Experimental validation of taxon-specific mini-barcode primers for metabarcoding of zooplankton.

AbstractMetabarcoding to determine the species composition and diversity of marine zooplankton communities is a fast-developing field in which the standardization of methods is yet to be fully achieved. The selection of genetic markers and primer choice are particularly important because they substantially influence species detection rates and accuracy. Validation is therefore an important step in the design of metabarcoding protocols. We developed taxon-specific mini-barcode primers for the cytochrome c oxidase subunit I (COI) gene region and used an experimental approach to test species detection rates and primer accuracy of newly designed primers for prawns, shrimps and crabs and published primers for marine lobsters and fish. Artificially assembled mock communities (with known species ratios) and unsorted coastal tow net zooplankton samples were sequenced and the detected species were compared to those seeded in mock communities to test detection rates. Taxon-specific primers increased detection rates of target taxa compared to a universal primer set. Primer cocktails (multiple primer sets) significantly increased species detection rates compared to single primer pairs and could detect up to 100% of underrepresented target taxa in mock communities. Taxon-specific primers recovered fewer false positive or negative results than the universal primer. The methods used to design taxon-specific mini-barcodes and the experimental mock community validation protocols shown here can easily be applied to studies on other groups and will allow for a level of standardization among studies undertaken in different ecosystems or geographic locations.The data added to figshare includes the Qiime2 bash script, raw sequence data generated for each mock community using next-generation sequencing and Sanger sequences downloaded from GenBank to create the local reference library.