Water

Herein processes follow lab SOP for the ‘Detection of Non-Indigenous Marine Species from Port Water Samples’ (Chaves et al., 2025) within Biodiversity Genomics Europe project. This is mirror for the workflow hosted in WorkflowHub (which hosts the downloadable PDF).

Sylphium dual filters

Samples for the following DNA extraction have been prepared according to guidelines here.

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Equipment and Consumables

Items	quantity	storage
Sylphium capsules filled with ATL buffer	3 mL per sample	-20°C
96% Ethanol (EtOH)	3 mL per sample	15-25ºC
5ml Syringes	1 per sample	15-25ºC
Extension Tubes (3 mL)	1 per sample	15-25ºC
Proteinase K (PK)	60 μL per sample	-20°C
Qiagen DNeasy Blood and Tissue kit	1 column per sample	15-25ºC
96-well 0.2 mL PCR plate		15-25ºC

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Procedure

Assuming that the samples have been prepared according to guidelines here:

• Set the thermoblock at 70ºC;

• Set the oven at 55ºC;

• Clean the working space and material with disinfectant (e.g. 5-10% bleach) and ethanol (to wipe off bleach) and leave the UV light on for at least 15 mins;

• Use filtered pipette tips at all steps;

• If AL buffer has precipitate, heat to 55°C for 5-10 min to dissolve.

• Follow the manufacturer’s instructions (for Qiagen DNeasy Blood and Tissue kit) to prepare the AW1 and AW2 buffers.

• AL buffer + ethanol (96%) could be used pre-mixed.

1. Mix each capsule by hand and transfer its content into a 15 mL tube using a syringe, through the inlet side.

2. Add 96% ethanol and AL Buffer to each tube in a 1:1:1 ratio (e.g. 2,000 μL ATL + PK : 2,000 μL AL buffer: 2,000 μL 96% ethanol). Vortex tubes immediately, for 20s, and short-spin them.

3. Capture and purify the eDNA following either option a) or b):

a. By centrifugation (based on the DNeasy Blood and Tissue standard protocol):

1. Load up to 650 μL of supernatant onto a Mini Spin Column. Centrifuge at 6,000 x g for 1 min.

2. Discard the 2-mL collection tube and replace it with a new collection tube (not provided). Repeat until all the supernatant has been processed.

3. Place the Mini Spin Column Filter into a clean 2-mL collection tube (provided).

4. Add 500 μL of AW1 Buffer and centrifuge at 6,000 x g for 1 min. Discard the 2-mL collection tube, and place the Mini Spin Column in a new collection tube (provided).

5. Add 500 μL of AW2 Buffer and centrifuge at 20,000 x g for 1 min. Discard the 2-mL collection tube.

b. Using the QIAvac 24 Plus vacuum manifold as an alternative:

1. Place the Mini Spin Columns in the QIAvac system.

2. Load 650 μL of supernatant onto a Mini Spin Column.

3. Turn on the vacuum pump at -80/-90 kPa.

4. Repeat the previous steps until all the supernatant has been processed.

5. Add 500 μL of AW1 Buffer.

6. Add 500 μL of AW2 Buffer.

7. Turn off the vacuum pump once all the volume has passed through.

4. Place the DNeasy Mini Spin Column in a new collection tube. Centrifuge at 20,000 x g for 2 min to completely dry the membrane. Discard the collection tube containing the flow-through.

5. Place the column in a clean 1.5 mL tube and add 100 μL of heated TE (at 70ºC) to the centre of the column membrane.

6. Incubate for 10 min at room temperature. Centrifuge at 6,000 x g for 1 min.

7. Repeat previous two steps above (5-6) using the same 1.5 mL tube to obtain maximum yield.

8. Transfer 50 μL eDNA extract to a 96-well plate (working plate) and archive the remaining at -20ºC or -80ºC. Leave at least two empty wells per plate for the PCR negative control (PNC).

9. Quantify the samples by spectrophotometry. Dilute samples with EB buffer into a new 96-well plate (if needed).

Proceed with amplicon library preparation.

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References

Chaves, C., Najera Cortazar, L. A., Martins, F., Veríssimo, J., Dunshea, G., & Price, B. (2025). Detection of Non-Indigenous Marine Species from Port Water Samples. WorkflowHub. https://doi.org/10.48546/workflowhub.sop.11.2

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