I’m really a COI guy when it comes to aquatic invertebrates: good references and species level resolution. However, there is a small but vocal community pushing the ribosomal marker 16S for DNA metabarcoding. The advantage is clear; highly conserved gene regions wich should be suited very well for designing universal primers (Deagle et al. 2014). So last year, we reached out to our colleges in France and they provided us with universal 16S primers to test on freshwater insects. We did test them with the same mock communities we used for the Folmer primers last year (Elbrecht & Leese 2015).

The preprint and raw data is now available here, and we are happy about any feedback which improves the manuscript! Let us know what you think = )

16S might show less primer bias, but taxonomic resolution remains un known as almost no reference data is available. Highly degenerated COI primer show equally good detection rates than the tested 16S marker, but here we tested only the Folmer primers which show high primer bias.
16S might show less primer bias, but taxonomic resolution remains unknown as almost no reference data is available. The tested standard COI barcoding Folmer primers show a strong primer bias (less consistent amplification).

COI vs 16S key findings

Pro 16S:

  • Less primer bias, could allow for rough estimation of biomass and decreased sequencing depth (cost reduction).
  • higher taxa detection rates than with COI Folmer primers (at least for insect taxa).

Contra 16S:

  • Little 16S reference data is available, generation of custom references necessary (Sanger sequencing).
  • Unknown known taxonomic resolution on species level.
  • This 16S marker was only compared against the standard Folmer primers, which are not ideal metabarcoding markers (improved COI primers might give equally good results, as first tests suggest).

So over all, if local reference data bases can be generated, the 16S marker can be a really good choice. Once more (hopefully complete mitochondrial genome data) are generated, the taxonomic resolution of the 16S marker can be investigated as well.

Personally, we will however continue using the COI barcoding region in the future. It’s just not feasible for our lab to generate a 16S database for thousands of taxa and our new COI primers optimised for freshwater invertebrate taxa show good results and detection rates of 100% for insects as well (preprint in the next 2 month). Never the less, it’s good thing that we were able to confirm the potential of the 16S marker, and the concerns raised in (Deagle et al. 2014).


  • Deagle, Jarman, Coissac, Pompanon, Taberlet (2014). DNA metabarcoding and the cytochrome c oxidase subunit I marker: not a perfect match. Biology Letters.
  • Elbrecht, Leese (2015). Can DNA-Based Ecosystem Assessments Quantify Species Abundance? Testing Primer Bias and Biomass—Sequence Relationships with an Innovative Metabarcoding Protocol. PloS one.
  • Elbrecht, Taberlet, Dejean, Valentini, Usseglio-polatera, Beisel, Coissac, Boyer, Leese (2016). Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects. PeerJ Preprints
New PrePrint: Testing 16S for DNA metabarcoding!

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