Mar 03, 2026

Public workspaceWildinSync-eDNA sampling of rivers

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm1549v3p/v1
  • Loïc Pellissier1,2,
  • Camille Albouy1,2,
  • Andrea Polanco3,
  • Arnaud Lyet4,
  • Maria Mutis Martinezguerra5,
  • Abby Hehmeyer4,
  • Jean-Baptiste Juhel6,
  • Karma Sherub1,2
  • 1Ecosystems and Landscape Evolution, Department of Environmental Systems Science, ETH Zürch, Zürich;
  • 2Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Birmensdorf, Switzerland, Switzerland;
  • 3Fundación Biodiversa Colombia, Bogota, Colombia;
  • 4World Wildlife Fund, Washington, USA;
  • 5Universidad Austral de Chile;
  • 6Université de Guyane, CNRS, IFREMER, Cayenne, France
  • WildinSync
Loïc Pellissier
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm1549v3p/v1
Protocol Citation: Loïc Pellissier, Camille Albouy, Andrea Polanco, Arnaud Lyet, Maria Mutis Martinezguerra, Abby Hehmeyer, Jean-Baptiste Juhel, Karma Sherub 2026. WildinSync-eDNA sampling of rivers. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm1549v3p/v1
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: February 01, 2026
Last Modified: March 03, 2026
Protocol Integer ID: 242384
Keywords: Freshwater, peristaltic, large volume, high sensitivity, environmental DNA, standardized freshwater edna sampling, dna traces from aquatic animal, edna sampling of river, freshwater sampling, term global biodiversity monitoring under the wildinsync initiative, environmental dna, global biodiversity monitoring, water sampling, dna extraction, aquatic animal, edna sampling, dna trace, detection of species presence, diverse habitat, distribution across diverse habitat, habitat type, species presence, ecosystem, wildinsync initiative, river, sequencing
Abstract
This protocol was developed to support long-term global biodiversity monitoring under the WildinSync initiative, which uses environmental DNA (eDNA) metabarcoding to assess the effects of nature-positive actions on ecosystems. Freshwater sampling is performed to capture DNA traces left by plants or animals, enabling detection of species presence and distribution across diverse habitats. The method consists of standardized Freshwater eDNA sampling to obtain DNA traces from aquatic animals. It provides guidance on required materials, field sampling steps, and preparation of samples for later molecular analyses (e.g., DNA extraction and sequencing). The protocol is applicable across a wide range of habitat types and environmental conditions worldwide. Applicability may be limited in environments where water sampling is not feasible or safe.
Materials
Metadata sheet to collect the information about the site.

Durable equipment
- Naturelink peristaltic pump (2)
- USB-C Wire (1)
- GPS (1)
- Battery Lithium USB type-C 12 V, 22 Ah (2)
- Timer (or a watch) (1)
- Easily cleanable lab tray dedicated only to (2)
- Rope Ø3 mm, length 3 m (to attach the peristaltic pump to the boat) (2)
- Dry bag to protect the pump and batterie during filtration (2)

Note: The peristaltic pump model manufactured recommended (Naturelink) is powered by a USB-PD circuit, allowing it to operate from any USB power supply that supports a power profile of 15 or 12 V, 20 W or more (9 V optional, but with reduced power). (It has a speed controller that allows the power and pumping volume to be regulated using a dimmer switch).

Consumable equipment
- eDNA filtration Kit (gloves, capsule, tube) (2)
- Buffer kit (gloves, Buffer syringe) (3 per Site)
- Box of nitrile gloves (1)
- Duct tape (9)
- Sample ID label stickers (2 per site)
- Pencil (2)
- Permanent marker (2)
- Paper tissues (1 package)
- Garbage bags

Chemicals
- Ethanol 70% (500 ml)
- Commercial Bleach (500 ml)
- Bleach wipes / OH 70% (1 package)
Troubleshooting
Personnel Required
A team of two people is more efficient for the good execution of the procedure on a boat, even if the procedure can be done with one person. Sampling from the shore only requires one person.

For very large rivers, samples should ideally be collected from a boat along a transect. If no boat is available or if environmental conditions do not allow navigation at the selected site (small stream or river), samples may be collected from the shore.
Safety
During eDNA filtration, personnel must be aware of field hazards such as slips on wet surfaces, strong currents, waves, and boat movement, and must wear non-slip footwear, weather-appropriate clothing, and a personal flotation device when on a vessel. Use personal flotation device (mandatory when working from a boat) and when around large river with strong currents.

Use nitrile gloves at all times to prevent DNA contamination and avoid touching sterile components or sediment with bare hands. Handle bleach and preservatives carefully, using gloves and safety glasses to prevent irritation or spills.

