I’m running and analysis of environmental DNA of soil samples from a wetland-like ecosystem in South America (it’s called páramo, if you want to see pictures) using the V3 region of the 16S rRNA, in order to see Archaeal communities. This analysis was done at different depths of a soil core of 150 cm in depth, and there were 6 replicates for each sample to be amplified and sequenced. In each PCR “batch” I included a positive control to test everything was working in the reaction; this positive control was DNA from a Haloferax chudinovii. Before sequencing all the soil samples, I sequenced only this Archaea to confirm its identity and that the primers were working well and, as expected, it turned out to be OK. Then I sent my samples alongside the PCR controls (positive and negative) for sequencing in an Illumina MiSeq PE 150. After demultiplexing I followed the moving Pictures Tutorial for quality filtering and Taxonomic Assignment, but the positive control is not even close to being an Archaea, in fact, the taxonomy shows that it has Polaromonas sp. For the classification I have trained my own naive-bayes classifier on SILVA 132 and SILVA 138 and have also used
classify-consensus-blast on NCBI refSeqs using the primers for V3 region I used, but I get the same results. In addition, I couldn’t find any Archaea in the samples either.
I feel that having this result on my positive control invalidates the results in the other samples
(regardless of presence/absence of Archaea), since it is completely different from what I was expecting, but I want to know if someone has encountered this issue in their analysis and what they have done to overcome this. I imagine that the best possible solution is sequencing all the samples again, but doing everything all over again is not feasible at the moment. Maybe somebody has a better opinion about how to approach this problem, either in the lab work processing or the bioinformatic analysis.
Thanks again and sorry for the long post.