I was using the MiSeq Sop to analyze the available example Mock fastq files. After analysis, I can’t find the Propionibacterium OTU although this bacterium has been reported to be within this mock mixture (Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. (2013): Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and Environmental Microbiology. 79(17):5112-20.).
Are we sure this bacterium is in this example dataset?
If you look at the primers and the V4 region for Propionibacterium, you’ll see that there is a mismatch to the primer at the 3’ end of the sequence. We occasionally get a few Prop. sequences through but there’s a clear bias. If people are interested in skin, I’d strongly suggest modifying the primer.
Thanks Pat for the quick response. As discussed by many others, universal primers just don’t seem to exist. An other possible solution to solve this problem is to use our micelle PCR amplification approach that we have recently published: http://www.nature.com/articles/srep14181. The compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, including those that amplify poorly due to primer mismatches. Also, it drastically reduce chimera formation. Sorry for the advertisement, but it could be of help for many of us.