Yesterday, a new publication by Vasco und Florian entitled “Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment“has been published in Frontiers in Environmental Science (Link). In the study we demonstrate problems with several of the currently available metabarcoding primers and tested four newly developed COI primers targeting aquatic macroinvertebrates. Our analyses show that the new primers (BF/BR) are highly suitable and amplify very equally the analysed taxa. Most importantly, however, our study shows how important careful design and validation of primers for a certain region, habitat or target taxon group is. With growing data bases there is still much potential for future improvement.
Last year we used a freshwater invertebrate mock community to test the standard COI barcoding Folmer primers in a metabarcoding setting and demonstrated that primer bias makes estimation of biomass impossible. Together with our colleagues from France we now tested the same mock samples with a ribosomal marker, which is supposed to show less primer bias due to more conserved primer binding sites. As we show in the new article which was just published in PeerJ, the 16S marker indeed leads to more consistent amplification results than the Folmer primer set when used in DNA metabarcoding.
However, primer bias is still substantial (two orders of magnitude) and thus estimation of biomass still impossible. Furthermore, we are currently lacking comprehensive 16S reference databases for animals, which are available for the COI barcoding marker. First tests show that the COI marker performance can be greatly improved by using metabarcoding optimised primers (we will publish a PrePrint soon). Thus, while the 16S marker might have potential, we focus on using the COI maker for DNA metabarcoding of insects but just not with Folmer primers.
Figure: The tested 16S marker shows less variation in read numbers than the COI Folmer maker, thus allows for better detection of freshwater taxa. However, with improved COI primers better detection rates are possible as well, as first tests demonstrated.