Dec 19, 2025

Public workspaceFarm slurry/wastewater sample collection, preparation and storage for mNGS and MSSPE

This protocol is a draft, published without a DOI.
  • Matt Parker1
  • 1Opendream
  • Opendream
Matt Parker
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Protocol Citation: Matt Parker 2025. Farm slurry/wastewater sample collection, preparation and storage for mNGS and MSSPE. protocols.io https://protocols.io/view/farm-slurry-wastewater-sample-collection-preparati-hg7yb3zpx
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: December 08, 2025
Last Modified: December 19, 2025
Protocol Integer ID: 234456
Keywords: Metagenomics, wastewater surveillance, pathogen surveillance, One Health, viral sequencing, next-generation sequencing, genomic epidemiology, livestock disease, zoonotic disease, pandemic prevention, rna viral pathogens from agricultural wastewater, farm slurry, agricultural wastewater, msspe protocol, wastewater sample collection, swine slurry, msspe, viral pathogen, storage for mng, mng, rna, priority dna
Abstract
Protocol developed for a non-invasive, MSSPE-based approach for detecting low-abundance, high-priority DNA and RNA viral pathogens from agricultural wastewater and swine slurry.
Materials
- Ziploc bag with *decontaminated or RNase-free 50mL falcon tube + collection spoon
- 70% ethanol spray solution
- Paper towels
- Extra gloves
- Red trash bag for hazardous material
- Large-medium cooler box with frozen ice packs

* Decontamination protocol (if collection kit not RNase-free):
1. Scrub all falcon tubes, lids, and spoons with brush to remove particles
2. Rinse well with dH2O (deionized water, distilled if not available)
3. Soak with 10% bleach solution for 5 minutes
4. Rinse well (again) with dH2O
5. Keep tube closed until sampling
Troubleshooting
Safety warnings
Don full PPE including non-powdered latex/nitrile gloves, N95 mask, face shield, and PPE suit
  • After donning gloves, spray new gloves with 70% ethanol before each collection
  • Wear plastic shoe covers if boots from farm are not available
Before start
Goal: To obtain a sample that will retain the integrity of nucleic acids.

Workflow:
  1. Collect samples using sterile techniques and minimizing environmental contamination, into a nuclease-free container.
  2. Concentrate samples via PEG precipitation.
  3. Store samples in DNA/RNA Shield (or other nucleic acid stabilizer) if cold-chain is not reliable.
  4. Freeze samples at -20°C (-80°C if possible) until ready to process.

Considerations:
  • PEG precipitation must be performed on the SAME day as sample collection.
  • Bead Bashing and Proteinase K Treatment must be performed on SAME day as extraction.
  • Nucleic acid degradation can come from many places (DNAses, RNAses, heat, freeze/thaws, metal ions, contaminating microbes). Flash freezing is the preferred choice (liquid nitrogen or ethanol & dry ice bath).
  • RNA is more fragile than DNA
  • Only 1 person should be physically handling samples during collection to minimize contamination
i.e. a different person should be taking photos and collecting sample notes
  • Do NOT touch the inner part of the 50mL tube at any time – even WITH gloves!

Prepare sample collection kit in advance:
  • Per sample: Ziploc bag with *decontaminated or RNase-free 50mL falcon tube + collection spoon
  • 70% ethanol spray solution
  • Paper towels
  • Extra gloves
  • Red trash bag for hazardous material
  • Large-medium cooler box with frozen ice packs

