Environmental DNA Metabarcoding- Full Pipeline Collection

This is a collection of protocols used to analyze environmental DNA (eDNA) of seawater samples.

As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly from 0 m to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015. These protocols are developed to work across all domains of life, from viruses to prokaryotes to eukaryotes, allowing for both amplicon sequencing and shotgun sequencing.

This pipeline includes the following steps:

1. Seawater filtration: Our standard protocol for filtering seawater on sterivex filters. Alternatives may be used.

2. DNA extraction from sterivex filters: Here we offer two alternatives:

• The robust Phenol Chloroform extraction protocol is used in our long-term monitoring program.
• A kit alternative requires less handling of toxic chemicals, and are used for stand-alone projects.

3. DNA metabarcoding, library prep , and sequencing: With the options of analyzing different marker genes:

• 18S rRNA nuclear gene: Diversity of Eukaryotes. (Balzano et al 2015)
• COI mitochondrial gene: Diversity of marine invertebrates. (Leray et al 2013)
• 12S rRNA mitochondrial gene: Diversity of fish. (Miya et al 2015)

4. Bioinformatic analyses

Examples bioinformatic analysis for the different genes can be found on our GitHub page: https://github.com/hakaigenomics

More information to be found under each protocol: