Limitations of MiFish primers
High numbers of unidentified ASVs are a common feature of barcoding and
metabarcoding studies (e.g. Leray & Knowlton, 2017). These unidentified
ASVs are typically attributed to incomplete reference databases (Curd et
al., 2019; Ransome et al., 2017; Schenekar et al., 2020) and/or novel
biodiversity (Barber & Boyce, 2006; Boussarie et al., 2018). However,
given that FishCARD includes 82.8% of all California Current fishes,
and the remaining 15.6% are mostly rare species unlikely to be found in
a kelp forest, it was extremely surprising that half of all ASVs and a
quarter of all sequences generated in our eDNA test datasets could not
be assigned.
The vast majority of these sequences and ASVs did not belong to
vertebrates, but instead uncultured marine bacteria, specifically
matching to 16S , rather than 12S loci. Since mitochondria
represent the capture of microbial endosymbionts by ancient eukaryotes
(Roger, Muñoz-Gómez, & Kamikawa, 2017) and that this capture occurred
in the sea, it perhaps is not surprising that primers designed to target
vertebrate 12S might also capture marine prokaryotes. Similarly,
the homology between vertebrate 12S and prokaryotic and bacterial16S genes is well known (Crews & Attardi, 1980) suggesting
capturing microbial 16S with vertebrate 12S primers is
also not surprising. However, this particular feature of the MiFish
primer set was previously unreported, potentially impacting the
interpretation of unidentified ASVs in fish metabarcoding studies.
These findings highlight the importance of accurate universal
metabarcoding primer design, especially in outlining both target and
non-target sequences. In the design of the MiFish Teleost 12Sprimers, uncultured marine microbe 16S sequences were not
considered as potential alternative targets for the primer set,
resulting in the selection of a metabarcoding locus with a high degree
of non-target amplification (Miya et al., 2015). This is an important
finding for the marine vertebrate eDNA community which has recently
converged on the MiFish 12S primers (Closek et al., 2019;
O’Donnell et al., 2017; Valsecchi et al., 2019; Yamahara et al., 2019)
as these results suggest that the MiFish 12S primer set will
generate substantial quantities of non-target eDNA reads. At best, this
non-target amplification will lead wasted sequencing effort, as every
microbial sequence generated reduces the number of vertebrate sequences.
At worst, it could result in incorrect interpretation of unidentified
ASVs. This problem is of particular concern in environments with high
relative abundance of marine bacterial communities and low relative
abundance of vertebrate biomass such as in some pelagic midwater and
deep sea habitats where recent eDNA sample collection efforts have
struggled to detect vertebrate sequences (K. Pitz personal
communication).