I have a problem with my ITS data. When I analyzed the data I got a lot of unassigned sequences (more than 50%) and I don't know where is the problem. This is my current code but I have changed it a lot looking for an answer for this issue (I haven't found anything).
qiime tools import
qiime quality-filter q-score
I think that here there is the big problem, because when I use cat for forward and reverse only I can put 1 primer...
--> Adapters Forward: GTGAATCATCGAATCTTTGAA Reverse: TCCTCCGCTTATTGATATGC
qiime cutadapt trim-single
--p-front GTGAATCATCGAATCTTTGAA \
qiime dada2 denoise-single
I know that if I use closed-reference I won't have the problem with Unassigned sequences but I need to understand why it happens.
qiime vsearch cluster-features-open-reference
qiime feature-classifier classify-consensus-blast
qiime taxa barplot
Perhaps the name of a file does not correspond to the previous one... I've tried so many things that it's crazy but in reality I correct it before executing it.
I have tried pairing/simple forward/ cat forward and reverse (I did it with qiime 1 and had no problem). More stuff... Don't delete adapters/delete adapters, use quality filter/do not use it,,,,, and many more things. Maybe this is the problem that I use so many plugins....
I also have a doubt... Let's suppose that I have indeed done it right (I doubt it) and I have many Unassigned sequences. To get the biodiversity there is no problem, I use everything. I think, sometimes too much, that if the taxonomics does not reflect the biodiversity, the interpretation of the data is wrong, isn't it?
Sorry for this long-long post but I have read a lot about this topic (in this forum and others places) but I am not making progress....