TITLE: W-2 WATER PROCESSING 
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
OBJECTIVE

To outline procedures for total nucleic acid extraction from water samples. 

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on procedures for total nucleic acid extraction from water samples to allow downstream library preparation and sequencing for pathogen detection. 

[BEFORE_START]

1. Water samples can be stored at 4 °C for 1 week, -20 °C for 1 month, and -80 °C for longer periods of time.
2. Make sure the water inlet tube and Magnetic Filter Funnel and glass bottles  are properly clean and dry. If autoclave is not available, the parts in the filter system that directly contacted to the water samples needs to be fully rinsed by 10% bleach, followed by water and 70% ethanol then dry.  
3. If the water samples were collected at a high temperature sampling site (≥25 °C) with visible floating plants, microalgae, and sediments, use 250 mL water sample for downstream filtration, otherwise, use 750 mL water sample for filtration.  
4. If water sample is frozen, fully thaw it at Room temperature then process it when it is still cold.  When water sample frozen in a plastic sample bag, wipe the bag surface with 70% ethanol to remove dusts and  sanitize the surface. Place three bags of 250 mL water samples from the same sampling location in a new 1190 ml sterile sample bag, then put the bagged samples in a suitable-sized container for thawing. After samples are fully defrosted, pour the water samples into the 1190 ml outer bag, and discard the original 250 ml sample bags. Hold the whole bag in a 1 L beaker.
 
5. Prepare 40% PEG-8000 solution for microbe aggregation. Check Appendix 3 for the recipe.
6. Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
7. 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.
8. For the first time use of IndiMag pathogen kit, add 96—100% ethanol to Buffer AW1 and AW2, and add 100% Isopropanol to ACB as indicated on the bottles.
9. Buffer ATL may form precipitates upon storage. If necessary, warm to 56 °C until the precipitates have fully dissolved. Prepare buffer ATL-DX: add 100 µLReagent DX to 15 mLBuffer ATL. If smaller amounts are needed, transfer 1.5 mL of Buffer ATL into a sterile 2 ml vial and add 10 µLReagent DX. Mix well, after the addition of Reagent DX. After preparation, the mixture is stable for 6 months at Room temperature (15 °C—25 °C).
10. MagAttract Suspension G from the IndiMag pathogen kit needs to be vortexed thoroughly for 3 min (before first use) or 1 min (before subsequent uses) to ensure that the magnetic silica particles are fully resuspended.
11. Binding beads need to be vortexed thoroughly before each use. 
12. Prepare a few 15 mL or 50 mL conical centrifuge tubes with nuclease-free water for preparing TNA elution in KingFisher Flex or KingFisher Duo Prime to avoid cross-contamination.

[GUIDELINES]

APPENDIX 3. Preparation of 40% PEG-8000 Solution

 
The final concentrations: 40% PEG-8000 and 1.2 Molarity (M) NaCl.

Procedure: 
Add stir bar, PEG-8000 and NaCl into an empty 1L sterile bottle (plastic or glass, autoclavable).
Add sterile 1xPBS to final volume 1L.
Autoclave the whole bottle with lid at 121 °C for 30 min. After autoclave, place the hot solution on a stirring hot plate and stir the solution until it cools down to Room temperature. 
If autoclave is not available, stir the solution on a stirring hot plate until the crystals are fully dissolved and filter the solution through 0.45 µm Corning Disposable Vacuum Filter/Storage Systems.
Store the 40% PEG-8000 solution at 4 °C.    

APPENDIX 4. Measuring Spoon for 0.1 mm Beating Beads
The spoon (Next Advance, MSP01-RNA) is used for 0.1 mm beating beads measurement. The step is described on step 40 the preparation before sample homogenization. One spoon equals to 100 µL.

 
APPENDIX 5. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
DNA quantification: 
According to the volume of sample used, add the 1xHS dsDNA Qubit Assay for a final volume of 200 µL (i.e., if using 3 µL of sample, add 197 µL of 1x HS dsDNA Qubit Assay. 

RNA Quantification:
1. In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay: 

 

2. In a new 0.6 mL tube, mix 197 µL of Qubit HS RNA Assay working solution and 3 µL of the sample. Incubate for 1 min at Room temperature before reading. 

