I am not sure whether this is the correct platform to ask, as my query is related to dada2 (which I am using in R) but I found a thread in this forum which seems to come very close to my problem:
Just like the user in this threat, I have complete separation of ASVs for my different treatment groups when plotting a Venn diagram, which seems biologically impossible
Unlike the user in the thread I do not have different runs and I experience the same separation when looking at other treatments (Surface vs Deep, North vs South etc). I also feel like I end up with a lot more ASVs than I should (24k).
A quick overview of my data:
Illumina PE 300bp
18S V9 region
Amplicon size ~260 bp
I also got a lot of "Unknown Eukaryotes" in my data, which I thought was normal for V9 data but now I am not so sure. I had basically finished all my analysis already and was about to submit a manuscript draft when I noticed the issue with the Venn diagram...
I have already played around (a lot!) with the trimming parameters but it didn't seem to make any difference. The reads are generally high quality and when I trim a lot I get a high proportion of reads outside my expected amplicon size.
I appreciate any kind of advice, and even if it's just the directions to a more appropriate forum! I have no idea where to go from here as it makes me question my entire data analysis.
Thanks in advance!