TITLE: Soil Viromics Protocol - Emerson Lab v1
AUTHORS: Joanne   Emerson, Sara  Geonczy, Christian  Santos Medellin, Anneliek  Ter Horst, Jane  Fudyma

[DESCRIPTION]
As of April 28, 2022, this is the soil viromics laboratory protocol used in the Emerson group at UC Davis to generate viral DNA from a soil sample for shotgun metagenomic sequencing. By no means did we 'invent' this protocol! It is a mash-up of several previously published protocols, influenced most substantially by Göller et al. 2020, Trubl et al. 2019, Williamson et al. 2005, and, for the ultracentrifugation step, Emerson et al. 2012 (see references below). This version of the protocol appears in Santos-Medellin et al. 2022, which would be an appropriate citation if you follow the procotol precisely, but we recommend citing at least Göller et al. 2020 as well, and for any deviations, please consult and reference the original publications as appropriate.

As part of this protocols.io page, you can find the laboratory protocol (under Steps), list of supplies and reagents (under Materials), and an extensive FAQ document (downloadable PDF under Guidelines & Warnings). Please consult the FAQ document before running the protocol. If you are inclined to deviate from the protocol, please consult the FAQ document first, as we have a number of suggestions in there, as well as info about what we have and have not tried. We will update/clarify as often as we can. 

Please note that there is another soil viromics protocol by Trubl et al. available on protocols.io, which has some great suggestions/clarifications in the discussion: https://www.protocols.io/view/soil-viral-extraction-protocol-for-ssdna-dsdna-vir-q26g75qklwz1/v3

References:

Göller et al. 2020 Microbiome: https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-020-0795-2

Trubl et al. 2019 PeerJ: https://peerj.com/articles/7265/

Williamson et al. 2005 AEM: https://journals.asm.org/doi/full/10.1128/AEM.71.6.3119-3125.2005

Emerson et al. 2012 AEM (especially the supplementary methods): https://journals.asm.org/doi/10.1128/AEM.01212-12 

Santos-Medellin et al. 2022 bioRxiv: https://www.biorxiv.org/content/10.1101/2022.03.24.485562v1.full

[GUIDELINES]
See FAQ document:

  


[STEPS]
SECTION: Preparation
1. Prepare PPBS buffer (https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-020-0795-2) (2% BSA, 10% PBS, 1% K-citrate, 150 mM MgSO4)




SECTION: Preparation
2. Clean ultracentrifuge tubes - scrub, fill with NaOH for at least 1 hour (also soak black caps, but NOT metal red caps), 
rinse 3x with MilliQ water, rinse with 100 μl of 0.02 μm filtered UltraPure water
SECTION: Preparation
1.1. Add PBS, K-citrate, MgSO4 to a 1 L bottle and fill to 1 L with milliQ water. Adjust pH to 6.5 (about 50 μl of 6N 
HCl or about 100 μl of 2N HCl). Leave a stirring bar inside the bottle. 




SECTION: Preparation
1.2. Autoclave bottle with buffer + empty bottle. 




SECTION: Preparation
1.3. Once buffer bottle reaches room temperature, place it on a magnetic stir plate and slowly add BSA directly into the bottle (avoid foaming, don’t agitate too much). Add ~5 g of BSA at a time. 




SECTION: Preparation
1.4. Filter through a 0.22 μm vacuum filter into the autoclaved empty bottle. 
SECTION: Preparation
1.5. Store in 4°C fridge (above freezing, not stable long-term, ~3 months, make sure buffer remains clear and 
uncontaminated by aliquoting only the amount needed). 
SECTION: Preparation
3. Pre-cool the ultracentrifuge rotor: 50.2 Ti (12 samples) or 70Ti (8 samples) at 4 °C (overnight the night before), i.e., leave the rotor in the fridge or cold room. 
SECTION: Preparation
4. Fresh soil is recommended, but if using frozen soil samples stored at -80°C, place samples in -20°C freezer overnight the night before. 
SECTION: Virion Purification and Concentration
6. Per sample, weigh 10 g soil into a 50 mL conical tube. 
SECTION: Virion Purification and Concentration
7. Add 9 mL PPBS to 10 g soil. 
SECTION: Virion Purification and Concentration
8. Shake (orbital shaker) at 300 rpm at 4°C for 10 min. If necessary, the shaking can be done at room temperature.
SECTION: Virion Purification and Concentration
9. 4000 rpm, 10 min, 4 °C Centrifuge at 4000 RPM at 4°C. 

