Nov 22, 2021

Public workspaceProtocols for eDNA/eRNA extraction from marine samples V.2

DOI
dx.doi.org/10.17504/protocols.io.bz95p986
  • 1Galway Mayo Institute of Technology
Luca Mirimin
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DOI: dx.doi.org/10.17504/protocols.io.bz95p986
Protocol Citation: Luca Mirimin, Dulaney Miller, Sara Fernandez 2021. Protocols for eDNA/eRNA extraction from marine samples. protocols.io https://dx.doi.org/10.17504/protocols.io.bz95p986Version created by Luca Mirimin
Manuscript citation:
Sara Fernandez, Dulaney L. Miller, Luke E. Holman, Arjan Gittenberger, Alba Ardura, Marc Rius, Luca Mirimin, Environmental DNA sampling protocols for the surveillance of marine non-indigenous species in Irish coastal waters, Marine Pollution Bulletin, Volume 172, 2021, 112893, ISSN 0025-326X, https://doi.org/10.1016/j.marpolbul.2021.112893
License: This is an open access protocol 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 protocol and it’s working
Created: November 22, 2021
Last Modified: November 22, 2021
Protocol Integer ID: 55325
Keywords: eDNA, eRNA, extraction, environmental samples, protocol
Abstract
This document provides a series of protocols used to extract eDNA or eRNA from marine environmental samples such as small and large volume (filtered) water, sediment or (fine mesh net) plankton. Specifically, these protocols are recommended for use following sampling as described in Mirmin et al. (2021; Environmental DNA sampling protocols for the surveillance of marine non-indigenous species.protocols.iohttps://dx.doi.org/10.17504/protocols.io.by7rpzm6).
Guidelines
While these protocols can be adapted and used for a wide range of sources and sample types, these protocols are recommended for samples collected using the methods described in:

Luca Mirimin, Dulaney Miller, Sara Fernandez2021.Environmental DNA sampling protocols for the surveillance of marine non-indigenous species.protocols.iohttps://dx.doi.org/10.17504/protocols.io.by7rpzm6
Materials
PROTOCOL 1 - eDNA extraction from water samples (low volume water)

Micropipette set (1-1000ul) and relevant filter tips
Microcentrifuge to accommodate 1.5-2.0mL tubes
Bead beater
Incubator (at last up to 60C)
Vortexer for 1.5-2.0mL tubes

Ethanol (96-100%) (not denaturated alcohol)
ReagentQIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
ReagentOakton™ Glass beads for MillsFisher Scientific
ReagentDNA-ExitusPlusApplychemCatalog #A7089
ReagentBuffer ATL (tissue lysis buffer)QiagenCatalog #19076
ReagentProteinase K, 2mLQiagenCatalog #19131
10mL tubes suitable to beat beater
1.5mL microtubes


PROTOCOL 2 - eDNA extraction from water samples (high volume water)

Micropipette set (1-1000ul) and relevant filter tips
Pipettes 1-10mL
Centrifuge to accommodate 1.5-2.0mL microtubes
Centrifuge capable of spinning 50 ml tubes at 2500 x g using swing-out rotor
Rocker/shaker for HV filter capsules
Incubator (at last up to 60C)
Vortexer for 1.5-2.0mL tubes with adapter for 50mL

Reagent PowerMax® Soil DNA Isolation KitMobioCatalog #12988-10

PROTOCOL 3 - eDNA/eRNA co-extraction from water samples (high volume water)

Micropipette set (1-1000ul) and relevant filter tips
Pipettes 1-10mL (not needed if extracting from small volume samples)
Centrifuge to accommodate 1.5-2.0mL microtubes
Centrifuge capable of spinning 50 ml tubes at 2500 x g using swing-out rotor (not needed if extracting from small volume samples)
Rocker/shaker for HV filter capsules (not needed if extracting from small volume samples)
Incubator (at last up to 60C)
Vortexer for 1.5-2.0mL tubes with adapter for 50mL (adapter not needed if extracting from small volume samples)

ReagentZR-Duet™ DNA/RNA MiniPrep PlusZymo ResearchCatalog #D7003
ReagentSuperScript II reverse transcription kit Life Technologies

PROTOCOL 4 - eDNA extraction from water samples (high volume tow net)

Micropipette set (1-1000ul) and relevant filter tips
Microcentrifuge to accommodate 1.5-2.0mL tubes
Incubator (at last up to 60C)
Vortexer for 1.5-2.0mL tubes

ReagentQIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
ReagentBuffer ATL (tissue lysis buffer)QiagenCatalog #19076
ReagentProteinase K, 2mLQiagenCatalog #19131
1.5mL microtubes

PROTOCOL 5 - eDNA extraction from sediment samples

Micropipette set (1-1000ul) and relevant filter tips
Microcentrifuge to accommodate 1.5-2.0mL tubes
Incubator (at last up to 60C)
Vortexer for 1.5-2.0mL tubes
Weighing scale (up to 10g)

