Jul 27, 2023
Version 2
Coastal Environmental DNA Sampling & Gravity Filtration Protocol V.2
- 1Stanford University
Protocol Citation: Meghan M. Shea, Alexandria B Boehm 2023. Coastal Environmental DNA Sampling & Gravity Filtration Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69y7klqe/v2Version created by Meghan M. Shea
Manuscript citation:
TBD
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: July 05, 2023
Last Modified: July 27, 2023
Protocol Integer ID: 84542
Keywords: environmental DNA, metabarcoding, water sampling, gravity filtration, coastal
Abstract
This is a protocol for collecting coastal environmental DNA (eDNA) samples and gravity filtering them on site, using a set-up with single-use enteral feeding pouches first described in Curd et al. 2019 (see Appendix S6) and further elaborated in Gold et al. 2021.
Image Attribution
Callie Chappell
Materials
Laboratory Preparation Supplies:
| Material | Amount Needed | Source | Link | Approx. Cost | |
| UV Crosslinker (e.g. UVP CL-1000 Ultraviolet Crosslinker) | 1 | UVP | NA | Unknown | |
| RNase Away | Enough to clean all materials | Unknown | NA | Unknown |
Field Blank Supplies:
| Material | Amount Needed | Source | Link | Approx. Cost | |
| 1000 mL Nalgene bottles | 1 per field blank | Unknown | NA | Unknown | |
| MilliQ water | 1000 mL per field blank | NA | Unknown |
Sampling Infrastructure & Transport:
| Material | Amount Needed | Source | Link | Approx. Cost | |
| Clothes rack | 1 | Amazon | https://www.amazon.com/dp/B08QM97RDG?psc=1&ref=ppx_yo2_dt_b_product_details | $87 | |
| Bricks for stability | 2 | NA | NA | NA | |
| Shade structure | 1 | Amazon | https://www.amazon.com/dp/B09785H5DY?psc=1&ref=ppx_yo2_dt_b_product_details | $61.1 | |
| Beach cart | 1 | Amazon | https://www.amazon.com/dp/B008DJDUPM?ref=ppx_yo2_dt_b_product_details&th=1 | $118.6 | |
| Plastic bins for transporting supplies (ideally sized to fit in beach cart) | 4-6 | Amazon | https://www.amazon.com/dp/B00CQGTGZQ?psc=1&ref=ppx_yo2_dt_b_product_details | $7/bin |
Tools:
| Material | Amount Needed | Source | Link | Approx. Cost | |
| Tube cutter | 1 | Amazon | https://www.amazon.com/gp/product/B0756D2T73/ref=ppx_yo_dt_b_asin_title_o00_s00?ie=UTF8&psc=1 | $8.72 | |
| Flathead screwdriver | 1 | Unknown | NA | Unknown | |
| Lab marker | 1 | VWR (52877-310) | https://us.vwr.com/store/product/4597364/vwr-chemical-resistant-laboratory-marker | $25.22/10 markers | |
| Temperature and salinity probe (e.g. Orion Model 1230 meter) | 1 | Orion Research Inc., Beverly, MA, USA | NA | Unknown |
Sampling Materials:
| Material | Amount Needed | Source | Link | Approx. Cost | |
| Zip ties | At least 1/sample plus additional for securing field set-up infrastructure | Unknown | NA | Unknown | |
| 1000 mL Covidien single-use enteral feeding pouches | 1/sample | Amazon | https://www.amazon.com/Kangaroo-ePump-Enteral-Feeding-Pump/dp/B01DME25J4 | $104.92/30 bags | |
| Sterile 0.22 μm Sterivex filters | 1/sample | MilliporeSigma via Fisher Scientific (SVGV010RS) | https://www.fishersci.com/shop/products/emd-millipore-sterivex-sterile-pressure-driven-devices-12/SVGV010RS | $284.44/50 filters | |
| Male luer lock to hose barb adapter (1/8'') | 1/sample | Avantor Masterflex, via Fisher Scientific (NC9668309) | https://www.fishersci.com/shop/products/male-luer-w-lock-ring-x1-8-in/NC9668309 | $15.3/25 adapters | |
| Dual male-female luer lock cap | 1/sample | Amazon | https://www.amazon.com/dp/B09MJQLHYB?ref=ppx_yo2_dt_b_product_details&th=1 | $17.99/100 caps | |
