with the fewest mismatching positions
02The mismatching positions numbered from the oligo's own 5′ end, and the per-position counts that separate one fixed substitution from scattered drift
03Mismatches inside the 3′ bases a polymerase has to extend through, counted apart, with probes excluded because a probe is not extended
04A mismatch rate per oligo over the genomes it could be scored on, with an exact (Clopper–Pearson) 95% interval, and the oligo whose rate decides the assay
05IUPAC codes in an oligo read as the bases they cover; a site over ambiguous bases in a submitted assembly left out of the denominator rather than counted either way

What it runs on

Every oligo's best site and mismatch count is checked against EMBOSS 6.6.0 fuzznuc on every scored pair of 37 published oligos against 400 genomes — 14,763 pairs, with no disagreement. The panel runs on @ports/workflow.

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

Questions

Does a mismatch mean the assay has failed?: No. Where it sits is what matters. A mismatch in the last few 3′ bases is the one a polymerase has to extend through; one further away is usually carried by an oligo whose melting temperature is above the annealing temperature. The panel counts the two apart and does not model the reaction.
What does the rate actually describe?: The genomes you gave it. A corpus drawn from one country, one month or one lineage answers that narrower question, and the interval beside the rate says how firmly the corpus supports it.
My probe has an R or a Y in it. Is that counted as a mismatch?: No. A designer who wrote R meant both bases, so a position the code covers is never a mismatch. That is what makes a pan-species probe readable here.
Is anything uploaded?: No. Every calculation runs in the browser on the records in your project.

Also searched as

primer mismatch against current genomes · PCR assay still works · in-silico inclusivity · 3 prime mismatch primer · primer binding site variation · assay dropout · published assay against new variants · degenerate primer IUPAC mismatch