We have a problem trying to demultiplex MinION sequences with custom barcodes. Do you have any software recommendations we can try for demultiplexing or how to demultiplex these custom barcodes with Albacore? We have tried using albacore but it only recognizes barcodes provided by Nanopore Technologies.


2 Answers 2


My semi-automatic workflow for demultiplexing nanopore reads using LAST can be found here - pay attention to the comments, where I've identified bits in the steps which might trip up people trying to follow them:


While this protocol is designed around using the ONT barcodes, the barcode fasta file can be replaced by another file [using barcode ID in the header] without changing the protocol. Make sure that this file only includes the unique parts of the barcodes (with a few bases of context if necessary to exclude random matches), so that reads won't be matched to the non-unique part of the wrong barcode sequence.

  • $\begingroup$ gringer, thanks for the workflow. It would tremendously help if you included a link to where to find LAST. Thanks! $\endgroup$ Dec 2, 2019 at 0:00
  • $\begingroup$ Thanks for the comment; I've added a link in the comments of the protocol, and in the answer for this question. I'll put a LAST link in the method once I do my next update of the protocol. $\endgroup$
    – gringer
    Dec 2, 2019 at 20:10
  • $\begingroup$ Thank you! I am using minibar, works great and very easy to add custom barcodes. L.Y. $\endgroup$ Dec 4, 2019 at 2:28

There are two other ways in which you could demultiplex reads if guppy doesn't have the config file for the kit you are using.

  1. Minibar: This works for dual index barcodes specifically
  2. Porechop: This is intended for the ONT sets, but you can also replace them with your own. However, it is designed to work with single index barcodes. That means, you will have to run it once with your forward indices, then take that output and run again with the reverse, and then do some gymnastics to combine it all together
  • $\begingroup$ What do you mean with run it twice, with forward and reverse indices? $\endgroup$
    – F.Lira
    Apr 20, 2021 at 7:35

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