Nanopore cDNA sequencing - primary and complementary strand in one read

Question

[ovovo]

Hello all, have somebody similiar experience with Nanopore cDNA sequencing (kit used was Direct cDNA Sequencing V14 with SQK-LSK114) with Dorado basecaller used. For mapping we used minimap2. The problem is following as you can see on images: we have reads that many of them have second part soft clipped. But this part of read is complementary sequence to the first part of read - supplementary alignment. Does somebody know what can cause this? Between first paired read (primary sequence) and complementary sequence there is no additional bases coresponding to primers used in protocol. It just seems that these two sequences were ligated one after another.

[GenoMax]

Dorado basecaller used

Were these samples barcoded? Can you confirm that the default of trim adapters/barcodes option was not changed for dorado run?

[ovovo]

samples weren't barcoded and only adapters were trimmed, not primers.

Answer 1

[GenoMax]

I guess I missed this

But this part of read is complementary sequence to the first part of read - supplementary alignment. Does somebody know what can cause this?

Just to confirm. The part of read that is soft-clipped above is complement of the part that has aligned? This is likely a duplex read. Have you looked at the number of reads that were reported as duplex when you did dorado basecalling. You may want to try: https://github.com/nanoporetech/duplex-tools

[ovovo]

Yes, as you say. Thanks, I will look into it.