SAMPLING PROTOCOL

MAXI AQUATIC eDNA KIT – PUMP FILTRATION

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KIT BACKGROUND

KIT CONTENTS

MAXI Aquatic eDNA Kits are designed to allow clients to filter more water from a single sample (and overall site), and subsequently obtain more DNA for additional analyses. Kit contents are based on one person collecting an eDNA sample. If two or more people will be handling kit contents and collecting an eDNA sample, please contact us about inclusion of extra gloves in kits. Extra gloves are not provided as standard to minimise waste.

MAXI Aquatic eDNA Kits come with a 3.5 L sampling bag and two filters, thus we recommend collecting 3 L of water from freshwater environments to be split equally across filters (1.5 L of water per filter). When collecting samples in marine environments, at least 2 L should be passed through each filter (see Appendix for sampling advice), so multiple water samples taken with the same sampling bag or sampling device (e.g. Kemmerer or Niskin sampler) may be required. This protocol is for processing larger volumes of water using pump filtration.

The Vampire pump is a transportable, peristaltic hose pump for liquids, that works independent of mains power and can sample directly from the water, sample container, or sampling device. The pump head creates a vacuum which draws the water sample through the tube and pushes the water through the eDNA filter attached to the end of the tube. Using a pump, instead of a manually operated syringe, reduces the labour and time required to filter each sample, which is particularly important when sample volumes are high.

1x pair of nitrile gloves

2 x resealable bag, each containing 1 x small syringe filled with 1.5 mL of DNA preservative solution (sealed with reusable Luer Lock cap) and 1 x separate Luer Lock cap

2 x enclosed filterS (0.8 μm pore size, polyether-sulfone)

1 x 3.5 L sampling bag
1 x specimen bag

1 x sampling datasheet

1 x 50 cm silicone hose with adaptor

VAMPIRE PUMP

This can be hired from NatureMetrics.

1 x pump head (A)

1 x drive unit (B)

USER PROTOCOL

PART 1: COLLECTING THE WATER

Ideal sampling strategies will vary among habitats and monitoring contexts. We recommend that you speak to a member of our team to plan your survey. We also recommend the inclusion of field negative controls (blanks) to detect possible contamination introduced during sampling. NatureMetrics provide a free kit to be used as a field blank if 20 or more Pump Aquatic eDNA Kits are purchased.

Download and log into the NatureMetrics app before arriving in the field. Avoid introducing DNA (from yourself, soil or other waterbodies) into the water and prevent spread of disease or invasive species by wearing the provided gloves and minimising contact with the water.

PART 2: SETTING UP THE PUMP

If operating the pump alone, it is recommended to set up the hose with the drive unit detached. If working in a team, one person can hold the drive unit, while another mounts the hose.

1. To remove the drive unit, turn the locking ring towards the unlock sign and remove the pump attachment, setting the drive unit safely aside.

2. Open the pump head by pulling up the white part at the top. Insert the silicone hose into the pump head as shown in image below, making sure there is at least 5 cm of hose between where it leaves the pump head and the hose adapter.

3. Close the pump head by pressing down the white part so that the silicone hose is securely fixed in place.

OPEN

CLOSED

4. If it has been removed, re-mount the pump head to the drive unit and turn the locking ring towards the lock sign until it clicks.

5. Check that the drive unit is set to pump water towards the filter. As you hold the drive unit with the pump head facing away from you, water will be pumped from left to right when the orange button is pushed in on the right-hand side (forward arrow). When the orange button on the left-hand side is pushed in (backwards arrow), water will be pumped from right to left.

6. Set the drive unit to gear 1 (orange slider on top of unit).

PART 3: FILTERING THE WATER AND PRESERVING THE DNA

1. Depending on the sample collection method you have used, hang the end of the hose directly in the sampling bag or container, or attach the end of the hose to the outlet of the sampling device (contact NatureMetrics for advice on different sampling devices).

2. Run the pump slowly by pressing the drive unit trigger slowly to fill the hose with water.

3. Once the hose is filled, attach the filter inlet (narrow end) to the hose adapter. They should twist together easily.

4. Run the pump slowly to begin with, while holding the hose securely just behind the adaptor. You can increase the speed once water is flowing freely.

5. When the flow of water leaving the filter outlet (wide end) slows, decrease pump speed to reduce the build-up of pressure. If too much pressure builds up it will make removing the filter harder or eject the adapter and filter from the hose.

6. Once all water has passed through the filter, or the filter is fully clogged, remove the hose from the sample and run the pump until the hose is cleared of any water. Continue to run the pump until no more water exits from the filter, to expel as much water as possible. It helps to gently shake the filter as you do this.

