The design and testing of taxon-specific mini-barcode markers for metabarcoding of marine zooplankton

DNA metabarcoding of zooplankton samples is well-suited to surveys of marine pelagic ecosystems, however consistent amplification across zooplankton groups cannot be assumed when using universal miniCOI primers. Under-representation of some groups may result from primer bias or inadequate coverage in barcode reference libraries. We designed taxon-specific mini-barcode primers to improve the representation of copepods, euphausiids, chaetognaths and hydrozoans in metabarcoding outputs of mixed zooplankton samples from the Southwest (SW) Indian Ocean. In silico analyses of downloaded sequences per group identified the most informative mini-barcode region, and barcode gap analyses confirmed that the selected regions could distinguish among species within each group. In vitro analyses of DNA extracts from individual specimens per group showed that the new mini-barcode primers outperformed the standard and universal miniCOI primers by consistently recovering higher Phred scores. Metabarcoding of four in situ mixed zooplankton samples collected with plankton tow nets and processed with a combination of taxon-specific and universal primers, identified 220 species. Use of the primer cocktails increased the proportionate representation of the target groups by three- to five-fold, compared to a previous study. Read counts were dominated by copepods and euphausiids, implying that they had the highest relative biomass in samples. We conclude that a combination of universal and taxon-specific primers in metabarcoding assays will achieve a more comprehensive assessment of biodiversity by enhancing species richness estimates across different groups.