Reagent PowerMax® Soil DNA Isolation KitMobioCatalog #12988-10

Protocol materials
ReagentBuffer ATL (tissue lysis buffer)QiagenCatalog #19076
ReagentOakton™ Glass beads for MillsFisher Scientific
Reagent PowerMax® Soil DNA Isolation KitMobioCatalog #12988-10
ReagentDNA-ExitusPlusApplychemCatalog #A7089
ReagentZR-Duet™ DNA/RNA MiniPrep PlusZymo ResearchCatalog #D7003
ReagentSuperScript II reverse transcription kit Life Technologies
ReagentQIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
ReagentQIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
ReagentProteinase K, 2mLQiagenCatalog #19131
ReagentBuffer ATL (tissue lysis buffer)QiagenCatalog #19076
ReagentProteinase K, 2mLQiagenCatalog #19131
Reagent PowerMax® Soil DNA Isolation KitMobioCatalog #12988-10
ReagentQIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
ReagentOakton™ Glass beads for MillsFisher Scientific
ReagentDNA-ExitusPlusApplychemCatalog #A7089
ReagentBuffer ATL (tissue lysis buffer)QiagenCatalog #19076
ReagentProteinase K, 2mLQiagenCatalog #19131
ReagentBuffer AEQiagenCatalog #19077
Reagent PowerMax® Soil DNA Isolation KitMobioCatalog #12988-10
ReagentZR-Duet™ DNA/RNA MiniPrep PlusZymo ResearchCatalog #D7003
ReagentSuperScript II reverse transcription kit Life Technologies
ReagentQIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504
ReagentProteinase K, 2mLQiagenCatalog #19131
ReagentBuffer ATL (tissue lysis buffer)QiagenCatalog #19076
ReagentBuffer AEQiagenCatalog #19077
Reagent PowerMax® Soil DNA Isolation KitMobioCatalog #12988-10
Safety warnings
Make sure to establish a Risk Assessment to mitigate any adverse effect on users while carrying out any of these protocols. Useful information (e.g. Safety Data Sheets) should be sourced from the relevant suppliers who procured the materials.
Before start
To minimize risk of sample contamination, note that all protocols should be carried out in dedicated eDNA extraction laboratories/rooms.

Ensure that each protocol and list of materials is checked before starting any of the procedures. Specifically, make sure that all key materials(e.g. kits) have not been modified or discontinued by the relevant supplier.
INTRODUCTION
INTRODUCTION
Note that these nucleic acid extraction protocols have been adapted and tested in conjunction with sampling protocols as detailed in:
Protocol
Environmental DNA sampling protocols for the surveillance of marine non-indigenous species
NAME

Environmental DNA sampling protocols for the surveillance of marine non-indigenous species

CREATED BY
Luca Mirimin

See also the relevant peer-reviewed publication here:

CITATION
Fernandez S, Miller DL, Holman LE, Gittenberger A, Ardura A, Rius M, Mirimin L (2021). Environmental DNA sampling protocols for the surveillance of marine non-indigenous species in Irish coastal waters.. Marine pollution bulletin.
LINK
https://doi.org/10.1016/j.marpolbul.2021.112893

Overview of protocols included in this document:

AB
Protocol titleDescription/purpose
PROTOCOL 1 - eDNA extraction from water samples (low volume water)Extracting eDNA from filter membranes following filtration of low voume marine water (e.g. 1L)
PROTOCOL 2 - eDNA extraction from water samples (high volume water)Extracting eDNA from High Volume filter capsules (e.g. 1 μm polyethersulfone filter membrane with an Effective Filtration Area of 1,300 cm2)
PROTOCOL 3 - eDNA/eRNA co-extraction from water samples (high volume water)Co-extracting eDNA and eRNA from filter membranes following filtration of marine water
PROTOCOL 4 - eDNA extraction from water samples (high volume tow net)Extracting eDNA from marine samples collected with a fine mesh (e.g. 50um) plankton net
PROTOCOL 5 - eDNA extraction from sediment samplesExtracting eDNA from marine sediment

PROTOCOL 1 - eDNA extraction from water samples (low volume water)
PROTOCOL 1 - eDNA extraction from water samples (low volume water)
15m
15m
Extract eDNA using the ReagentQIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504 following manufacturer's instructions on “Purification of Total DNA from Animal Tissues (Spin-Column Protocol)”, with the following modifications:

Following sample collection as per "Protocol for collection of water samples (low volume water)", cut each filter membrane in half with scissors and place it in a Amount10 mL tube containing 0.25 g of 0.1 mm glass beads and 0.25 g of 0.5 mm glass beads ReagentOakton™ Glass beads for MillsFisher Scientific or equivalent. Both scissors and tweezers used for cutting and handling the membranes should be decontaminated with ReagentDNA-ExitusPlusApplychemCatalog #A7089 (or equivalent) prior to use.

