TITLE: eDNA sample collection method comparison
AUTHORS: Anya Kardailsky, Ryan East

[DESCRIPTION]
There is an urgent global need for novel approaches to monitor our freshwater ecosystems. Current biomonitoring tools are time-consuming, costly, and dependent on taxonomic specialists. Therefore, traditional methods are falling short of fulfilling the growing public and policy obligations to monitor these vital, and fragile, ecosystems. Molecular approaches have the potential to revolutionize freshwater biomonitoring but are limited by incomplete reference databases. Furthermore, method development and testing has been lacking for benthic macro-invertebrate and fish environmental DNA (eDNA) surveys. A comparison of eDNA collection and extraction methods is initiated.

[STEPS]
SECTION: Sample collection and preservation
2. Wilderlab sampling kits with 0.45µm cellulose nitrate filter columns https://www.wilderlab.co.nz/order

SECTION: Sample collection and preservation
8. Physico-chemical water tests were done using YSI Pro2030 Dissolved Oxygen and Conductivity Meter and collecting 50ml falcon tubes for nitrate/nitrite analysis.
SECTION: Sample collection and preservation
3. Sterivex-GP capsule 0.22µm filters (Sterivex)
SECTION: Sample collection and preservation
2.1. Push one litre of water from the bucket through the filter using the provided collection tubes and record if the pores become too clogged to filter an entire litre.
If there are not enough collection tubes, 2ml sterivex collection tubes are also fine.
SECTION: Sample collection and preservation
7. Whirl-Pak‱ Speci-Sponge‱ were deployed at the same sampling sites and left overnight
SECTION: Sample collection and preservation
6. Sediment samples were collected at the same sites as water samples
SECTION: Sample collection and preservation
3.3. Add Longmire buffer to preserve the DNA in the filter and cap and parafilm the ends of the filter casing. Immediately label the tubes and place into a chilly bin for travel before storage in -4°C 
SECTION: Sample collection and preservation
6.2. Immediately label the tubes and place into a chilly bin for travel before storage in -4°C.
SECTION: Sample collection and preservation
7.2. These were then collected, and all samples were kept at -4°C until extraction. 
SECTION: Sample collection and preservation
1. Prior to sample collection, all equipment possible was sterilized using a 10-min exposure to 10% bleach solution and then rinsed with ultrapure water (UltraPure‱ DNase/RNase-Free Distilled Water, Invitrogen‱) twice to remove any DNA contamination.
Making sure to not handle any equipment involved with sampling without gloves.
At each site for each sampling method collect 5 samples at each site. For water it is prefereable that all the water is collected at once in one bucket.
SECTION: Sample collection and preservation
1.1. Water samples must be collected first to minimise sediment in the water column which will clog the filters
SECTION: Sample collection and preservation
2.2. Once one litre of water is filtered reset the collection tube with no water inside and push air through the filter.
SECTION: Sample collection and preservation
2.3. Add the provided storage buffer to the filter and cap both ends of the filter casing. Immediately label the tubes and place into a chilly bin for travel before storage in -4°C 
SECTION: Sample collection and preservation
1.2. We then added negative filtration controls by filtering 500 mL ultrapure water and extracted two extraction blanks consisting of 500 µL ultrapure water. We filtered and extracted all negative controls alongside the samples. All laboratory work prior to amplification was performed in a PCR-free designated clean room.
SECTION: Sample collection and preservation
4. Vacuum filtration 1.0µm cellulose nitrate filters by a vacuum pump 
SECTION: Sample collection and preservation
4.1. Pump one litre of water from the bucket through the filter using an active filtration pump and record if the pores become too clogged to filter an entire litre.
SECTION: Sample collection and preservation
2.4. Repeat steps 4 times to have 5 replicates at each site
SECTION: Sample collection and preservation
3.2. Once one litre of water is filtered reset the collection tube with no water inside and push air through the filter.
SECTION: Sample collection and preservation
3.1. Push one litre of water from the bucket through the filter using the provided collection tubes and record if the pores become too clogged to filter an entire litre.
SECTION: Sample collection and preservation
3.4. Repeat steps 4 times to have 5 replicates at each site
SECTION: Sample collection and preservation
4.2. Once one litre of water is filtered, filters are rolled up using decontaminated tweezers, cut into ~1 mm slices and placed in 2 mL Eppendorf tubes.
