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.
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A barcoded experiment fails in a way the data cannot show you. If two tags in the pool are one substitution apart, a single read error moves one construct's counts onto another's row, and nothing downstream can detect that it happened — the totals still add up, the plot still looks like a result. The defence is a minimum edit distance across the whole set: if every pair differs by at least d edits, then up to (d−1)/2 errors can be corrected and up to d−1 detected, which is the same coding theory that protects a barcode on a parcel. This designs to that constraint directly rather than filtering afterwards, so a GC window, a homopolymer cap and the restriction sites you clone with are all honoured inside the search. The distance it reports is re-derived over the finished pool rather than assumed from the search that produced it. Choose Levenshtein if your reads come off a long-read platform, where insertions and deletions are the dominant error; Hamming is faster and adequate where errors are substitutions, but it offers no protection against an indel shifting one barcode into another, and the tool says so rather than letting the faster option look equivalent. If you already have a set — inherited, ordered years ago, or assembled by hand — the audit mode names the closest pair, which is the thing you can actually act on.
Choose how many barcodes you need and press Design.
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 only means “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. So distance 3 corrects one and detects two, which is the usual choice for short Illumina indices; distance 5 corrects two, which is worth it for long-read platforms or when a sample is irreplaceable. 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, which 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 says 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 you should rather than 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.
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