SECTION: Virion Purification and Concentration
10. Carefully pour supernatant into a new 50 mL conical & store supernatant at 4°C. 
SECTION: Virion Purification and Concentration
11. Repeat steps 7-10 so that samples have gone through a total of 3x shaking (may need to vortex prior to shaking to loosen up soil; if so, reduce the shaking time by the amount of vortexing time) & 3x centrifuging, and continue to pool supernatant into the same tube as in step 10. Should accumulate ~27 mL supernatant total (less if soils are high in organic matter). 
SECTION: Virion Purification and Concentration
12. Centrifuge conical containing accumulated supernatant for 8 min at 10,000 RCF (g) (7840 RPM) at 4°C.
SECTION: Virion Purification and Concentration
13. Transfer supernatant to new 50 mL conical tube. 




SECTION: Virion Purification and Concentration
14. Centrifuge the 50 mL conical tube containing supernatant (from step 13) for 8 min at 10,000 RCF (g) (7840 RPM) at 4°C. 
SECTION: Virion Purification and Concentration
15. (*optional*) Avoiding any pellet at the bottom of the tube, pre-filter the supernatant through a 5 μm filter into a new 50 mL conical tube. 
SECTION: Virion Purification and Concentration
16. Avoiding any pellet at the bottom of the tube, filter the supernatant through a 0.22 μm filter into an ultracentrifuge tube. Depending on the soil, you might need multiple filters, and/or this might be easier if you have pre-filtered (optional step 15 above).




SECTION: Virion Purification and Concentration
17. Check the mass of all tubes, tare the heaviest one, and add PPBS to the others to equalize weight (within a 0.1 g 
margin of error). 




SECTION: Virion Purification and Concentration
18. Ultracentrifuge at 35,000 rpm & 4°C under vacuum. Depending on the rotor, the length of spin will be different (according to sediment coefficient (s) = 58): 70Ti rotor (maximum 8 samples) = 3 hrs, 50.2Ti rotor (maximum 12 samples) = 2 hr 25 min.
SECTION: Viral Pellet Resuspension and DNase Treatment
20. Carefully pour or remove supernatant by pipette, taking care not to disturb the viral pellet (which may or may not be visible, but you will often see a 'hole punch-sized' brown or yellow pellet).
SECTION: Viral Pellet Resuspension and DNase Treatment
21. DNase treatment is recommended, especially for fresh soil, but it may not be possible for some soils (e.g., very dry soil or soils stored frozen). If not DNase treating samples, go to first step of DNA Extraction below (and follow special instructions). 




SECTION: Virion Purification and Concentration
19. Before removing tubes from the ultracentrifuge, pay attention to their orientation in the rotor so that you know where to expect the pellet of viral particles (on the lower outside of the tube).
SECTION: Viral Pellet Resuspension and DNase Treatment
22. If DNAse treating samples, resuspend pellet in 100 μl of 0.02 μm filtered UltraPure water into a labeled 2 mL 
microcentrifuge tube. Set heat block to 37°C. 




SECTION: Preparation
5. Keep a record of how your ultracentrifuge tubes are labeled and which tube will go with which sample.
SECTION: Viral Pellet Resuspension and DNase Treatment
22.1. Make master mix (per sample: 10 μl of DNase buffer + 10 μl of DNase).
SECTION: Viral Pellet Resuspension and DNase Treatment
22.2. Add 20 μl of master mix to resuspended virions. Mix by pipetting.
SECTION: Viral Pellet Resuspension and DNase Treatment
22.3. Incubate at 37°C for 30 min.