Immediately label the tubes and place into a chilly bin for travel before storage in -4°C 
SECTION: Sample collection and preservation
6.1. Use 50ml falcon tubes to collect 3 samples of sediment at each site.
SECTION: Sample collection and preservation
5. Only once all water samples have been collected can sediment be then collected to avoid clogging of the filters due to sediment in the water column.
SECTION: Sample collection and preservation
7.1. Using decontaminated chicken wire mesh the sponges were attached using zip ties and deployed at the sites so they were completely submerged and held down with rocks overnight.
SECTION: Sample collection and preservation
4.3. Repeat steps 4 times to have 5 replicates at each site
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9. Wilderlab samples and negative controls extracted as in commerial supplier's instructions
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.1. Use syringe to take out storage buffer from the inlet (don’t push the liquid through the outlet!).
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.2. Transfer buffer to a 1.5 mL Eppendorf tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.3. Add 180 µL ATL buffer and 20 µL Proteinase K.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.4. Vortex at maximum speed for 5 seconds.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.5. Pulse-spin to bring down all liquid to bottom of tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.6. Incubate samples at 56°C overnight.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.7. Vortex at maximum speed for 5 seconds.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.8. Pulse-spin to bring down all liquid to bottom of tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.9. Add 200 µL AL buffer.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.10. Vortex at maximum speed for 15 seconds
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.11. Pulse-spin to bring down all liquid to bottom of tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.12. Add 200 µL 100% ethanol (best to use ethanol stored at -20°C).
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.13. Vortex at maximum speed for 15 seconds.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.14. Pipet the mixture into a DNeasy Mini spin column placed in a 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.15. Centrifuge at 6,000 x g for 1 minute.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.16. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.17. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.18. Add 500 µL Buffer AW1.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.19. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.20. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.21. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.22. Add 500 µL Buffer AW2.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.23. Centrifuge samples for 3 minutes at 20,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.24. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.25. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.26. Centrifuge samples for 1 minute at 20,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.27. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.28. Transfer the spin column in a 1.5 mL Eppendorf tube with caps removed.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.29. Add 200 µL AE buffer to the center of the membrane (preheat AE buffer to 56°C).
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.30. Incubate for 1 minute at room temperature.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.31. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.32. Transfer eluate to a new 1.5 mL Eppendorf tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
9.33. Store DNA at -20°C.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10. Sterivex samples and negative controls extracted using the protocol developed by Spens et al. (2017)
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11. Vacuum filtration samples and negative controls were extracted as in Jeunen et al. (2019)
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12. The sediment samples were extracted using a modified extraction protocol without saturated phosphate buffer developed by Olmedo-Rojas et al. (2023)
SECTION: DNA extractions sponge samples
13. The extraction protocol for sponges has not been optimised and so two different extraction techniques were investigated. For each of the extractions the sponges were first cut into ten pieces that would each fit into 2ml Eppendorf tubes and then further cut into 3 pieces while in the tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.1. Use 5 mL syringe to take out Longmire’s buffer from the inlet (don’t push the liquid through the outlet!).
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.2. Transfer buffer to a 1.5 mL or 2 mL Eppendorf tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.3. Spin buffer at 6,000 x g for 30-45 minutes.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.4. Discard supernatant (be careful not to disturb the pellet!)
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.5. Add 180 µL ATL buffer and 20 µL Proteinase K.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.6. Vortex at maximum speed for 5 seconds.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.7. Pulse-spin to bring down all liquid to bottom of tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.8. Incubate samples at 56°C overnight.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.9. Vortex at maximum speed for 5 seconds.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.10. Pulse-spin to bring down all liquid to bottom of tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.11. Add 200 µL AL buffer.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.12. Vortex at maximum speed for 15 seconds
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.13. Pulse-spin to bring down all liquid to bottom of tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.14. Add 200 µL 100% ethanol (best to use ethanol stored at -20°C).
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.15. Vortex at maximum speed for 15 seconds.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.16. Pipet the mixture into a DNeasy Mini spin column placed in a 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.17. Centrifuge at 6,000 x g for 1 minute.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.18. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.19. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.20. Add 500 µL Buffer AW1.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.21. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.22. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.23. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.24. Add 500 µL Buffer AW2.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.25. Centrifuge samples for 3 minutes at 20,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.26. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.27. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.28. Centrifuge samples for 1 minute at 20,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.29. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.30. Transfer the spin column in a 1.5 mL Eppendorf tube with caps removed.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.31. Add 200 µL AE buffer to the center of the membrane (preheat AE buffer to 56°C).
