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21 months ago
JC • 0

I prepared a library to run on illumina Hiseq but ended up sequencing it on a MinION sequencer (Oxford Nanopore Technologies). Since the output does not have barcodes in the header, I am having problems demultiplexing.

I did try using grep to search for my adapters/barcodes but i get few reads. I also used agrep with ambiguities and i get a lot more reads. but i cannot also retain the quality scores (the lines before the sequences.)

Any advice or direction to any already established pipelines for this would be greatly appreciated. Thanks!

sequencing next-gen • 1.7k views
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Demultiplexing for nanopore sequencing is done with guppy, and it does support custom barcodes. But with such short Illumina barcodes I hope you realized you're setting yourself up for a messy situation.

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21 months ago
GenoMax 121k

Illumina sequencing libraries are going to have this structure (one more link). You are going to need to find P5/P7 sequence in addition to the indexed adapters in your data. I don't think there are any defined pipelines for this but if there are then someone is bound to chime in.

In any case, you may need to wrangle with your data to see if you can make this work.

Edit: Take a look at bbduk.sh in filter mode. A guide is available here. It may work. Full sequences of Illumina index primers (for TruSeq and Nextera) are in adapters.sh file in resources directory of BBMap suite download. You will need those.

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21 months ago
camppatrick ▴ 10

You could try Porechop. Setting up a custom barcode search on that is not too difficult.