Hi @MiriamGorostidi,
great detective work so far, I did not know that ITSxpress is not meant for IonTorrent sequences.
I think your plan should work, and more or less your pipeline will be the following:
I) Denoise each run with dada2 pyro, using the same parameters
II) Merging the obtained feature tables
III) Before merging the rep-seq for each run, reorient the sequences using your database as reference
This is important because sklearn expect all reads in same orientation and it can be confused if it is not the case. You can use RESCRIPt plug in to do that (please look at 'orient sequence by alignment to reference' in the tutorial Processing, filtering, and evaluating the SILVA database (and other reference sequence data) with RESCRIPt )
IV) Plot the rarefaction curve using the merged feature table, and use it to define the rarefaction threshold comparing all the samples. That should avoid any question form the reviewer on your doubt ;).
Oh I forgot, all the above after removing the PCR primers using cutadapt!
Let us know how is going!
Cheers
Luca