Add Amount720 µL of ReagentBuffer ATL (tissue lysis buffer)QiagenCatalog #19076 , Amount950 µL of distilled water and 100 μL of Proteinase KReagentProteinase K, 2mLQiagenCatalog #19131 (2 mg/L final concentration).

Bead beat the mixture for Duration00:15:00 at half speed using a
Equipment
Mixer Mill MM 400
NAME
Retsch
BRAND
MM400
SKU
https://www.retsch.com/products/milling/ball-mills/mixer-mill-mm-400/function-features/
LINK
(or equivalent)

15m
Follow subsequent steps as per manufacturer's recommendations

During the final elution phase, add Amount100 µL ofReagentBuffer AEQiagenCatalog #19077 and spin through the column.
Repeat the step above for a final total volume of Amount200 µL .
Store extract at Temperature-20 °C .

PROTOCOL 2 - eDNA extraction from water samples (high volume water)
PROTOCOL 2 - eDNA extraction from water samples (high volume water)
Extract eDNA using the Reagent PowerMax® Soil DNA Isolation KitMobioCatalog #12988-10 following manufacturer's instructions.

Note
Note that some initial modifications should be incorporated when extracting from samples collected using a semi-automated eDNA sampler.
Equipment
Mark II InDepth eDNA sampler
NAME
Semi-automated water sampler
TYPE
Applied Genomics
BRAND
Mark II
SKU
https://appliedgenomics.co.uk/
LINK
Further details are available at Biodiversity and eDNA Survey, Analysis and Monitoring | Applied Genomics

PROTOCOL 3 - eDNA/eRNA co-extraction from water samples (high volume water)
PROTOCOL 3 - eDNA/eRNA co-extraction from water samples (high volume water)

Note
The following protocol was adapted from Pochon et al. (2017)

CITATION
Pochon X, Zaiko A, Fletcher LM, Laroche O, Wood SA (2017). Wanted dead or alive? Using metabarcoding of environmental DNA and RNA to distinguish living assemblages for biosecurity applications.. PloS one.
LINK
https://doi.org/10.1371/journal.pone.0187636

Co-extract eDNA and eRNA using ReagentZR-Duet™ DNA/RNA MiniPrep PlusZymo ResearchCatalog #D7003 following manufacturer's recommendations.

Store eDNA at Temperature-20 °C and eRNA at Temperature-80 °C .

Note
To monitor posible crosscontamination in the extraction proccess, include a negative control in the DNA isolation step, consisting of the same components but no starting material, to be processed alongside the eDNA/eRNA samples throughout all subsequent steps. In case there is a contamination, it can be detected after sequencing.

Reverse Transcribe eRNA into cDNA using ReagentSuperScript II reverse transcription kit Life Technologies or equivalent kit.

cDNA can be stored at Temperature-20 °C .
PROTOCOL 4 - eDNA extraction from water samples (high volume tow net)
PROTOCOL 4 - eDNA extraction from water samples (high volume tow net)
Extract eDNA using the ReagentQIAgen DNeasy Blood and Tissue Kit, 50 rxnQiagenCatalog #69504 following manufacturer's instructions on “Purification of Total DNA from Animal Tissues (Spin-Column Protocol)”, with the following modifications:
Following sample collection as per "Protocol for collection of water samples (high volume tow net)", mix by briefly vortexing and place a Amount15 mL sub-sample into a new clean Amount50 mL falcon tube (conical bottom). Centrifuge at max speed to form a pellet. Remove supernatant by pipetting.

Add Amount1350 µL of ReagentBuffer ATL (tissue lysis buffer)QiagenCatalog #19076 , Amount100 µL ReagentProteinase K, 2mLQiagenCatalog #19131 and follow manufacturer's recommendations for all subsequent steps. Including an overnight incubation step.
During the final elution phase, add Amount100 µL ofReagentBuffer AEQiagenCatalog #19077 and spin through the column.
Repeat the step above for a final total volume of Amount200 µL .
Store extract at Temperature-20 °C .
PROTOCOL 5 - eDNA extraction from sediment samples
PROTOCOL 5 - eDNA extraction from sediment samples
Extract eDNA from a Amount5 g sub-sample using Reagent PowerMax® Soil DNA Isolation KitMobioCatalog #12988-10 following manufacturer's recommendations.

Citations
Step 1
Fernandez S, Miller DL, Holman LE, Gittenberger A, Ardura A, Rius M, Mirimin L. Environmental DNA sampling protocols for the surveillance of marine non-indigenous species in Irish coastal waters.
https://doi.org/10.1016/j.marpolbul.2021.112893
Step 4
Pochon X, Zaiko A, Fletcher LM, Laroche O, Wood SA. Wanted dead or alive? Using metabarcoding of environmental DNA and RNA to distinguish living assemblages for biosecurity applications.
https://doi.org/10.1371/journal.pone.0187636