Barcode Designer — Sequencing Tags That Cannot Be Misread Into Each Other
Design a set of DNA barcodes that are pairwise far enough apart that a read error cannot turn one into another, or check a set you already have for the pair that will collide first.
Design a set of DNA barcodes that are pairwise far enough apart that a read error cannot turn one into another, or audit a set you already hold. If two tags are one substitution apart, a single read error moves one construct's counts onto another's row and nothing downstream can detect it. The defence is a minimum edit distance across the whole set: if every pair differs by at least d edits, up to (d−1)/2 errors can be corrected and up to d−1 detected. The search applies that constraint directly rather than filtering afterwards, so a GC window, a homopolymer cap and the restriction sites you clone with are honoured inside it, and the distance reported is re-derived over the finished pool rather than assumed from the search. Choose Levenshtein for long-read platforms, where insertions and deletions dominate; Hamming is faster and adequate where errors are substitutions, but it offers no protection against an indel shifting one barcode into another. Audit mode names the closest pair in an existing set, which is the thing you can act on.
Choose how many barcodes you need and press Design.
Design, edit, and verify complete constructsSeqStudio combines sequence editing and annotation, plasmid maps, primer/cloning/CRISPR design, Sanger verification, and GenBank/SnapGene files in one workspace.
How to use the Barcode Designer tool
- 1Pick how many barcodes you need and how long they can be. Longer barcodes make a given distance far easier to reach.
- 2Set the minimum distance. Three corrects one read error and detects two; one means only “not identical” and corrects nothing.
- 3Add the restriction sites you clone with as excluded motifs, and paste any barcodes already in use so the new ones stay clear of them.
- 4Read the achieved distance, not the requested one: a pool that fell short looks identical in the sequence list.
Frequently asked questions
What minimum distance do I actually need?
It follows from how many errors you want to survive. At distance d you can correct up to (d−1)/2 errors and detect up to d−1. Distance 3 corrects one and detects two, which is the usual choice for short Illumina indices; distance 5 corrects two, which suits long-read platforms or an irreplaceable sample. Distance 1 means only that no two barcodes are identical, and gives no protection at all.
Levenshtein or Hamming?
Hamming counts substitutions only. It is the right measure when the platform's errors are substitutions, as Illumina's mostly are, and it is faster. Levenshtein also counts insertions and deletions, which are the dominant error on nanopore and PacBio, and an indel is exactly what shifts a barcode into its neighbour. If you are not sure, use Levenshtein: a set built to a Levenshtein distance also satisfies that Hamming distance, but not the other way round.
Why did it give me fewer barcodes than I asked for?
Because the constraints left no room, and the alternative would be worse. A barcode of a given length at a given minimum distance has a finite number of admissible forms, and a narrow GC window or a tight homopolymer cap shrinks that further: 200 barcodes of length 5 at distance 4 do not exist. Rather than quietly relaxing the distance to hit your count, it stops and states which constraint to loosen. Lengthening the barcode is almost always the cheapest fix.
Can I add a second plate to barcodes I am already using?
Yes, and that is preferable to designing a fresh set. Paste the existing barcodes into the “already in use” box: they are held fixed, the new ones are kept the same distance from them, and only the new ones are returned. Designing a second set independently is how two plates end up with a colliding pair between them.
Does a good distance mean the pool will work?
No, and the tool's own gate lists what it does not check. Edit distance says nothing about whether the barcodes amplify evenly, whether they form dimers with your primers, or whether a homopolymer miscall breaks the independence the error-correction bounds assume. Run the finished oligos through the oligo pool screen for the dimer half.