Feature classifier fails to accurately classify one set of samples (works for other sets)

[SoilRotifer (Mike Robeson)]

@mike_kratz

Mixed orientations reads are due to forward and reverse reads not being properly separated into separate R1 and R2 files, correct (this leads to some sequences in the same file beginning with different primers)?

Correct.

@mike_kratz

I'm assuming it is better to orient before dada2 processing for the sake of the error model parameters and I'm also missing all of the forward reads from the R2 file

Yes, exactly.

@mike_kratz

If converting the demux.qza file is the right way to go, how would I go about doing that?

I had a lapse in my thinking when I recommended the orient-seqs command. Sorry to lead you astray!

I think the only tool we have implemented in QIIME 2 that can demux reads in mixed orientation is the qiime cutadapt demux-paired ... command using the --p-mixed-orientation command. But I think cutadapt expects the barcode to be within the sequence itself. Which you can do by concatenating the I1 sequence to the R1 as outlined here. But I think you'd still have to know something about orientation.

I'd ask your sequencing facility to help you out with this. I am sure they have code or tools available to help you get the data in the correct format.