* Decontamination protocol (if collection kit not RNase-free):
1. Scrub all falcon tubes, lids, and spoons with brush to remove particles
2. Rinse well with dH2O (deionized water, distilled if not available)
3. Soak with 10% bleach solution for 5 minutes
4. Rinse well (again) with dH2O
5. Keep tube closed until sampling
Sample collection
Scoop 30-40mL of sample into 50mL falcon tube and close firmly
  • Note color, flow (stagnant, fast moving, etc), consistency of wastewater, and location of collection (well, gutter, pool, etc)
  • Throw away used collection spoon into red trash bag
Spray and wipe down outside of 50mL tube with 70% ethanol
  • Throw away used paper towels into red trash bag
Carefully place tube into ziploc bag and close
Take photo of sample using mobile app KoboToolbox or OHTK
  • Make sure barcode is showing clearly
  • Press location button to make sure lat/long GIS coordinates are accurate
Place sample into cooler box with ice packs and shut lid immediately
  • Throw away used gloves into red trash bag
Secure cooler box in transport vehicle so samples do not move around during transit
Proceed to PEG precipitation immediately upon returning samples to the lab.
PEG precipitation
Add 3g of PEG to the 30mL sample
Add 0.68g NaCl (0.3 M, Millipore Sigma) to the 30mL sample.
Manually shake by hand for 30 seconds or until the PEG and NaCl is dissolved.
Centrifuge at 1200g*, 4°C for 2 hours*.
* every centrifuge is different so anywhere from 800-2000g
** Centrifuge at CoM does not go to 4°. Room temp is OK but less ideal
*** Can be shortened to 1 hour centrifuge

a. Slowly pour out supernatant and discard- Take care to NOT REMOVE the pellet
b. To remove remaining supernatant use a P1000
c. It is OK to leave ≤100ul of supernatant
Add 100-300 uL sterile PBS to the pellet, and pipet up and down to combine the pellets for each sample*

a. Amount of PBS depends on how 'dirty' the sample is. If the pellet is larger and thick add more PBS
b. Use the least amount of PBS possible
* if the sample is impossible to pipette, try scooping with the pipette end of a pipette tip if you don't have disposable sterile spatulas, or spatulas that you can sterilize between samples by dipping in ethanol and then flaming
Vortex 2 minutes at maximum speed on vortex adapter to completely resuspend pellet.
Transfer sample to a (locking) 1.5 Eppendorf tube OR cryovial.
Add DNA/RNA Shield (2x concentrate) at 1:1 ratio. Mix vigorously by vortexing.
Store at -20C (-80C if available) or proceed to Bead Bashing*.

*If sample is not sludgy and is easily pipettable - proceed without bead bashing to proteinase k.

Bead Bashing Protocol (for sludgy samples)
Samples can be frozen at -80°C or proceed with homogenization.
Add ZR Bashing beads (Zymo Cat. #S6012-50) to shield sample if not already in lysis tubes. Homogenize the sample using a high speed benchtop homogenizer. Typically, 1 min of vortexing followed by 1 min incubation on ice for a total of 3 cycles will thoroughly homogenize the sample.
Spin the sample to pellet the cell debris and bashing beads. Transfer the supernatant to a new tube.

a. If sample is still too solid to pipette or there is no supernatant centrifuge for longer, start with 1 minute centrifuge up to 5 minutes.
b. If after 5 min long centrifugation you still can’t pipette, add more shield (50ul at a time) mix and spin again for 5 min
After incubation, vortex sample and centrifuge at max speed for 2 minutes to pellet debris. Transfer the cleared supernatant to a new nuclease-free tube.
Proceed immediately to Proteinase K Treatment.
Proteinase K Treatment
For every 400 µl* of reagent/sample mixture, add 10 µl Proteinase K and mix thoroughly. Incubate at room temperature (20-30°C) for 30 minutes.
*Use ⅓ of sample (~200 uL) - save remaining sample for future extractions.
a. Take aliquot which is representative of the sample – include particulate matter as well. Doesn’t need to be precise; the most important thing is that aliquot is completely representative.
b. If sample is pipettable (not much particulate matter) – continue with extraction without bead bashing
Then do 1:1 lysis buffer
Purify with one of the following: Zymo Pathogen Magbead, Zymo Quick DNA/RNA Fecal/Soil Microprep Kit.
Proceed immediately to extraction.
Protocol references