AQUATIC KIT FOR THE DOT-NM AUTOSAMPLER

For use in all countries

• All kit users must familiarize themselves with data requirements set out in the Sample Manifest, prior to sampling.
• Please ensure all information sent to NatureMetrics is in English.
• Datasheets must be completed using a pencil only.

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KIT BACKGROUND

THIS KIT IS FOR USE WITH THE DOT AUTOSAMPLER

This protocol is for collecting eDNA samples using the DOT Autosampler, which can be bought from DOT (https://dartmouthocean.com/products/edna-sampler). Make sure to buy the autosampler that is compatible with NatureMetrics filters. The Autosampler is a transportable, autonomous sampler, which filters 9 independent samples and the sample volume and sample times of each sample can be programed. It performs internal cleaning between samples to minimize contamination. The autosampler enables autonomous sample collection which can reduce sampling cost when sample collection is resource intensive. The partnership between DOT and NM provides clients with an end-to-end service for autonomously collecting samples. Please contact DOT for autosampler reagents (RNAlater, miliq water, and low concentration HCl) because these are not included in the NM filter kit. This protocol details how to load filters into the autosampler and remove filters after sampling. DOT should be contacted for all other protocols for how to operate, program, clean and maintain the autosampler.

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YOU WILL NEED

• Device to capture GPS coordinates (lat / long) (e.g. GPS capable smartphone or standalone GPS device)
• Pencil (to complete the datasheet)
• DOT-NM Autosampler (This must be purchased from DOT).

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More than one person using the kit? Please purchase extra gloves.

To minimize waste we provide two pairs of gloves per kit, one for loading filters and one for removing filters. If you want new gloves for each filter, or two or more people will be handling the contents of the kit, you will need to purchase additional pairs of gloves. We recommend purchasing medical-grade nitrile gloves.

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TIPS FOR GETTING THE BEST RESULTS

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Avoiding Inconclusive Results:

To get the best results possible, to avoid inconclusive results, and to avoid situations where data cannot be reported from a sample, please read our HOW TO AVOID INCONCLUSIVE RESULTS guide before undertaking fieldwork. Please note that the autosampler uses a different preservative, so some recommendations about preservation are not applicable to autosampler samples.

In summary, when inconclusive results occur, it is because the target DNA was not detected, DNA was degraded, or PCR (a crucial part of the lab process) was inhibited.

To help avoid inconclusive results, do not leave your samples exposed to sunlight or heat, and return samples to NatureMetrics as soon as possible after collecting samples from the autosampler.

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Avoiding contamination:

To increase confidence in results, avoiding contamination of samples is highly important. Sealed kits are sterile until opened; do not open more than one kit bag at a time, keep kit contents inside the bag before use, and put on the provided gloves before touching other kit components. Whilst wearing gloves, avoid touching anything not necessary to sample collection to minimize introducing external DNA into the sample. Change gloves between each kit. If any reusable tools are used, these should be decontaminated, using a disinfectant wipe or a bleach solution, between each sample. Disinfectant wipes are provided to wipe gloved hands in between removing each filter from the autosampler after samples have been collected.

We also recommend the inclusion of field negative controls (blanks), as part of your sampling strategy, to detect possible contamination introduced during sampling. These are purified water (i.e., mineral, deionised, or MilliQ water) processed in the same way as water samples (see Advice Note for details). NatureMetrics can run one filter for every 20 normal filters as a field blank. Please talk with a NatureMetrics team member if you want field blanks. Note that contamination is assessed after amplification and the field blanks are not sequenced.

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Sampling strategy:

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 and discuss autosampler deployment and mounting. If planning your own survey, it is important to follow the guidance outlined in the Advice Note, as suitable sampling strategies differ for different waterbodies.

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USER PROTOCOL

PART 1: Inserting filters into the autosampler

Once a suitable sampling strategy has been decided and planned:

1. Rinse and dry autosampler. Set up clean area for autosampler and lay out kit. The clean area can be an area cleaned with 10% bleach or a surface that is sterile such as the inside of glove packaging.

2. Check DOT protocols to ready the autosampler for new filters. This may include running the cleaning routine.

3. Remove top cassette by following DOT protocols (unscrewing 3 bolts).

4. Remove plastic spacers from the cassette if present.

5. Open the kit bag. Kits should be used one at a time to minimize contamination between samples. Do not open more than one kit at a time, especially if you have more than one autosampler.

