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DNA extraction protocol for DNA-metabarcoding of marine phytoplankton using Zymobiomics DNA minprep kit (Zymo Research; D4300) v1

The DNA extraction protocol described in this document follows the standard protocol provided with the Zymobiomics DNA miniprep kit (https://files.zymoresearch.com/protocols/_d4300t_d4300_d4304_zymobiomics_dna_miniprep_kit.pdf) with a few modifications. The protocol is part of the project "DNA-metabarcoding of marine phytoplankton" funded by the Swedish Environmental Protection Agency and the Swedish Agency for Marine and Water Management. Phytoplankton play a key role in global biogeochemical cycles and form the basis of marine food webs. Quantitative assessment of the abundance and biodiversity of phytoplankton is therefore a natural component of environmental monitoring programs. Until now such monitoring has largely been based on microscopic counting. This is a time consuming process and requires personnel with high skill for identifying the organisms, and the smallest cells can hardly be identified with this method. There is hence an urge for faster, more precise and reproducible ways of quantifying plankton species in environmental samples. High-throughput sequencing of taxonomic marker genes (metabarcoding) has emerged as a popular alternative for studying plankton diversity. However, also this method has its limitations, and in order to be robustly implemented in monitoring programs, protocols need to be optimised and validated. The main goal of the project is to evaluate high-throughput sequencing as a tool to investigate the diversity of phytoplankon in the seas surrounding Sweden, with special focus on non-indigenous species and harmful algae. Specifically we want to test effects of storage of samples, sample volume, replication and background DNA in the water (“eDNA”), develop and evaluate an improved method for identifying phytoplankton based on sequencing a longer region of the rRNA operon and compare results from metabarcoding with results from microscope-based methods. The tests will be conducted on water samples collected as part of the Swedish National Marine Monitoring programme. This will provide a large data set covering all major sea areas surrounding Sweden and allow us to compare the metabarcoding results with taxonomic identifications obtained using microscopy as part of the monitoring programme. This collaborative project will be conducted by a team of researchers with complementary expertise in plankton ecology, marine monitoring, molecular biology and bioinformatics.