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.32. Incubate for 1 minute at room temperature.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.33. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.34. Transfer eluate to a new 1.5 mL Eppendorf tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
10.35. Store DNA at -20°C.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.1. Add 0.5 g of 0.5 mm Zirconia/Silica Beads to each 2ml tube from collection.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.2. Add 720 µL ATL buffer and 80 µL Proteinase K.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.3. Vortex at maximum speed for 10 minutes.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.4. Incubate samples at 56°C overnight.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.5. Vortex at maximum speed for 1 minute.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.6. Centrifuge samples at 6,000 x g for 1 minute.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.7. Transfer supernatant (600 µL) into a new 2 mL Eppendorf tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.8. Add 600 µL AL buffer.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.9. Add 600 µL 100% ethanol.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.10. Vortex at maximum speed for 1 minute.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.11. Pipet the mixture (620 µL) into a DNeasy Mini spin column placed in a 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.12. Centrifuge at 6,000 x g for 1 minute.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.13. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.14. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.15. Add 500 µL Buffer AW1.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.16. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.17. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.18. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.19. Add 500 µL Buffer AW2.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.20. Centrifuge samples for 3 minutes at 20,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.21. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.22. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.23. Centrifuge samples for 1 minute at 20,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.24. Discard the flow-through and collection tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.25. Transfer the spin column in a 1.5 mL Eppendorf tube with caps removed.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.26. Add 200 µL AE buffer to the center of the membrane (preheat AE buffer to 56°C).
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.27. Incubate for 1 minute at room temperature.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.28. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.29. Transfer eluate to a new 1.5 mL Eppendorf tube.
SECTION: DNA extractions water samples all done using the Qiagen DNeasy Blood &amp; Tissue Kit
11.30. Store DNA at -20°C.
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.1. Add 0.25 grams of soil sample to a PowerBead Tube
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.2. Vortex for 2 sec
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.3. Add 60 μL of solution C1 (if solution C1 is precipitated, heat solution to 60°C until dissolved before use)
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.4. Vortex in a horizontal position at maximum speed for 10 mins
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.5. Centrifuge at 10,000 x g for 30 s at ambient temperature
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.6. Transfer the supernatant to a clean 2 ml Collection tube
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.7. Add 250 μL of solution C2 and vortex for 5 s
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.8. Incubate at 4°C for 5 mins
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.9. Centrifuge at room temperature for 1 min at 10,000 x g
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.10. Transfer up to, but no more than, 600 μL of supernatant to a 2 ml Collection tube
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.11. Add 200 μL of Solution C3
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.12. Vortex
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.13. Incubate at 4°C for 5 mins
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.14. Centrifuge at room temperature for 1 minute at 10,000 x g
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.15. Transfer up to, but no more than, 750 μL of supernatant to a 2 ml Collection tube
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.16. Add 1200 μL of Solution C4 to the supernatant (shake Solution C4 before use)
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.17. Vortex for 5 mins
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.18. Load approximately 675 μL onto a Spin Filter
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.19. Centrifuge at 10,000 x g for 1 min at room temperature
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.20. Discard the flow-through
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.21. Add 675 μL of supernatant to the Spin Filter
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.22. Centrifuge at 10,000 x g for 1 min at room temperature
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.23. Load the remaining supernatant onto the Spin Filter
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.24. Centrifuge at 10,000 x g for 1 min at room temperature
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.25. Add 500 μL of Solution C5
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.26. Centrifuge at room temperature for 30 s at 10,000 x g
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.27. Discard the flow-through
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.28. Centrifuge at room temperature for 1 min at 10,000 x g
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.29. Place the Spin Filter in a 2 ml Collection Tube (avoid splashing Solution C5 onto the Spin Filter)
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.30. Add 100 μL of Solution C6 to the centre of the white filter membrane of the Spin Filter tube
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.31. Centrifuge at room temperature for 30 s at 10,000 x g
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.32. Discard the Spin Filter
SECTION: DNA extractions sediment samples done using the PowerSoil® DNA Isolation Kit
12.33. Store DNA at -20° to -80°C
SECTION: DNA extractions sponge samples
14. For the first extraction protocol the tubes were filled with longmire buffer and left to defrost and sit for at least 3 hours.
