TITLE: Protocols for eDNA/eRNA extraction from marine samples
AUTHORS: Luca    Mirimin, Dulaney Miller, Sara Fernandez

[DESCRIPTION]
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).

[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.

[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

[STEPS]
SECTION: INTRODUCTION
1. Note that these nucleic acid extraction protocols have been adapted and tested in conjunction with sampling protocols as detailed in:
 

See also the relevant peer-reviewed publication here:

 

SECTION: INTRODUCTION
1.1. Overview of protocols included in this document:

 

SECTION: PROTOCOL 1 - eDNA extraction from water samples (low volume water)
2. Extract eDNA using the   following manufacturer's instructions on “Purification of Total DNA from Animal Tissues (Spin-Column Protocol)”, with the following modifications:

SECTION: PROTOCOL 1 - eDNA extraction from water samples (low volume water)
2.1. 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 10 mL tube containing 0.25 g of 0.1 mm glass beads and 0.25 g of 0.5 mm glass beads  or equivalent. Both scissors and tweezers used for cutting and handling the membranes should be decontaminated with   (or equivalent) prior to use.

SECTION: PROTOCOL 1 - eDNA extraction from water samples (low volume water)
2.2. Add 720 µL of  , 950 µL of distilled water and 100 μL of Proteinase K  (2 mg/L final concentration).

SECTION: PROTOCOL 1 - eDNA extraction from water samples (low volume water)
2.3. Bead beat the mixture for 15 min at half speed using a 
 
(or equivalent) 

SECTION: PROTOCOL 1 - eDNA extraction from water samples (low volume water)
2.4. Follow subsequent steps as per manufacturer's recommendations

SECTION: PROTOCOL 1 - eDNA extraction from water samples (low volume water)
2.5. During the final elution phase, add 100 µL of  and spin through the column.
Repeat the step above for a final total volume of 200 µL.
Store extract at -20 °C .

SECTION: PROTOCOL 2 - eDNA extraction from water samples (high volume water)
3. Extract eDNA using the     following manufacturer's instructions.
SECTION: PROTOCOL 2 - eDNA extraction from water samples (high volume water)
3.1. 
 

SECTION: PROTOCOL 3 - eDNA/eRNA co-extraction from water samples (high volume water)
4. 
 

 

SECTION: PROTOCOL 3 - eDNA/eRNA co-extraction from water samples (high volume water)
4.1. Co-extract eDNA and eRNA using  following manufacturer's recommendations.

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

 

SECTION: PROTOCOL 3 - eDNA/eRNA co-extraction from water samples (high volume water)
4.2. Reverse Transcribe eRNA into cDNA using   or equivalent kit.

cDNA can be stored at -20 °C .
SECTION: PROTOCOL 4 - eDNA extraction from water samples (high volume tow net)
5. Extract eDNA using the   following manufacturer's instructions on “Purification of Total DNA from Animal Tissues (Spin-Column Protocol)”, with the following modifications:
SECTION: PROTOCOL 4 - eDNA extraction from water samples (high volume tow net)
5.1. Following sample collection as per "Protocol for collection of water samples (high volume tow net)", mix by briefly vortexing and place a 15 mL sub-sample into a new clean 50 mL falcon tube (conical bottom). Centrifuge at max speed to form a pellet. Remove supernatant by pipetting.

SECTION: PROTOCOL 4 - eDNA extraction from water samples (high volume tow net)
5.2. Add 1350 µL of  , 100 µL   and follow manufacturer's recommendations for all subsequent steps. Including an overnight incubation step.
SECTION: PROTOCOL 4 - eDNA extraction from water samples (high volume tow net)
5.3. During the final elution phase, add 100 µL of  and spin through the column.
Repeat the step above for a final total volume of 200 µL.
Store extract at -20 °C .
SECTION: PROTOCOL 5 - eDNA extraction from sediment samples
6. Extract eDNA from a 5 g sub-sample using   following manufacturer's recommendations.