APPENDIX 6. Expected Outcomes
 
 

RESPONSIBLE PERSON
Principal Investigator, Study Coordinator, Entomology Component Lead, Managers 

 

DATA MANAGEMENT

Document the following information in REDI-NET DCS W-2 Water Sample Processing including:
   Date of water filtration, Personnel-ID, and Lab-ID
   Date of extraction, Personnel-ID, and Lab-ID
   Project-ID and associated project code
   Sample-ID
   Volume of water sample used (in ml) 
   Number of membrane (10, 5, 0.45 µm) used to complete the water filtration. 
   Number of half membrane subjected to the extraction.
   Extraction kit and extraction instrument
   Volume of elution of total nucleic acids
   Well-ID
   Concentration of total nucleic acids in ng/μL
   Personnel signature 
  Any comments during the water filtration process or the TNA extraction 

Label samples according to REDI-NET SOP DE Data Entry. 

Water sample number is defined as follows:

 




 
MAINTENANCE OF EQUIPMENT

Caution on RNA handling:

RNases are very stable and difficult to inactivate and only minute amounts are sufficient to destroy RNA. 
Care should be taken to avoid inadvertently introducing RNases into the samples during or after the purification procedure. 
Sample handling and extraction should be performed under an extraction hood and respecting Good Laboratory Practices. 
Use filter tips all the time.

Cleaning filter compartments

Wipe magnetic filter funnel with 70% ethanol between each sample. If dirt accumulated, clean the unit with soapy water then completely rinse off the soap with deionized water. Magnetic filter funnel and 1L glass bottle are autoclavable (as well as tubing if it was made by silicon); autoclave them  after filtering 2-3 batches of water samples. If autoclave is not available, immerse the compartments in 1% bleach for 15 min then rinse off the bleach and air dry the whole unit. Make sure the water inlet tube is completely dried before processing a new batch filtration.    

 Storage of the buffers from IndiMag pathogen kit

Proteinase K is stable for at least 1 year after delivery when stored at Room temperature (15 °C—25 °C). To store for more than 1 year or if ambient temperature often exceeds 25 °C, storage at 2 °C—8 °C is recommended. Do not add Proteinase K directly to the Buffer VXL mixture! This can cause clogs or precipitates.
Precipitates may form after storage at low temperature or prolonged storage. To dissolve precipitate, incubate Buffer VXL or ACB for 30 min at 37 °C, with occasional shaking.
Reconstituted Buffer AW1 can be stored at Room temperature (15 °C—25 °C) for up to 1 year. Mix well after adding Ethanol.
Buffer AVE is RNase-free upon delivery. It contains sodium azide, an antimicrobial agent that prevents growth of RNase-producing organisms. However, as this buffer does not contain any RNase degrading chemicals, it will not actively inhibit RNases introduced by inappropriate handling. When handling Buffer AVE, take extreme care to avoid contamination with RNases. Follow general precautions for working with RNA, such as frequent change of gloves and keeping tubes closed whenever possible.

QUALITY CONTROL

This SOP is reviewed by the applicable supervisor annually or as required in order to maintain its relevance.



[STEPS]
SECTION: VACUUM PUMP SET UP
1. 
 
Wipe the surfaces with 70% ethanol to remove contaminants. 

SECTION: VACUUM PUMP SET UP
2. 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).
SECTION: VACUUM PUMP SET UP
3. 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.
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
4. 

 
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.

 

SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
5. If the water samples have high turbidity, settle water at 4 °C for 60 min.

SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
6. Wipe the filter holder of the magnetic funnel with 70% ethanol and let ethanol air dry.
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
7. Place a 30 µm filter membrane disc on the filter holder.
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
8. Attach the top funnel cup to the filter holder.
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
9. Pour the settled water sample to the magnetic funnel cup, avoid disturbing the precipitation as much as possible.
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
10. 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). 

SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
11. Discard the 30 µm filter membrane disc.
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
12. 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.

SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
13. 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).

SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
14. 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.

SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
15. 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.

SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
26. 
After overnight incubation with PEG-8000, water samples are ready for viral particle capturing filtration.
SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
27. Assemble the filtration system following the steps described in steps 4-6.
SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
28. 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.

SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
29. 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.

SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
30. 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.

SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
31. 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.
SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
32. 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.

SECTION: SAMPLE LYSIS
38. Pre-cool the Bullet Blender by adding dry on ice into the cooling compartment and running the cooling program.
SECTION: SAMPLE LYSIS
39. Clean the work surfaces with RNaseZap, then wipe the surfaces with 70% molecular biology grade ethanol to remove additional contaminants.
SECTION: SAMPLE LYSIS
40. 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.
SECTION: SAMPLE LYSIS
41. Add 500 µL of ATL-DX buffer and 135 µL VXL buffer to the Thermo Scientific Screw Cap 1.5 mL Micro Tubes containing 0.1 mm and 3.5 mm UFO beating beads.

