Another topic on negative controls

Hullo QIIME people,

Forgive me for yet another topic of negative controls. I have read most of the topics of negative control contamination and looked closely at my data. I included many different controls as I was preparing from this but of course I am still a little dissapointed that there is no clear answer. All of my negative controls had no detectable DNA concentraiton or amplification prior to being sent for sequencing. But naturally, after PCR (externally) and following sequencing many of my samples had feature count similar to my unknowns. Analysing the data after the taxonomy allocation step indicates that some of these sequences only appear in the negative -> However, some high abundances in neg controls are similar to the unknowns. I don’t think I will go the “delete” approach here. I can’t/don’t want to just minus the neg from unknown as in some cases they are higher.

I looked at Rstudio Decontam package but honestly the instructions were a bit confusing.

My questions- If there is a significant difference between negative controls and unknowns in the beta and alpha diversity analsyes… does it matter if you treat the negatives? My PCOA graphs show all negative controls clustering together out of my main other data.