fundamentals of eDNA

This week we discussed the study by Collins et al. (2019). The authors compared multiple different primer sets for targeting freshwater and marine fish. The authors constructed a reference library of both freshwater and marine fish in the British Isles and designed two new COI primers in-silico. Then, they tested out the primer sets on various fish tissues both in-silico and in-vitro. Twelve different primer sets were included in the in-silico PCR test that covered COI, cytochrome b, ribosomal 12S, and ribosomal 16S. For the in-vitro testing, the authors surveyed five different sites in the UK using both eDNA and traditional surveying methods during the Fall of 2016. At each site, they collected triplicate 2L water samples and filtered them through a 0.22µm filter. They used four primer sets for in-vitro testing (3 for COI and 1 for 12S). The authors found that the MiFish primer set that targets the 12S region worked best (detected more species than other primers tested). It seems to me that primer sets need to be tested before being implemented in a study to ensure that those primers are optimal for your target species(s) and sampling environment.

We discussed the following questions:

• How can the results here play into index site sampling and our Maine eDNA research? These results emphasize the fact that not all primer sets will work for all collaborators, and primers need to be tested to ensure that there is true positive detection for target organism(s). -What other questions do you have about degenerate or universal primers and their applications? I have no other questions about degenerate or universal primers or their applications.
• Have you seen examples where universal primers don’t work for certain taxa? Yes, during my master’s, I was focused on quantifying abundance of nitrogen fixing microbes. I found a few different primer sets in the literature, but none of them worked for our taxa. Most of the primers were designed to target nitrogen fixers in the water column, and we were interested in these taxa in the sediment.
• Thoughts on authors’ primer design and primer choice process? I thought that 18S was often used for eukaryotes, and I don’t know why they did not use any 18S primers.
• Applications for metazoans vs phytoplankton vs bacteria? For bacteria, archaea, and micrbial eukaryotes, we mostly use 16S and 18S primers.