| Round silicone rubber cap | 1/sample | McMaster-Carr (92805k32) | https://www.mcmaster.com/catalog/129/4296/92805K32 | $21.15/100 caps | |
| Sterile 3 mL luer lock syringes | 1/sample | BD, via Fisher Scientific (14-823-435) | https://www.fishersci.com/shop/products/bd-disposable-syringes-luer-lok-tips-3/14823435 | $21.60/200 syringes | |
| Small bungees | 1/sample | Amazon | https://www.amazon.com/dp/B09LHL4937?psc=1&ref=ppx_yo2_dt_b_product_details | $11.96/40 bungees | |
| Whirl-pak bags | 1/sample |
Note
On Sterivex filters: they are sold in two styles, one with a male luer lock outlet and one with a male nipple outlet (listed here). The nipple outlet is usually significantly cheaper, but requires a special cap solution to close (round silicone rubber cap). If using the luer lock outlet configuration, no silicone rubber caps would be needed, and the dual male-female luer lock cap could be used to secure both the inlet and outlet.
Preservation Materials:
| Material | Amount Needed | Source | Link | Approx. Cost | |
| 9 QT Insulated Cooler | 1 | Amazon | https://www.amazon.com/gp/product/B09HMZT1C5/ref=ppx_yo_dt_b_asin_title_o00_s00?ie=UTF8&psc=1 | $18.54 | |
| Ice packs | Enough to mostly fill cooler | Unknown | NA | Unknown |
Pre-Sampling Preparation
Pre-Sampling Preparation
Clean 1000 mL Nalgene bottles (as many as field blanks needed) with a 10% bleach rinse (leave for 10 minutes), then three rinses of DI water
Fill cleaned Nalgene bottles with 1000 mL of MilliQ water to use as field blanks
Sterilize luer lock to hose barb adapters, male-female luer lock caps, and silicone rubber cap
Fill a container (e.g. old pipette tip boxes, so you can easily drain the water with the lid) with 10% bleach solution, add adapters and caps, and let sit for 10 minutes
Example of cleaning set-up using a pipette tip box (Photo Credit: Meghan Shea)
Rinse adapters and caps 3 times with DI water
Diagram of rinsing procedure using a pipette tip box (Photo Credit: Meghan Shea)
Place adapters and caps in a single layer in UV Crosslinker for 15 minutes
Store sterilized caps and adapters in sterile containers
Wipe down plastic storage bins with RNase Away, and organize needed field supplies (wiping individual items with RNase Away when possible as well).
- Sampling Infrastructure & Transport: clothes rack, bricks for stability, shade structure, beach cart
- Tools: tube cutter, flathead screwdriver, lab marker, temperature and salinity probe (e.g. Orion Model 1230 meter), prepared field blanks
- Sampling Materials (1/sample): zip ties, 1000 mL Covidien single-use enteral feeding pouches, sterile 0.22 μm Sterivex filters, male luer lock to hose barb adapters, dual male-female luer lock caps, round silicone rubber caps, sterile 3 mL luer lock syringes, small bungees, whirl-pak bags
- Preservation Materials: insulated cooler, ice packs
Note
See MATERIALS list for more details on specific field supplies
Sampling Set-Up
Sampling Set-Up
Wheel cart with supplies to sampling location
Example of beach cart used to transport sampling supplies (Photo Credit: Callie Chappell)
Put together clothes rack for hanging enteral feeding pouches for gravity filtration and weigh down with bricks. Set up shade structure to cover clothes rack.
Example field set-up with clothes rack and shade structure (Photo Credit: Callie Chappell)
Note
Keeping samples in the shade was prioritized to avoid potential DNA degradation by sunlight (Andruszkiewicz et al., 2017).
