Testing eDNA extraction, amplification and metabarcoding methods for monitoring marine vertebrates
Environmental DNA (eDNA) metabarcoding is a powerful tool for assessing marine vertebrate biodiversity, yet methodological variables such as filter types, DNA extraction protocols, and amplification efficiency can significantly influence results. This study investigates the performance of three different filter-DNA extraction methods using both eDNA samples and mock community (MC) data, evaluating their effectiveness in capturing vertebrate biodiversity signals from marine environments. A short ~245 bp fragment of the mitochondrial 16S rRNA gene was amplified and reamplified using MarVer3(A) primers (Valsecchi et al., 2020), a modified version of the original MarVer3 set, optimized for greater specificity toward marine mammals, teleost fish, and other relevant taxa.  Three filtration and extraction workflows were tested: Method 1 involved filtering 4 L of seawater through mixed cellulose ester (MCE) filters with subsequent extraction using the DNeasy PowerWater Kit; Method 2 used Sterivex filters with the DNeasy PowerWater Sterivex Kit; and Method 3 applied a modified extraction protocol to Sterivex filters, incorporating a bead-beating pre-homogenization step prior to extraction with the standard PowerWater Kit. Filtration controls were processed to monitor potential contamination.  To further evaluate amplification performance, we compared two DNA polymerase enzymes with differing fidelity: the high-fidelity, proofreading Platinum SuperFi II DNA Polymerase (Invitrogen), and the non-proofreading Supreme NZYTaq 2x Green Master Mix (NZYTech). These enzymes differ in their ability to detect and correct replication errors; proofreading enzymes possess 3'–5' exonuclease activity that enhances taxonomic resolution by reducing amplification errors, while non-proofreading enzymes lack this activity but offer a more cost-effective solution for large-scale studies. By applying both enzymes to MC and eDNA samples, we assessed differences in amplification accuracy and taxonomic resolution, providing key insights into enzyme performance trade-offs in marine vertebrate metabarcoding.
Environmental DNA (eDNA) metabarcoding is a powerful tool for assessing marine vertebrate biodiversity, yet methodological variables such as filter types, DNA extraction protocols, and amplification efficiency can significantly influence results. This study investigates the performance of three different filter-DNA extraction methods using both eDNA samples and mock community (MC) data, evaluating their effectiveness in capturing vertebrate biodiversity signals from marine environments. A short ~245 bp fragment of the mitochondrial 16S rRNA gene was amplified and reamplified using MarVer3(A) primers (Valsecchi et al., 2020), a modified version of the original MarVer3 set, optimized for greater specificity toward marine mammals, teleost fish, and other relevant taxa.  Three filtration and extraction workflows were tested: Method 1 involved filtering 4 L of seawater through mixed cellulose ester (MCE) filters with subsequent extraction using the DNeasy PowerWater Kit; Method 2 used Sterivex filters with the DNeasy PowerWater Sterivex Kit; and Method 3 applied a modified extraction protocol to Sterivex filters, incorporating a bead-beating pre-homogenization step prior to extraction with the standard PowerWater Kit. Filtration controls were processed to monitor potential contamination.  To further evaluate amplification performance, we compared two DNA polymerase enzymes with differing fidelity: the high-fidelity, proofreading Platinum SuperFi II DNA Polymerase (Invitrogen), and the non-proofreading Supreme NZYTaq 2x Green Master Mix (NZYTech). These enzymes differ in their ability to detect and correct replication errors; proofreading enzymes possess 3'–5' exonuclease activity that enhances taxonomic resolution by reducing amplification errors, while non-proofreading enzymes lack this activity but offer a more cost-effective solution for large-scale studies. By applying both enzymes to MC and eDNA samples, we assessed differences in amplification accuracy and taxonomic resolution, providing key insights into enzyme performance trade-offs in marine vertebrate metabarcoding.
CIIMAR - Interdisciplinary Centre of Marine and Environmental Research