DNA metabarcoding is an emerging technique for enhancing microbial community monitoring programs that assess water quality and environmental health. However, comparability between studies is limited by a lack of standardized laboratory protocols. To contribute to protocol optimization and decision making, we compare seven commercially available DNA extraction kits and evaluate their impact on DNA yield, PCR inhibitor removal, and taxonomic resolution of microplankton, microalgae, and particle-associated bacterial communities using 18S V9 and 16S V3-V4 amplicon sequencing of samples from two contrasting estuarine environments varying in salinity and productivity. We observed greater DNA yields and detected PCR inhibition at the higher productivity, lower salinity site. The Qiagen DNeasy PowerSoil Pro and Omega E.Z.N.A. Tissue DNA kits both yielded greater amounts of DNA, but only the soil- and plant-specific kits effectively removed PCR inhibitors. Notably, differences in taxonomic richness between kits were statistically significant but small (often <10 taxa). Additionally, beta diversity was more strongly influenced by abundance than by the presence/absence of taxa. All extraction methods shared 84-90% of resolved taxa and detected all major eukaryotic divisions/phyla identified by microscopy, as well as cyanobacteria. Furthermore, all kits detected several potentially harmful taxa, including Akashiwo sanguinea and Vibrio sp. These results suggest that DNA extraction methods should be chosen based on environmental conditions, and we recommend the Qiagen PowerSoil Kit for obtaining high yields of uninhibited DNA from productive estuarine water samples. While alpha- and beta-diversity are largely similar between extraction methods, pilot studies should be performed to confirm the resolution of taxa of interest. 

DNA was extracted from filtered surface water samples at two sites using seven commercially available DNA extraction methods: Omega E.Z.N.A. Tissue DNA Kit, Omega E.Z.N.A. Tissue DNA Kit+ Bead-Beating, Qiagen DNeasy Blood & Tissue Kit, Omega E.Z.N.A. Soil DNA Kit, Qiagen DNeasy Blood & Tissue Kit+ Bead-Beating, Qiagen DNeasy Plant Pro Kit, and Qiagen DNeasy PowerSoil Pro Kit.

Extracted DNA was amplified using a two-step PCR method and sequenced using an Illumina MiSeq. Reads were demultiplexed using the Illumina MiSeq Software and quality control was performed using fastp. Primer trimming, denoising, ASV generation, and taxonomic assignments were performed using QIIME2.