Sampling
Sampling
Put on fresh gloves
Remove enteral feeding pouch from plastic wrapper
Image of enteral feeding pouch (Photo Credit: Meghan Shea)
Label pouch with unique sample site/time identifier
Walk pouch to designated sampling site, rinse pouch three times with sampling water, and directly fill pouch with water to 1000 mL mark while keeping the tubing out of the water
Researcher rinsing pouch before collecting water sample (Photo Credit: Callie Chappell)
Using temperature/salinity probe, record the temperature and salinity at the site the sample was collected
Secure the top of the pouch and return to clothes rack
Using a zip tie, secure the pouch to the top of the clothes rack
Researcher attaching pouches to clothes rack (Photo Credit: Callie Chappell)
Note
We found that reusable zip ties could not support the weight of the pouches after their first use. We also trialled shower curtain rings as another possibility for reusable pouch holders, but could not find a style that we were confident could consistently support the pouch weight.
Using a small tube cutter, cut the tubing attached to the pouch at the end of the less-rigid opaque section beneath the first purple joint
Image of enteral feeding pouch tubing annotated with an arrow to show where to cut the tubing (Photo Credit: Meghan Shea)
Put on fresh gloves, and grab one luer lock adapter and a Sterivex filter
Place the barb end of the luer lock adapter into the cut end of the tubing, remove the Sterivex filter, and lock the filter to the tubing
Label the filter with an ethanol-proof lab marker with a unique sample site/time identifier
Using a small bungee cord, secure the filter to the bottom of the clothes rack so that it does not blow in the wind or brush against other filters
Close-up view of the Sterivex filters secured with small bungee cords (Photo Credit: Callie Chappell)
Using a flathead screwdriver, twist the inside of the purple connector on the pouch tubing to start the flow of water
Diagram showing the location of the purple connector that controls the flow of water (Photo Credit: Meghan Shea)
Allow water to filter until all water has passed through (1-2 hours), or until drips slow to less than one/minute (clog); if the latter, record the amount of water that has passed through the filter
Remove the filter, and using a sterile 3 mL syringe, pass air through the filter until excess water has been removed
Cap the inlet end of the Sterivex filter with a male-female luer lock cap and the outlet end with a silicone rubber cap
Label a small Whirl-Pak bag with the unique sample site/time and place the filter inside
Place bag on ice until transporting to -15°C freezer at the conclusion of field sampling
Wrap-Up
Wrap-Up
If pouches need to be transported while still filtering:
Using a flathead screwdriver, twist the inside of the purple connector to stop the flow of water on any still-filtering bags
Carefully lift the clothes rack on top of the cart; secure with zip ties
Example of mobile filtration set-up (Photo Credit: Callie Chappell)
Using a flathead screwdriver, twist the inside of the purple connector to start the flow of water on any still-filtering bags. The cart can now be transported as pouches are still filtering.
Transport filters to -15°C freezer to await extraction
Protocol references
Gravity filtration using Covidien single-use enteral feeding pouches was adapted from:
Curd, E.E., Gold, Z., Kandlikar, G.S., Gomer, J., Ogden, M., O’Connell, T., Pipes, L., Schweizer, T.M., Rabichow, L., Lin, M., Shi, B., Barber, P.H., Kraft, N., Wayne, R., Meyer, R.S., 2019. Anacapa Toolkit: An environmental DNA toolkit for processing multilocus metabarcode datasets. Methods in Ecology and Evolution 10, 1469–1475. https://doi.org/10.1111/2041-210X.13214
Gold, Z., Sprague, J., Kushner, D.J., Marin, E.Z., Barber, P.H., 2021. eDNA metabarcoding as a biomonitoring tool for marine protected areas. PLOS ONE 16, e0238557. https://doi.org/10.1371/journal.pone.0238557
Motivation for preventing DNA degradation by sunlight:
Andruszkiewicz, E.A., Sassoubre, L.M., Boehm, A.B., 2017. Persistence of marine fish environmental DNA and the influence of sunlight. PLOS ONE 12, e0185043. https://doi.org/10.1371/journal.pone.0185043