Strandness of Isoseq HiFi reads?

Question

[compuTE]

Hi,

I'm starting to explore some long read cDNA sequencing data (Isoseq). When loading the aligned bam files to IGV (using hifi reads), I see that the reads are marked pointing towards a direction (I assume of transcription). However, I don't fully understand how we got that information as, as far as I know, the molecules are ligated with the same adapters in the 5' and the 3'. So, how is the strand information being kept?

My guess is that the aligners (I'm using minimap2) assume the direction of transcription using the position of the polyA in the transcript. But I'm new to this technology, i don't know... Maybe I'm misinterpreting something, so if someone could explain this in a bit more detail or refer me to a relevant resource from PacBio, I would very much appreciate it.

Best,

[Shiyi]

William Rowell , my alignment shows that my isoseq is not stranded, could you point me to which steps could have gone wrong for me? Thanks!! IGV alignment

[William Rowell]

If you reach out to [e-mail], they can point you in the right direction.

Answer 1

[William Rowell]

During the demultiplex step of Iso-Seq, transcript orientation is normalized based on the orientation and position of the primers. Any sequence downstream will be oriented in the direction of transcription.