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.
You feed the script a barcode file and input fastq file, and it creates a trained mismatch matrix, aligns to the barcodes, then demultiplexes the reads into separate files for each barcode:
./fastq-dental.pl -barcode barcode_full_PBK004.fa -mat bc.mat reads_all.fastq.gz
While the approach designed around using the ONT barcodes, the barcode fasta file can be replaced by another file [using barcode ID in the header] without change the protocol.
FWIW, there is also now some support for detecting strand direction (e.g. using the strand switch primer provided by ONT), but I haven't yet got it working properly doing both demultiplexing and strand orientation at the same time.
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.
- Minibar: This works for dual index barcodes specifically
- 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