Hello,
I don't know if it's too late but I found this topic " Fungal ITS analysis tutorial"
In fact there is a section in this tutorial which is exactly my problem in this topic:
"One issue with ITS (and other marker genes with vast length variability) is readthrough , which occurs when read lengths are longer than the amplicon itself!"
There is the command used like me but only in one step:
qiime cutadapt trim-paired \
--i-demultiplexed-sequences demux.qza \
--p-adapter-f AYTTAAGCATATCAATAAGCGGAGGCT \
--p-front-f AACTTTYRRCAAYGGATCWCT \
--p-adapter-r AGWGATCCRTTGYYRAAAGTT \
--p-front-r AGCCTCCGCTTATTGATATGCTTAART \
--o-trimmed-sequences demux-trimmed.qza
I tried downloading the data from this tutorial to see if my problem is with these data too, but the forward primers have already been trimmed in the raw reads.
So I think the command in the Fungal ITS analysis tutorial has the same problem that I had, I think we need to use the 2 step cutadapt solution.
Moreover, I detected an error in this tutorial: the forward primer and the reverse primer are swapped.
So, Instead of the command seen in the tutorial above the real command is (with the wildcard enabled!):
qiime cutadapt trim-paired \ --i-demultiplexed-sequences demux.qza \ --p-adapter-r AYTTAAGCATATCAATAAGCGGAGGCT \ --p-front-r AACTTTYRRCAAYGGATCWCT \ --p-adapter-f AGWGATCCRTTGYYRAAAGTT \ --p-front-f AGCCTCCGCTTATTGATATGCTTAART \ --p-match-read-wildcards --p-match-adapter-wildcards --o-trimmed-sequences demux-trimmed_swapped.qza
=> So to have the expected results: the 2 steps solution:
qiime cutadapt trim-paired
--i-demultiplexed-sequences demux-paired-end.qza
--p-adapter-f AGWGATCCRTTGYYRAAAGTT
--p-adapter-r AYTTAAGCATATCAATAAGCGGAGGCT
--p-match-read-wildcards
--p-match-adapter-wildcards
--verbose
--o-trimmed-sequences trimmed_remove_primers_wild.qza
qiime cutadapt trim-paired
--i-demultiplexed-sequences trimmed_remove_primers_wild.qza
--p-front-f AGCCTCCGCTTATTGATATGCTTAART
--p-front-r AACTTTYRRCAAYGGATCWCT
--p-match-read-wildcards
--p-match-adapter-wildcards
--p-discard-untrimmed
--verbose
--o-trimmed-sequences trimmed_remove_primers_wild_2.qza
How can I report this error on the tutorial Fungal ITS analysis tutorial?