Yes, you need to reverse complement of the degenerate bases. So instead of AGWGATCCRTTGYYRAAAGTT
I think you want AGSGATCCRTTGYYRAAAGTT
(as the RC of AACTTTYRRCAAYGGATCWCT
... looks like the W was not RC-ed in your example).
Also, --p-front-*
should be the primer on the 5' end of the read, then --p-adapter
should be the RC of the primer that may be found somewhere to the 3' end of the sequence if you have primer read-through. It looks like you may have switched these.
Don't follow the fungal ITS tutorial too litarally; that tutorial uses a mock community in which the reads were swapped around and there may be some other funny details. So you will need to double-check the direction of your reads, e.g., look at the first few sequences manually to see if you can detect the presence of your forward/reverse primers to determine the read direction and appropriate settings for those parameters.
I think not, unless if you switched the read order (to import forward as reverse and vice versa).
Try this and let me know how it goes:
qiime cutadapt trim-paired \
--i-demultiplexed-sequences Fungal-demux-paired-end.qza \
--p-adapter-f AYTTAAGCATATCAATAAGCGGAGGCT \
--p-front-f AACTTTYRRCAAYGGATCWCT \
--p-adapter-r AGWGATCCRTTGYYRAAAGTT \
--p-front-r AGCCTCCGCTTATTGATATGCTTAART \
--o-trimmed-sequences Fungal-demux-trimmed-new.qza
and if that doesn't work, try this (switching the forward and reverse read primers):
qiime cutadapt trim-paired \
--i-demultiplexed-sequences Fungal-demux-paired-end.qza \
--p-adapter-r AYTTAAGCATATCAATAAGCGGAGGCT \
--p-front-r AACTTTYRRCAAYGGATCWCT \
--p-adapter-f AGWGATCCRTTGYYRAAAGTT \
--p-front-f AGCCTCCGCTTATTGATATGCTTAART \
--o-trimmed-sequences Fungal-demux-trimmed-new.qza