W-2 WATER PROCESSING

Wipe the surfaces with 70% ethanol to remove contaminants.

Use tubing to connect a 3-liter Medi-Vac Canister with vacuum pump through the vacuum outlet on the lid. (If possible, the canister should be set up inside a biosafety cabinet).

Connect tubing with the 3-liter Medi-Vac Canister through the port for air-in (indicated as patient) on the lid. Close unused inlets. Turn on the pump to test the vacuum suction by feeling the airflow.

Assemble Magnetic Filter funnel, tubing, GL45 Screw Cap with 2-hose connector, 1L dry glass bottle (autoclaved or bleach rinsed) and the Medi-Vac Canister as Appendix 1.

If the water samples have high turbidity, settle water at 4°C for 1h 0m 0s.

Wipe the filter holder of the magnetic funnel with 70% ethanol and let ethanol air dry.

Place a 30 µm filter membrane disc on the filter holder.

Attach the top funnel cup to the filter holder.

Pour the settled water sample to the magnetic funnel cup, avoid disturbing the precipitation as much as possible.

Turn on the pump (<15 psi) to allow the water sample to pass through the filter and be collected in the bottle (if the pump flow rate cannot be controlled, put a tube clip on the air outlet tube to control the flow rate avoiding the water splash in the bottle). Turn off the vacuum pump after the water sample runs out (If clogging happens, replace the membrane disc filter with a new one and collect all the filtrates in the same bottle).

Discard the 30 µm filter membrane disc.

Pour the filtrate to a sterile sampling bag with flat-wire closure or a clean bottle and connect the bottle back to the filtration system.

Place a new 10 µm filter membrane on the filter holder and filter the filtrate as step 9-10. Avoid using multiple 10 µm filter membranes to finish the filtration (the plastic bag can be reused to contain the same sample in the second round of the filtration).

Store the 10 µm filter membrane in a sterile 60 mm Petri dish and keep the dish On ice. Label the Petri dish with sample ID, filtration date, and membrane size.

Place a 5 µm filter membrane on the membrane holder then repeat step 9-10. Avoid using multiple 5 µm filter membranes to finish the filtration.

Store the 5 µm filter membrane in a sterile 60 mm Petri dish and keep the dish On ice. Label the Petri dish with sample ID, filtration date, and membrane size.

Evenly distribute 250µL DNA/RNA Shield Reagent on the membranes in the Petri dishes, seal the Petri dishes with parafilm and store at -20°C for short-term and -80°C for long-term until DNA/RNA extraction.

Add 187mL of PEG-8000 solution to a 750mL water sample filtrate (or 62.5mL PEG-8000 solution to a 250mL filtrate, the final concentration of PEG-8000 is 8%). Mix well by shaking.

Rinse the magnetic funnel, water inlet tube, and 1L glass bottle with 10% bleach, then wash away the bleach with deionized water.

Rinse the inlet tube and 1L glass bottle with 70% ethanol, shake off the residuals and allow to air dry. Wipe dry the magnetic funnel with 70% ethanol.

Repeat steps from 4 to 19 for another sample.

To speed up the water filtration, prepare multiple sets of tubes and clean 1L bottles to avoid the waiting time for the air dry. The magnetic funnel can be used right after the 70% ethanol wipe.

After finishing all the sample filtration for the day, prepare a positive control for the batch: add 100µL EBV and 50µL DENV-1 standard into 180mL sterile 1x PBS then add 45mL of PEG-8000 solution.

Negative control for the batch of sample filtration: 180mL sterile 1x PBS then add 45mL of PEG-8000 solution.

Store PEG-8000-added samples and controls at 4°C for more than 4h 0m 0s or 4h 0m 0s for the next filtration round (DO NOT store the PEG-8000-added samples at 4°C more than 24h 0m 0s that will compromise the sample stability).

After overnight incubation with PEG-8000, water samples are ready for viral particle capturing filtration.

Assemble the filtration system following the steps described in steps 4-6.

Place a new 5 µm filter membrane on the filter holder and filter the filtrate as steps 9-10. Avoid using multiple 5 µm filter membranes to finish the filtration.

Store the 5 µm filter membrane in a sterile 60 mm Petri dish and keep the dish On ice. Label the Petri dish with sample ID, filtration date, and membrane size.

Pour the filtrate to a sterile sampling bag with flat-wire closure or a clean bottle and connect the bottle back to the filtration system.

Place a new 0.45 µm filter membrane on the membrane holder and filter the filtrate as steps 9-10. Avoid using multiple 0.45 µm filter membranes to finish the filtration.

Store the 0.45 µm filter membrane in a sterile 60 mm Petri dish and keep the dish on ice. Label the Petri dish with sample ID, filtration date, and membrane size.

Evenly distribute 250µL DNA/RNA Shield Reagent on the membranes in the Petri dishes, seal the Petri dishes with parafilm and store at -20°C for short-term and -80°C for long-term until DNA/RNA extraction.

Discard the filtrate.

Rinse the magnetic funnel, water inlet tube, and 1L glass bottle with 10% bleach, then wash away the bleach by running under deionized water.

Rinse the inlet tube and 1L glass bottle with 70% ethanol, shake off the residuals, and allow to air dry.

Wipe dry the magnetic funnel with 70% ethanol. Repeat steps from 26 to 34 for another sample.

Pre-cool the Bullet Blender by adding dry On ice into the cooling compartment and running the cooling program.

Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.

Transfer 0.1 mm zirconium oxide beads (2 spoons, Appendix 4) and four 3.5 mm UFO beads to Thermo Scientific Screw Cap 1.5 mL Micro Tubes. Each water sample needs two bead tubes. Can be prepared in advance as described in Before Start.