 


SECTION: SAMPLE LYSIS
42. 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)
SECTION: SAMPLE LYSIS
43. Use 70% ethanol to wipe forceps and surgical scissors (or use new razor blade).

SECTION: SAMPLE LYSIS
44. Trim the outer circle of the membrane that had no water sample flowing through off, discard the outer circle (see Figure 1).
SECTION: SAMPLE LYSIS
45. Cut the membranes into 2 halves.
SECTION: SAMPLE LYSIS
46. 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).
SECTION: SAMPLE LYSIS
47. 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). 
SECTION: SAMPLE LYSIS
48. Add 20 µL Proteinase K from IndiMag kit and incubate the tube at 56 °C in the heat block shaker set up at 1400 rpm, 20 min (if heat block shaker is not available, vortex the tube every 5 min).

SECTION: INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to section &quot;SET UP SAMPLE PLATE AND ELUTION STRIP&quot;
52. Confirm 96 deep-well magnetic head and 96 well deep-well heat block are being used. 
SECTION: INSTRUMENT SET UP (KingFisher Flex only, if using KingFisher Duo Prime, go to section &quot;SET UP SAMPLE PLATE AND ELUTION STRIP&quot;
53. Ensure the program IndiMag_Pathogen_KF_Flex_4wash has been downloaded and loaded onto the KingFisher Flex instrument. 
SECTION: SET UP THE PROCESSING PLATES
54. Set up the Wash, Elution, and Tip Comb Plates outside the instrument according to the following table. 

 

     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   

SECTION: EXTRACTION
55. Centrifuge the bead tubes with lysate from step 51 for 12000 x g, 5 min. 

SECTION: EXTRACTION
56. Transfer 425 µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
SECTION: EXTRACTION
57. Add 540 µL Buffer ACB, and 20 µL 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 2 min). Add 560 µL mixture to each sample.

SECTION: EXTRACTION
58. Select the program IndiMag_Pathogen_KF_Flex_4wash on the instrument.
SECTION: EXTRACTION
59. Start the run, then load the prepared plates into the positions when prompted by the instrument.
SECTION: QUANTIFICATION AND STORAGE
60. After the running protocol is completed (~35 min), 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. 

 

SECTION: QUANTIFICATION AND STORAGE
61. In a 0.6 mL microcentrifuge tube, use 3 µL 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). 
SECTION: QUANTIFICATION AND STORAGE
62. 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).


SECTION: INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to section &quot;SET UP THE PROCESSING PLATES&quot;
63. 
Confirm 12-tip magnetic head and 12 deep-well heat blocks are being used.
SECTION: INSTRUMENT SET UP (KingFisher Duo Prime only, if using KingFisher Flex, go to section &quot;SET UP THE PROCESSING PLATES&quot;
64. Ensure the program IndiMag_Pathogen_KF_Duo_4wash has been downloaded and loaded onto the KingFisher Duo Prime instrument.
SECTION: SET UP SAMPLE PLATE AND ELUTION STRIP:
65. Set up the Sample Plate according to the table below:  
     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   

SECTION: SET UP SAMPLE PLATE AND ELUTION STRIP:
66. Set up the Elution Strip according to the table below: 
 

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

SECTION: EXTRACTION
67. Centrifuge the bead tubes with lysate from step 51 for 12000 x g, 5 min.

SECTION: EXTRACTION
68. Transfer 425 µL supernatant without any particle carryover to the wells of the Deep-well plate. This plate becomes the Sample Plate.
SECTION: EXTRACTION
69. Add 540 µL Buffer ACB, and 25 µL 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 565 µL mixture to each sample. 

SECTION: EXTRACTION
70. Select the program IndiMag_Pathogen_KF_Duo_4wash on the instrument.
SECTION: EXTRACTION
71. Start the run, then load the prepared plates into position when prompted by the instrument.
SECTION: EXTRACTION
72. 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.
SECTION: QUANTIFICATION AND STORAGE
73. After the running protocol is completed (~35 min), 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.
 

SECTION: QUANTIFICATION AND STORAGE
74. In a 0.6 mL microcentrifuge tube, use 3 µL 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).

SECTION: QUANTIFICATION AND STORAGE
75. 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).


SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None.  
     PEG-8000      400 g        NaCl      70 g        Add 1x PBS to final volume         1 L   
 The final concentrations: 40% PEG-8000 and 1.2 Molarity (M) NaCl.

SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. Add stir bar, PEG-8000 and NaCl into an empty 1L sterile bottle (plastic or glass, autoclavable).
SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. Add sterile 1xPBS to final volume 1 L.

SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. Autoclave the whole bottle with lid at 121 °C for 30 min. After autoclave, place the hot solution on a stirring hot plate and stir the solution until it cools down to Room temperature.

SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. If autoclave is not available, stir the solution on a stirring hot plate until the crystals are fully dissolved and filter the solution through 0.45 µm Corning Disposable Vacuum Filter/Storage Systems.
SECTION: APPENDIX 3. Preparation of 40% PEG-8000 Solution
None. Store the 40% PEG-8000 solution at 4 °C. 

SECTION: APPENDIX 5. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
None. DNA quantification: 
According to the volume of sample used, add the 1xHS dsDNA Qubit Assay for a final volume of 200 µL (i.e., if using 3 µL of sample, add 197 µL of 1x HS dsDNA Qubit Assay. Vortex for 5 -10 seconds, then incubate for 2 min at Room temperature before reading.

SECTION: APPENDIX 5. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
None. RNA Quantification: 
SECTION: APPENDIX 5. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
None. In a new microcentrifuge tube/falcon tube (depending on the number of samples processed), prepare a working solution of the Qubit HS RNA Assay: 
     Reagents      Volume/sample      Volume   for n+1 sample        Qubit RNA HS Assay buffer      199   µL      ….   µL        Qubit RNA HS Assay Dye      1   µL      ….   µL   
 
SECTION: APPENDIX 5. DNA and RNA Measurement using QUBIT FLUOROMETER 4.0
None. In a new 0.6 ml tube, mix 197 µL of Qubit HS RNA Assay working solution and 3 µL of the sample. Vortex for 5 s, then incubate for 2 min at Room temperature before reading.

SECTION: APPENDIX 1.  Example of the water filtration system assembly 0m
76. 
 

 

 

 
SECTION: APPENDIX 2. Reference of Water Filtration Speed
77. APPENDIX 2. Reference of Water Filtration Speed

 

 

 
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
16. 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. 
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
17. Evenly distribute 250 µL 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. 
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
18. Add 187 mL of PEG-8000 solution to a 750 mL water sample filtrate (or 62.5 mL PEG-8000 solution to a 250 mL filtrate, the final concentration of PEG-8000 is 8%). Mix well by shaking.
 

SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
19. Rinse the magnetic funnel, water inlet tube, and 1L glass bottle with 10% bleach, then wash away the bleach with deionized water. 
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
20. 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.  
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
21. Repeat steps from 4 to 19 for another sample.
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
22. 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.
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
23. After finishing all the sample filtration for the day, prepare a positive control for the batch: add 100 µL EBV and 50 µL DENV-1 standard into 180 mL sterile 1x PBS then add 45 mL of PEG-8000 solution. 
SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
24. Negative control for the batch of sample filtration: 180 mL sterile 1x PBS then add 45 mL of PEG-8000 solution.

SECTION: WATER SAMPLE FILTRATION FOR CAPTURING BACTERIAL AND EUKARYOTIC TARGET
25. Store PEG-8000-added samples and controls at 4 °C for more than 240 min or 240 min for the next filtration round (DO NOT store the PEG-8000-added samples at 4 °C more than 1440 min that will compromise the sample stability). 
SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
33. Evenly distribute 250 µL 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. 
SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
34. Discard the filtrate.
SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
35. Rinse the magnetic funnel, water inlet tube, and 1L glass bottle with 10% bleach, then wash away the bleach by running under deionized water.
SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
36. Rinse the inlet tube and 1L glass bottle with 70% ethanol, shake off the residuals, and allow to air dry.
SECTION: POTENTIAL VIRAL PARTICLE COLLECTION
37. Wipe dry the magnetic funnel with 70% ethanol. Repeat steps from 26 to 34 for another sample.

SECTION: SAMPLE LYSIS
49. Load the sample/bead tubes in the Bullet Blender.
SECTION: SAMPLE LYSIS
50. Set the speed at 12 and time at 3. Press Start.
SECTION: SAMPLE LYSIS
51. Let the samples settle for 1 min and then repeat step 50.
STOPPING POINT: lysed samples can be stored at 4 °C .