qPCR was performed on the same environmental DNA samples, first using a primer pair targeting the archaeal 16S gene, and subsequently, another qPCR was performed using primer pairs targeting the 16S gene of Lokiarchaeota, Bathyarchaeota, and Woesearchaeota, respectively. BLAST confirmed that the primer designed to target the common archaeal 16S gene also indeed binds to the 16S site for Loki-/ Bathy-/ Woese- archaeota. I want to know if it is okay to process this data as follows:
Total Remaining Archaeal gene copies = Archaeal gene copies - Lokiarchaeota gene copies - Bathyarchaeota gene copies - Woesearchaeota gene copies
Robert Adolf Brinzer at the very least, I would assume any biases in amplification to be conserved for the specific taxa. But yes, in theory, I am "assuming equal amplification". There were adequate technical replicates, but unfortunately not adequate biological replicates. Even if I am not getting equal amplification for the remaining taxa, the general point that I am trying to convey has not much to do with the remaining taxa, but more to do with the taxa that I am subtracting. What I want to show is that one of the archaeal taxa is selectively enriched in a particular sample. This means that if the relative abundance of Total Remaining Archaeal is rather low, and the relative abundance of one of the specific taxa (Loki, Bathy, Woese) is quite high, then one can conclude that of the total observable archaeal abundance, the composition is skewed towards one of the taxa.
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