6. Put on the gloves provided and minimize contact with any objects other than filters to avoid introducing contaminant DNA into the samples.

7. To create a clean area, we suggest laying out paper glove packaging.

8. Remove nine filters from the kit bag and put them on the clean space (glove paper). Leave luer lock caps in the bag (for use after sample collection).

9. The filter number must correspond with the number on the autosampler cassette. Start with filter number one, screw in the small plastic connecter piece (may be attached to plastic spacer or provided by DOT – see picture below) and then screw the other end of the filter into the number one cassette location. Repeat for filters 2-9. Verify again that the filter number matches the number on the cassette.

10. After nine filters are screwed into the cassette, insert the cassette back into the autosampler. Make sure all connector pieces (in picture below) are inserted into the correct hole in the body of the autosampler. Re-attach the 3 screws that hold the cassette onto the body of the autosampler.

11. Close the plastic kit bag and keep all contents.

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PART 2: Considerations for sample collection

1. Check the amount of the three reagents (RNAlater, miliq water, and low concentration HCl) and replace if necessary by following DOT protocols.

2. Set sample times and volume filtered using the DOT software. Follow DOT protocols.

3. The recommended volume of water to sample is dependent on environment. We recommend 3L for freshwater environments and 5L for marine environments. Keep in mind that turbidity can clog filters and reduce the amount of water filtered. The autosampler will automatically stop filtering if the filter clogs.

4. Follow DOT protocols to make sure autosampler is ready for deployment.  Deploy as recommended by DOT.

PART 3: Remove filters after sampling.

1. Rinse and dry autosampler. Set up clean area to set autosampler and lay out kit.  

2. Remove top cassette with the nine filters by following DOT protocols (unscrewing 3 bolts).

3. Open the kit bag. Kits should be used one at a time to minimize contamination between samples.

4. Put on the gloves provided and minimize contact with any objects other than filters to avoid introducing contaminant DNA.

5. Create a clean space either by cleaning an area with bleach or laying out paper glove packaging.

6. Remove 18 luer locks from the small bag and put them on the clean space (glove paper). Space out pairs of luer locks on the clean area so that you are touching only two luer locks for each filter to reduce contamination between filters.

7. Unscrew one filter. Note that black knobs may turn with filter and you may need to hold the black knob when unscrewing the filter.

8. Unscrew the small connector piece from the filter you just removed. If you cannot unscrew by hand you can use the needle nose pliers provided in your autosampler tool kit. Use one hand to hold the filter and the other hand to hold the pliers. Gently grasp the connector piece with the pliers and unscrew.

9. Cap both ends of the filter with 2 luer locks and place the filter inside a specimen bag and seal.

10. Use the disinfectant wipe provided and clean gloved hands and pliers (if used).

11. Repeat steps 7-10 for filters 2-9. Use a new disinfectant wipe every 2 or 3 filters, or when the wipe is dry.

12. Depending on next steps, you may need to put plastic spacers into the cassette before attaching the cassette back to the autosampler body. This is needed to run the cleaning program before putting new filters into the cassette.

PART 4: RETURNING YOUR SAMPLES

Once you have collected and filtered your samples, follow our guidelines below to safely transport your samples to the NatureMetrics laboratory. For the best results, please return samples to us within 24 hours, using the fastest possible service (next day delivery if available). 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 (-20 ̊C) 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 returning samples within 2 weeks after collection or long-term freezing is not possible.

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1. Complete the sampling datasheet using a pencil (do not use pen as ink can easily run, to the point where it becomes unreadable).

2. Place the bagged filters and sampling datasheet inside the 5 L bag that was included in the kit bag.

3. Complete the sample manifest, which was attached to the email confirming shipping of your kits. Please record your sampling data on paper and on the sample manifest so there is a physical and electronic copy of your data.

4. For sample returns, visit our customer support desk here. Use the Logistics Request form to upload your completed sample manifest and book your sample return. 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 analyze samples that are returned using the correct NatureMetrics logistics procedure.

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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 bags containing the filters and the sampling datasheet. However, all kit contents can be sent back to NatureMetrics for recycling.

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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.

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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.

Health & Safety information for this kit can be viewed here.

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