Add 500µL of ATL-DX buffer and 135µL VXL buffer to the Thermo Scientific Screw Cap 1.5 mL Micro Tubes containing 0.1 mm and 3.5 mm UFO beating beads.

Each water sample has 4 filter membranes from different filtrations (before PEG-8000 treatment: membrane of pore size 10 and 5, one of each; after PEG-8000 treatment: membrane of pore size 5 and 0.45, one of each)

Use 70% ethanol to wipe forceps and surgical scissors (or use new razor blade).

Trim the outer circle of the membrane that had no water sample flowing through off, discard the outer circle (see Figure 1).

Cut the membranes into 2 halves.

Place 4 halves of the filter membranes from different filtrations of the same water sample in a new Petri dish and store the unused half membranes in the original Petri dish at -20°C for future use (see Figure 1).

Use the forceps to stack the 2 half membranes, then fold the stacked halves into a smaller sector and cut it ( smaller than 1 mm x 3 mm, see example in Figure 1) into the a tube prepared in step 40 (Suggest collecting the 2 half membranes before PEG-8000 in tube A and the 2 half membranes from after PEG-8000 treatment in tube B).

Add 20µL Proteinase K from IndiMag kit and incubate the tube at 56°C in the heat block shaker set up at 1400rpm (if heat block shaker is not available, vortex the tube every 0h 5m 0s).

Load the sample/bead tubes in the Bullet Blender.

Set the speed at 12 and time at 3. Press Start.

Let the samples settle for 0h 1m 0s and then repeat step 50.

STOPPING POINT : lysed samples can be stored at 4°C.

Confirm 96 deep-well magnetic head and 96 well deep-well heat block are being used.

Ensure the program IndiMag_Pathogen_KF_Flex_4wash has been downloaded and loaded onto the KingFisher Flex instrument.

Set up the Wash, Elution, and Tip Comb Plates outside the instrument according to the following table.

A	B	C	D	E
Plate ID	Plate position	Plate type	Reagent	Volume per well
Tip comb	7	Place a 96 Deep-well Tip comb in a deep-well plate
Elution	6	Deep-Well	Nuclease-free water	75 µL
Wash 4	5	Deep-Well	100 % ethanol	750 µL
Wash 3	4	Deep-Well	80% ethanol	750 µL
Wash 2	3	Deep-Well	Buffer AW2	700 µL
Wash 1	2	Deep-Well	Buffer AW1	700 µL
Sample	1	Sample Lysate	Lysate and lysis buffer	985 µL

Centrifuge the bead tubes with lysate from step 51 for 12000x g.

Transfer 425µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.

Add 540µL Buffer ACB, and 20µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming (can be mixed on Hula mixer for 0h 2m 0s). Add 560µL mixture to each sample.

Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.

Start the run, then load the prepared plates into the positions when prompted by the instrument.

After the running protocol is completed (~0h 35m 0s), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.

In a 0.6 mL microcentrifuge tube, use 3µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions(Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit) (see Appendix 5 and Appendix 6).

Proceed with sample testing following the REDI-NET SOP W-4 Water Testing or store at -20°C for less than 2 weeks (for long-term storage the sample needs to be stored at -80°C following the REDI-NET SOP W-3 Water Storage).

Confirm 12-tip magnetic head and 12 deep-well heat blocks are being used.

Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.

Set up the Sample Plate according to the table below:

A	B	C	D
Row ID	Plate Row	Reagent	Volume per well
Sample row	A	Lysate and lysis buffer	985 µL
Wash 1	B	Buffer AW1	700 µL
Wash 2	C	Buffer AW2	700 µL
Wash 3	D	80 % ethanol	750 µL
Wash 4	E	100 % ethanol	750 µL
Tip Comb Wash 2	F	12-Tip comb
	G	Empty
	H

Set up the Elution Strip according to the table below:

A	B	C	D
Row ID	Plate Row	Reagent	Volume per well
Elution	A	Nuclease-free water	75 µL

Centrifuge the bead tubes with lysate from step 51 for 12000x g.

Transfer 425µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.

Add 540µL Buffer ACB, and 25µL MagAttract Suspension G to each sample in the sample plate. For multiple samples, make a master mix with 10% overage. Invert slowly to mix the master mix, avoid foaming. Add 565µL mixture to each sample.

Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.

Start the run, then load the prepared plates into position when prompted by the instrument.

Keep the door open while extraction. The chamber of the KingFisher Duo Prime is small. Closing the door makes the ethanol vapor restrained inside the chamber and increases the ethanol contamination.

After the running protocol is completed (~0h 35m 0s), immediately remove the elution plate from the instrument and cover the plate or transfer the eluate to the final tube or plate of choice for final storage.

In a 0.6 mL microcentrifuge tube, use 3µL total nucleic acid for DNA and RNA concentration measurement using Qubit 4 Fluorometer following manufacturer instructions (Kits needed: Qubit 1X dsDNA HS Assay Kit and Qubit RNA HS Assay Kit). (see Appendix 5 and Appendix 6).

Proceed with sample testing following the REDI-NET SOP W-4 Water Testing or store at -20°C for less than 2 weeks (for long-term storage the sample needs to be stored at -80°C following the REDI-NET SOP W-3 Water Storage).

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APPENDIX 2. Reference of Water Filtration Speed

A	B	C	D	E	F	G	H
		Before add PEG-8000			After add PEG-8000
Sample type	Water volume	10 µm	5 µm	Filtration time	5 µm	0.45 µm	Filtration time
Summer pond water with floating plants, high turbidity	250 ml	16 s	1 h 24 m	1 hr 25 m	35 s	4 m 14 s	5 m 49 s
Winter pond water with little floating plants	250 ml	10 s	14 s	24 s	29 s	3 m 07 s	3 m 36s