7. Detach the filter from the hose by unscrewing it slowly.

8. Uncap the small syringe (already filled with preservative solution) and twist it on to the filter inlet. Do not discard the Luer Lock cap – hold the cap in one hand as it will be needed in step 9. The solution is non-hazardous to aquatic life but release into the environment should be avoided. We advise performing this step away from the water’s edge and avoiding spills.

9. Hold the filter so that outlet points upwards. We advise you to do this step away from anyone’s eyes. Carefully and slowly press the plunger to push the preservative solution into the filter. This must be done slowly to allow the preservative solution to spread out over the filter surface. Stop when the first drop can be seen emerging from the filter outlet, but do not remove the small syringe with preservative solution. Cap the filter outlet using the Luer Lock cap that was on the small syringe in step 8.

10. If it has been removed, re-mount the pump head to the drive unit and turn the locking ring towards the lock sign until it clicks.

11. Place the filter into the specimen bag and seal. If storing samples at ambient temperatures, samples should be received by the lab within 2 weeks of sampling. If this is not possible, we recommend that samples are frozen as soon as possible after sampling to enhance DNA preservation until samples are sent back to us. Cold shipping is not necessary for frozen samples. It should be noted that although freezing is recommended as best practice for optimal storage, we understand that it is not always feasible. Storing at ambient temperatures is a tried and tested method that yields excellent data. The addition of freezing is to further safeguard the samples against potential DNA degradation, which can affect species detection when samples are stored at high temperatures for long periods. Please contact NatureMetrics if return within 2 weeks or long-term freezing is not possible.

You can view a video demonstration of filtration with the Standard Aquatic eDNA Kit here.

PART 4: RETURNING YOUR SAMPLES

Once you have collected and filtered your sample, follow our guidelines below to safely transport your sample to the NatureMetrics laboratory.

1. Complete the sampling datasheet and use the NatureMetrics app to scan the barcode on the sampling datasheet to complete your online submission. Please record your sampling data on paper and through the app so there is a physical and electronic copy of your data.

2. Place the bagged filter and sampling datasheet in the bag that the kit contents arrived in.

3. For UK sample returns, simply use the standard postal service with recorded delivery. Follow the packaging notes and send to: Nature Metrics, 1 Occam Court, Surrey Research Park, Guildford, GU2 7HJ, United Kingdom.

4. For international sample returns, contact your NatureMetrics Business Development Manager or our Logistics team (logistics@naturemetrics.co.uk) for further information. NatureMetrics is fully compliant with current legislation on the transport of biological material and our operations team are on hand to ensure that all return shipments meet the required specification. We are only able to analyse samples that are returned using the correct NatureMetrics logistics procedure.

PACKAGING NOTES AND QUICK LINKS

  • Return your samples using the box that kits were sent in, where possible.

  • If using an alternative box, ensure that it is in good condition and remove any old labels.

  • Avoid excess spacing by adding cushioning material (e.g. bubble wrap, newspaper) to avoid the product shifting inside the box.

  • Use good quality sealing tape and seal along box edges in a H pattern.

  • The only essential thing to send back to us is the specimen bag containing the filter and the sampling datasheet. However, all kit contents can be sent back to NatureMetrics for recycling.

  • Register for your my.naturemetrics account here.

  • Please find our Terms and Conditions here.

CLIENT RESPONSIBILITIES

Important considerations for your sampling

  • The absence of a detection does not necessarily imply the absence of a taxon from a location.

  • Samples from some locations might have compounds that inhibit analyses resulting in reduced data generation – while we routinely test for inhibition, it is not always possible to overcome.

  • It is the responsibility of the client to ensure that all efforts have been made to avoid contamination from external sources and between samples.

  • Handling samples without gloves can increase the content of human DNA, reducing data generation.

  • After sampling, movement of samples across borders without permission is not allowed as they are classed as biological samples.

Important considerations for interpreting your results

It should be noted that DNA can enter an ecosystem via many routes (e.g. wastewater from commercial and domestic sources). While DNA from a given taxon may be present and detectable, it is not possible to discern the source of the DNA. Results from common food items, domestic species and livestock species need to be interpreted with caution.

Disclaimer: Safe sample collection is the responsibility of the Client. NatureMetrics accepts no liability associated with the use of the kits and sample collection. The Client is solely responsible for the quality of the samples and the representativeness of the samples received by NatureMetrics. The information contained within the Final Report provided by NatureMetrics to the Client is not intended to be advisory, it is informational. Interpretation and decisions are the sole responsibility of the Client. NatureMetrics does not accept any liability whatsoever for any reliance placed on any information contained within, or any use that may be made of, the Final Report by the Client. Please read the full limitation of liability statement in the Terms and Conditions.

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