SECTION: DNA extractions sponge samples
14.1. The longmire buffer was then transferred to a new tube, squeezing out the liquid as much as possible
SECTION: DNA extractions sponge samples
14.2. Transfer buffer to a 1.5 mL or 2 mL Eppendorf tube.
SECTION: DNA extractions sponge samples
14.3. Spin buffer at 6,000 x g for 30-45 minutes.
SECTION: DNA extractions sponge samples
14.4. Discard supernatant (be careful not to disturb the pellet!)
SECTION: DNA extractions sponge samples
14.5. Add 180 µL ATL buffer and 20 µL Proteinase K.
SECTION: DNA extractions sponge samples
14.6. Vortex at maximum speed for 5 seconds.
SECTION: DNA extractions sponge samples
14.7. Pulse-spin to bring down all liquid to bottom of tube.
SECTION: DNA extractions sponge samples
14.8. Incubate samples at 56°C overnight.
SECTION: DNA extractions sponge samples
14.9. Vortex at maximum speed for 5 seconds.
SECTION: DNA extractions sponge samples
14.10. Pulse-spin to bring down all liquid to bottom of tube.
SECTION: DNA extractions sponge samples
14.11. Add 200 µL AL buffer.
SECTION: DNA extractions sponge samples
14.12. Vortex at maximum speed for 15 seconds
SECTION: DNA extractions sponge samples
14.13. Pulse-spin to bring down all liquid to bottom of tube.
SECTION: DNA extractions sponge samples
14.14. Add 200 µL 100% ethanol (best to use ethanol stored at -20°C).
SECTION: DNA extractions sponge samples
14.15. Vortex at maximum speed for 15 seconds.
SECTION: DNA extractions sponge samples
14.16. Pipet the mixture into a DNeasy Mini spin column placed in a 2 mL collection tube.
SECTION: DNA extractions sponge samples
14.17. Centrifuge at 6,000 x g for 1 minute.
SECTION: DNA extractions sponge samples
14.18. Discard the flow-through and collection tube.
SECTION: DNA extractions sponge samples
14.19. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions sponge samples
14.20. Add 500 µL Buffer AW1.
SECTION: DNA extractions sponge samples
14.21. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions sponge samples
14.22. Discard the flow-through and collection tube.
SECTION: DNA extractions sponge samples
14.23. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions sponge samples
14.24. Add 500 µL Buffer AW2.
SECTION: DNA extractions sponge samples
14.25. Centrifuge samples for 3 minutes at 20,000 x g.
SECTION: DNA extractions sponge samples
14.26. Discard the flow-through and collection tube.
SECTION: DNA extractions sponge samples
14.27. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions sponge samples
14.28. Centrifuge samples for 1 minute at 20,000 x g.
SECTION: DNA extractions sponge samples
14.29. Discard the flow-through and collection tube.
SECTION: DNA extractions sponge samples
14.30. Transfer the spin column in a 1.5 mL Eppendorf tube with caps removed.
SECTION: DNA extractions sponge samples
14.31. Add 200 µL AE buffer to the center of the membrane (preheat AE buffer to 56°C).
SECTION: DNA extractions sponge samples
14.32. Incubate for 1 minute at room temperature.
SECTION: DNA extractions sponge samples
14.33. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions sponge samples
14.34. Transfer eluate to a new 1.5 mL Eppendorf tube.
SECTION: DNA extractions sponge samples
14.35. Store DNA at -20°C.
SECTION: DNA extractions sponge samples
15. For the second extraction protocol the tubes were left to defrost and the water was removed from the tubes using pipettes leaving the sponge in the tube before continuing with the extract protocol