Ensure the pump and battery remain dry and well secured. Use proper lifting techniques for heavy equipment and remain alert to changes in water flow.
Training Requirements
Follow the specific protocol for the procedure.
Time Needed to Execute the Procedure
About 80 minutes if the two sample replicates are collected with two pumps running in parallel. If filtration is done in parallel with two pumps, avoid placing them too close to each other. Keep at least five meters between the two set-ups.
Preparation
  • Define a dedicated workspace where you will perform the filtration away from possible source of DNA contamination. Get the equipment out of the sampling bag.

Caution: Make sure no one goes in the water before or during the filtration to avoid contamination.

  • Set up the peristaltic pump on a dedicated place at height to prevent exposure to direct water, such as a flat stone. While the pump is water-resistant, it is not fully waterproof. Use snap hooks and rope to secure it if you are in a boat.
  • Branch the battery to the pump.
  • Remove the superior part of the peristaltic head and lay it next to the pump. Make sure that it is placed in a secured location.
Naturelink peristaltic pump recommended for the sampling.
  • Prepare 1 tube kit and one capsule kit on hand. In some version of the kits, those can be in the same plastic package.
  • Use bleach wipes to clean the scissors/penknife and to clean the external part of the plastic packages of the kits. Use the knife to open the packaging of tubing kit without taking out the content. Open on the side that allows to take easily the gloves out of the package.
  • Also use the scissors/penknife to open the bag containing the capsule (if they are not in the same bag).
Tubing kit containing the hose with the sterile strainer and the screw coupling as well as the gloves. Note that a different version of the kits contains the capsules and the tubing together.

Caution: Do not touch the strainer or the screwed coupling end.

  • Remove only the gloves. Put the gloves on. In a version of the kit, the gloves are provided separately.

Caution: After putting the gloves on, it is imperative not to touch contamination source (skin, other equipment). The pump is also not clean, so refrain from touching it.

  • Keep the strainer and screwed coupling end inside the bag.
  • Open the bag with the capsule.
Sampling capsule from top to bottom, with the arrow indicating the flow direction.


Caution: Be careful, together with the capsule, there are the corresponding labels (stickers) with unique sample ID. Keep them secure during the procedure. They will be required to label the sample after the filtration is complete.

  • Identify the top and lower end of the capsule. Identify the caps of the capsule inside the plastic bag and make sure to keep them at the bottom of the bag.

Caution: Be careful not to lose the caps during the procedure. Otherwise the sample will be lost as you will not be able to close the capsule at the end of the filtration.

  • Without removing the entire capsule from the bag, peek out the upper part.
  • Screw the threaded end of the hose onto the top end of the capsule. SECURE WELL.

Caution: The strainer must not touch ANYTHING.
Caution: The lower end of the capsule (drain end) must not touch ANYTHING.

  • Position the hose inside the pump, keeping the appropriate distance for filtering/drainage on both sides.

Caution: The arrow on the pump indicates the direction of the water flow, so that the capsule is positioned in the direction of the arrow. Adjust the tubing in the right direction.

  • Lock the head of the peristaltic pump.
  • Take out the capsule off the bag and keep the capsule drain vertical. Keep the empty filter kit bag as it will receive the capsule when the filtration is done.

Caution: The strainer should be SUSPENDED in the water, it should not touch substrate to avoid sediment disturbance. If possible, keep at least 10cm between the strainer and the sediments.
Caution: During the filtration, the capsule must be kept in vertical position (check arrow on capsule, indicating downward position).
Caution: The drain of the capsule must always be kept clear and must not touch anything to avoid DNA contamination.
Caution: Pay attention to the water current. Our setup should align with the direction of the water current to prevent the discharge from the pump from being filtered out.
Caution: If the filter clogs, the capsule might be ejected. Do not place the capsule directly over the river. Find a place where the capsule would fall in a secure location if it is ejected.

Note: If you sample from a boat, consider using a clean fishing line. Attach one end to the lead sinker and the other end to the boat. Then, attach the tube along the fishing line using duct tape. This will prevent the strainer from coming out of the water when the boat moves.

  • At this point, the workspace is completely set up. The filtration can begin.
  • Do not forget to note the start time of the filtration and the coordinates on the field sheet. Also record the width and depth of the river.
Sampling
Note: If you carry out the filtration from a boat, initiate the filtration while maintaining a speed of approximately 3 knots to perform a transect parallel to the shore or following the habitat of interest.

  • For the Naturelink pump, turn on and set up the pump according to the specifications, recommended 50% speed for 1h time. If the flow continues without reduction after 10 minutes, increase the speed to 70%.

Caution: If the water passing through the capsule is turbid the membrane can become clogged. Check regularly that the water flows well through the capsule. If it is not the case, stop the filtration, note the volume that the capsule has filtered and proceed with the next steps.

