Which Sequencing Primers to Order - Plan Reads by Discrimination
Choose the smallest set of sequencing primers that can tell your candidate molecules apart, and find out which pairs nothing will separate - before you pay for the reads.
🔒 Nothing you paste is logged or stored on our servers
The usual way to pick sequencing primers is coverage: tile the construct until every base is read. For a verification that answers the wrong question. You are not trying to read the construct, you are trying to find out which molecule you actually have - and the molecules a cloning reaction can produce are enumerable before you sequence. Against a known candidate set, a read is worth exactly the pairs of candidates it separates, so a read covering 700 new bases on which every candidate agrees is worth nothing, and a short read across a junction is worth everything. This returns the smallest set of primers that separates every pair any of them can, and names the pairs none of them separates - which is the real answer to "will three reads be enough", and a number nobody currently gets before the reads are paid for.
0 candidates. The intended construct plus what else the reaction could give you.
0 primers to choose between.
Paste the molecules your reaction could produce and the primers you could use.
How to use the Sequencing Read Planner tool
- 1Paste your candidate molecules as FASTA - the intended construct plus the alternatives the reaction could give you. The assembly outcomes tool enumerates those for you.
- 2Paste the sequencing primers you could use, as FASTA.
- 3Read the plan: which primers to order, and which candidate pairs no primer here can separate.
- 4Order the reads, then bring the traces back to the mixture deconvolution tool.
Frequently asked questions
Why not just tile the whole construct?
Because coverage and discrimination are different objectives. If every candidate molecule carries the same 700 bases, a read across them tells you nothing about which one is in your tube - and it costs the same as the short read across the junction that would settle it.
Where do the candidate molecules come from?
The assembly outcomes tool enumerates what a given reaction can produce: the intended construct, empty backbone, double insert, flipped part and so on. You can also paste your own.
What does it mean when a pair cannot be separated?
That no primer in the set you gave produces a read that differs between those two molecules. It is the most useful thing on the page: it tells you to add a primer, or to accept that sequencing will not distinguish them and use a digest instead.
Does it model read quality?
It models the read window - a Sanger read does not start at the primer's 3' end and does not run forever, and both are parameters with documented defaults. It does not model whether a particular read will fail.