SECTION: DNA extractions sponge samples
15.1. Add 0.5 g of 0.5 mm Zirconia/Silica Beads.
SECTION: DNA extractions sponge samples
15.2. Add 720 µL ATL buffer and 80 µL Proteinase K.
SECTION: DNA extractions sponge samples
15.3. Vortex at maximum speed for 10 minutes.
SECTION: DNA extractions sponge samples
15.4. Incubate samples at 56°C overnight.
SECTION: DNA extractions sponge samples
15.5. Vortex at maximum speed for 1 minute.
SECTION: DNA extractions sponge samples
15.6. Centrifuge samples at 6,000 x g for 1 minute.
SECTION: DNA extractions sponge samples
15.7. Transfer supernatant (600 µL) into a new 2 mL Eppendorf tube.
SECTION: DNA extractions sponge samples
15.8. Add 600 µL AL buffer.
SECTION: DNA extractions sponge samples
15.9. Add 600 µL 100% ethanol.
SECTION: DNA extractions sponge samples
15.10. Vortex at maximum speed for 1 minute.
SECTION: DNA extractions sponge samples
15.11. Pipet the mixture (620 µL) into a DNeasy Mini spin column placed in a 2 mL collection tube.
SECTION: DNA extractions sponge samples
15.12. Centrifuge at 6,000 x g for 1 minute.
SECTION: DNA extractions sponge samples
15.13. Discard the flow-through and collection tube.
SECTION: DNA extractions sponge samples
15.14. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions sponge samples
15.15. Add 500 µL Buffer AW1.
SECTION: DNA extractions sponge samples
15.16. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions sponge samples
15.17. Discard the flow-through and collection tube.
SECTION: DNA extractions sponge samples
15.18. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions sponge samples
15.19. Add 500 µL Buffer AW2.
SECTION: DNA extractions sponge samples
15.20. Centrifuge samples for 3 minutes at 20,000 x g.
SECTION: DNA extractions sponge samples
15.21. Discard the flow-through and collection tube.
SECTION: DNA extractions sponge samples
15.22. Place the spin column in a new 2 mL collection tube.
SECTION: DNA extractions sponge samples
15.23. Centrifuge samples for 1 minute at 20,000 x g.
SECTION: DNA extractions sponge samples
15.24. Discard the flow-through and collection tube.
SECTION: DNA extractions sponge samples
15.25. Transfer the spin column in a 1.5 mL Eppendorf tube with caps removed.
SECTION: DNA extractions sponge samples
15.26. Add 200 µL AE buffer to the center of the membrane (preheat AE buffer to 56°C).
SECTION: DNA extractions sponge samples
15.27. Incubate for 1 minute at room temperature.
SECTION: DNA extractions sponge samples
15.28. Centrifuge samples for 1 minute at 6,000 x g.
SECTION: DNA extractions sponge samples
15.29. Transfer eluate to a new 1.5 mL Eppendorf tube.
SECTION: DNA extractions sponge samples
15.30. Store DNA at -20°C.
SECTION: qPCR
16. Metabarcoding library preparation followed the protocol described in Jeunen et al. (2022). Using universal primers developed by Leray et al. (2013) a qPCR of a dilution series of neat, 10-fold and 100-fold dilution of the input DNA in duplicate must be carried out to ensure adequate amplification and correct for potential PCR inhibitors.
 
 
  
 

SECTION: Invertebrate metabarcoding sequencing
17. Once optimal DNA concentrations are found. Samples can be sequenced using invertebrate specific primers the BF1 and BR2 primers developed by Elbrecht et al. (2017).
Similar to the protocol detailed in Jeunen et al. (2022), a one-step amplification protocol was conducted to generate the amplicons for library preparation using fusion primers containing a modified Illumina sequencing adapter, a barcode tag (6 bp in length), and the template-specific primer. 
 
Barcode sequences were selected with at least three base pair differences to minimize issues with barcode hopping. Each sample was amplified in duplicate and assigned a unique barcode
combination to allow for pooling of samples.
SECTION: Fish metabarcoding sequencing
18. 
SECTION: Invertebrate metabarcoding sequencing
17.1. Pool samples into minipools of similar concentrations with the negative controls in a separate minipool. 
Measure the DNA concentration of each pool with a high sensitivity qubit assay.
Pool the minipools into one pool at an equimolar concentration. Except the negatve controls which just had 1ul added to the final pool.
SECTION: Invertebrate metabarcoding sequencing
17.2. Measure the DNA concentration of the final library pool with a high sensitivity qubit assay.
Dilute the final library to sequencing specifications.
SECTION: Invertebrate metabarcoding sequencing
17.3. Sequence!!!