Caution: During the filtration, the water should completely cover the membrane surface within the capsule. If it is not the case, slow down the water flow on the pump and very carefully unscrew the overpressure valve on the top of the capsule until the water covers the membrane. Be mindful that this operation entails risk, the valve is under pressure and could be ejected.

Note: If you wish to calibrate the pump, we recommend timing how long it takes for the filtered water discharged from the pump to fill a 0.5/1 L container. The optimal operation of the pump targets a flow rate of less than 1 L·min^−1. You can then adjust the strength of the pump. When this allotted time period ends, the pump will automatically switch off.

  • Once the system has filtered 30 L of water and/or the filtering time has ended, you need to purge the remaining water from the capsule. This is done with the pump on, WITHOUT TOUCHING THE strainer remove the strainer from the water and allow only air to pass through the hose, this will serve to empty all the water from the capsule (this may take a few minutes). Switch off the pump.

Caution: The strainer must not touch anything during this step. You can touch the hose.

  • Unscrew the head and remove the hose.

Caution: The unprotected lower end of the capsule must not touch ANYTHING.

  • Ensure that the capsule is completely emptied of filtrate water.
Sample Preservation
  • With CLEAN scissors/penknife open the buffer kit bag containing the gloves and the kit.
  • Put on the new gloves that are inside the buffer kit bag.
Buffer kit containing the preservation buffer. Several models of kits exist including a squeezing bottle.

  • Remove the screw cap from the tip of the syringe.
  • Unscrew the end of the hose and capsule, and immediately screw the end of the syringe onto the capsule.
  • Unscrew the pressure valve of the capsule.

Caution: SLOWLY unscrew the valve, be careful that the valve cap may separate, fall off and become contaminated/lost.

  • Always keeping the capsule in an upright position, SLOWLY start pouring the contents of the syringe into the capsule. Regulate the inflow with the valve cap.

Caution: The contents of the syringe should be poured COMPLETELY into the capsule.

  • Proceed to secure the top of the capsule with the remaining cap, making sure to still keep the gloves on.
  • Once the capsule is closed and the sample is secured, vigorously move and shake the capsule up and down to ensure that the preservative/buffer permeates the entire filter inside for at least 30 s.
  • For transportation, we recommend to use a parafilm band to provide an extra sealing of the caps and avoid that they unscrew during transport.
  • Identify the capsule with its corresponding sticker (1). Put the capsule in a plastic bag (preferably the numbered bag) and store it inside the box. Store it in a dark place at room temperature, avoiding large temperature variations.
  • Identify the box with the corresponding sticker (2).
  • Remember to write in your notebook/field form the metadata associated with the sample in the field sheet.
  • Identify the metadata with its corresponding sticker (3).
Storage
  • Back to the lab, store samples in a dark place at room temperature, avoiding large temperature variations.

The use of a refrigerator or freezer for storage is strictly prohibited.

Note: It is recommended to send the samples for processing as quickly as possible to the lab that will process them.
Quality Control
All samples have a replica sample, which follows the same process either at the same time if two peristaltic pumps are available, or one filtration followed by the other at the same site if only one pump is available.
Problem solving
• The pump suddenly stops

This could be due to either the battery running out of power or the connection between the pump and the battery being lost. Check the battery charge, then test each electrical connection by applying pressure to it.

• Water does not flow through the capsule

Ensure that the arrow on the capsule is pointing downwards. Check that the tube is not bent inside the pump head.

• The tube is ejected from the filter capsule during filtration

If there is a lot of sediment in the water, the filter in the capsule can quickly become saturated, causing excess pressure that will eject the tube from the lower end of the capsule. In this case, it is advisable to hold the tube at the lower end and reduce the filtration flow rate. If the ejection continues, stop filtration and note the filtration time.

• When filling the capsule with buffer (last steps 14 to 17), the buffer overflow

Try opening the pressure relief valve slightly to release the air and allow the buffer to go back in. Be careful not to drop this part. Close again once finished.

• Water does not purge from the capsule at the end of the filtration

Try putting the capsule with the outlet on looking up and shaking the capsule slightly to facilitate the process.

Protocol references
Lyet, A., Pellissier, L., Valentini, A., Dejean, T., Hehmeyer, A., & Naidoo, R. (2021). eDNA sampled from stream networks correlates with camera trap detection rates of terrestrial mammals. Scientific Reports, 11(1), 11362.

Sherub, K., Thurnheer, S., Lüthi, M., Marques, V., Lyet, A., Dhendup, T., Dorji, L. Albouy C. & Pellissier, L. (2026). Comparing Watershed‐Based eDNA Sampling and Camera Trapping for Assessing Mammal Diversity in North‐Western Bhutan. Environmental DNA, 8(1), e70243.
Acknowledgements
We thank all the partners and collaborators who made this initiative possible. The protocol was developed and tested within the framework of several scientific projects supported by the Swiss National Science Foundation.