Collection Citation: Colleen Kellogg, Matt Lemay, rute.carvalho Carvalho, Andreas Novotny 2024. Environmental DNA Metabarcoding- Full Pipeline Collection. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lywxwpvx9/v1
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: June 19, 2024
Last Modified: June 20, 2024
Collection Integer ID: 102123
Keywords: dna extraction from sterivex filter, seawater sample, seawater filtration, filtering seawater, seawater on sterivex filter, environmental dna, plankton communities in the northern salish sea, dna metabarcoding, dna extraction, plankton community, hakai institute ocean observing program, part of the hakai institute ocean observing program, coi mitochondrial gene, diversity of marine invertebrate, diversity of fish, rrna mitochondrial gene, marine invertebrate, biomolecular sample, rrna nuclear gene, northern salish sea, robust phenol chloroform extraction protocol, bioinformatic analysis for the different gene, sterivex filter, analyzing different marker gene, diversity of eukaryote, mitochondrial gene, fish, prokaryotes to eukaryote, eukaryote, prokaryote, different gene, dna
Abstract
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)
