Trimming adapters and primer on ONT nanopore sequencing

Question

[l.gallucci]

Hi all!

I'm dealing with a specific nanopore run...for background:

we are doing pcr on extracted samples to target 16S with long reads primers already barcoded. Then following the normal steps nanopore protocol for 16S, we are going to ligate two different pool of DNA samples with barcode 1 and barcode 2 respectively and the native adapter.

Now, my question is... if I'm doing the demultiplexing/trimming through dorado in a first step just the barcode added at last step will be removed, giving demultiplexed pool 1 and pool 2, I'm right?

dorado basecaller sup ./ --emit-fastq -v --kit-name SQK-NBD114-24 --trim all 

So, in theory, I need to additionally demultiplex in another round the 2 pool, with the primer used during the PCR?

Do you have suggestions? If some details are missing, please ask!

Answer 1

[GenoMax]

if I'm doing the demultiplexing/trimming through dorado in a first step just the barcode added at last step will be removed, giving demultiplexed pool 1 and pool 2, I'm right?

Correct. dorado will only recognize ONT index/barcode and use it to demultiplex the data and remove that index. If you have internal barcodes that your amplicons have then you will need to demultiplex those yourself.

[l.gallucci]

Hi GenoMax thank you for the reply! The ONT-barcoded primers used are similar to this: ONT-BC_F01-GM3F CCGTGACAAGAAAGTTGTCGGTGTCTTTGTGAGAGTTTGATCMTGGC They are from biomers.

I have the entire structure (Padding+Barcode+Primer) for all. Do you have any suggestions how to proceed?