I would like to know if anyone else has had experience using the 18S rRNA primers from the Earth Microbiome Project protocol.
I have used the 1391f and EukBr primers (with mammalian blocking primer) for a human faecal microbiome project, where our goal was to detect microbial eukaryotes. I adpoted a different sequencing strategy from the one in the protocol - instead of ordering unique primers with barcodes in them, I ordered the primers with Illumina adapters attached for the Nextera XT dual-indexing strategy.
However, when analysing my data I found that these primers actually amplified the V9 region of bacterial 16S rRNA as well as the V9 region of 18S rRNA - 19-99% (mean 76%, median 86%) of the reads in each sample was simply “Eukaryota” classified no further. There were several ASVs with thousands of counts each, and when I BLASTed these sequences they all hit 16S rRNA. (I aligned against the SILVA 18S database only so I expect this is why they were still called “Eukaryota” - no bacteria in there).
I aligned these 18S rRNA primers to the E. coli 16S rRNA gene in silico and found that 1391f aligns to it perfectly with no mismatches, and EukBr aligns with 4 mismatches. In the EMP protocol, it says:
Relative to the 1389F primer of Amaral-Zettler et al. (2009), our 1391f primer’s degeneracy and its 3′ end were modified to minimize amplification of bacteria and archaea.
I’m struggling to see how this can be the case when they align to E. coli quite well. Additionally, the 1389f primer in Amaral-Zettler et al. is described as “universal”, not “eukaryotic”.
So I am wondering - has anyone else used these primers and found that they amplify bacterial 16S rRNA really well? Does anyone have any suggestions as to why this may have happened in my situation, but apparently has not been an issue for the EMP?