SECTION: Viral Pellet Resuspension and DNase Treatment
22.4. Add 10 μl of Stop solution. Briefly vortex. Proceed to first step of DNA Extraction (and follow special instructions).
SECTION: Viral DNA Extraction
23. In principle, many DNA extraction protocols would work at this point. You can try your favorite. We use the QIAGEN PowerSoilPro DNeasy kit.
SECTION: Viral DNA Extraction
24. To prepare, aliquot an appropriate amount of each solution. Keep CD2 at 4°C. 
SECTION: Viral DNA Extraction
25. Prepare Sample
SECTION: Viral DNA Extraction
25.1. Turn on heat block to 65°C (ours takes about 15 min to reach temperature) 
SECTION: Viral DNA Extraction
25.2. Spin the PowerBead Pro Tube briefly to ensure that the beads have settled at the bottom.
SECTION: Viral DNA Extraction
25.3. Add up to 800 μl of CD1 to bead tubes. 
SECTION: Viral DNA Extraction
25.4. (1)  No DNase step: Scrape pellet from ultracentrifuge tube and place in bead tube. Take 100 μl of CD1, which is already in bead tube, and use it to resuspend whatever is left of the pellet and put into bead tube. 

(2)  DNase step: After stop solution has been added, transfer resuspended pellet to bead tube. 




SECTION: Viral DNA Extraction
25.5. Vortex briefly to mix.
SECTION: Viral DNA Extraction
25.6. Incubate on heat block at 65°C for 10 min.
SECTION: Viral DNA Extraction
26. Cell Lysis 
SECTION: Viral DNA Extraction
26.1. Vortex tubes at maximum speed for 10 min.
SECTION: Viral DNA Extraction
26.2. Centrifuge tubes at 15,000 x g (RCF) for 1 min. 
SECTION: Viral DNA Extraction
27. Inhibitor Removal
SECTION: Viral DNA Extraction
26.3. Transfer supernatant to new microcentrifuge tube. (expect 500-600 μl)
SECTION: Viral DNA Extraction
27.1. Add 200 μl CD2 
SECTION: Viral DNA Extraction
27.2. Vortex 5 sec
SECTION: Viral DNA Extraction
27.3. Centrifuge at 15,000 x g (RCF) for 1 min. Avoiding the pellet, transfer up to 700 μl of supernatant to a new 
microcentrifuge tube. (expect 500-600 μl).
SECTION: Viral DNA Extraction
28. Bind DNA
SECTION: Viral DNA Extraction
28.1. Add 600 μl CD3
SECTION: Viral DNA Extraction
28.2. Vortex for 5 sec.
SECTION: Viral DNA Extraction
28.3. Load 650 μl of lysate to an MB Spin Column.
SECTION: Viral DNA Extraction
28.4. Centrifuge at 15,000 x g (RCF) for 1 min.
SECTION: Viral DNA Extraction
28.5. Discard the flow-through and repeat previous steps to ensure that all the lysate has passed through the MB 
Spin Column.
SECTION: Viral DNA Extraction
28.6. Carefully place the MB Spin Column into a new collection tube (provided).
SECTION: Viral DNA Extraction
29. Wash
SECTION: Viral DNA Extraction
29.1. Add 500 μl EA to the MB Spin Column.
SECTION: Viral DNA Extraction
29.2. Centrifuge at 15,000 x g (RCF) for 1 min.
SECTION: Viral DNA Extraction
29.3. Discard the flow-through and place the MB Spin Column back into the same 2 ml collection tube.
SECTION: Viral DNA Extraction
29.4. Add 500 μl of C5 onto spin column.
SECTION: Viral DNA Extraction
29.5. Centrifuge at 15,000 x g (RCF) for 1 min.
SECTION: Viral DNA Extraction
29.6. Discard the flow-through and place the MB Spin Column into a new 2 ml collection tube (provided).
SECTION: Viral DNA Extraction
29.7. Centrifuge at 16,000 x g (RCF) for 2 min. Carefully place the MB Spin Column into a new 1.5 ml elution tube 
(provided).
SECTION: Viral DNA Extraction
30. Elute DNA
SECTION: Viral DNA Extraction
30.1. Add 50-100 μl of C6 to center of the white filter membrane.
SECTION: Viral DNA Extraction
30.2. Centrifuge at 15,000 x g for 1 min. Discard the MB Spin Column. The DNA is now ready for downstream 
applications.
SECTION: DNA Quantification
31. Quantify DNA w/ method of choice (e.g., Qubit, use 2 μl, https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Qubit_dsDNA_HS_Assay_UG.pdf). Store short-term (up to a few weeks) at -20°C after quantification, longer term at -80°C.