Materials and Reagents

Stool aliquoting and pulverization
1. Versi-dry sheets (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 74018-00 )
2. Large Kim-wipes (KCWW, Kimberly-Clark, catalog number: 34721 )
3. Extra heavy-duty aluminum foil (VWR, catalog number: 89107-734 )
4. Dry ice
5. Liquid nitrogen and a Dewar along with Styrofoam cup
6. Pre-labeled screw capped tubes (STARSTEDT) in a freezer box (4 pre-labeled tubes per sample)
7. Stool scrapers (autoclaved in sets of 6) (Fisher Scientific, catalog number: 21-401-25B )
8. Sterilization pouches (small)
9. Autoclave bags
10. Bleach (5.25% solution of sodium hypochlorite)
11. 75% EtOH
Virus-like particle (vlp) preparation
1. Sterile 1.5 ml and 2.0 ml screw-cap tubes
2. 1 ml luer-lok syringes (BD, catalog number: 309628 )
3. 0.45 µm filters 13 mm diameter (EMD Millipore, catalog number: SLHV013SL )
4. 0.22 µm filters 13 mm diameter (EMD Millipore, catalog number: SLGV013SL )
5. 1 M Tris, pH 7.5 (Fisher Scientific, catalog number: MT-46-030-CM ) (Tris 1 M pH 7.5, DNase-, RNase-, protease-free [6 x 1 L bottles])
6. 5 M NaCl (Promega, catalog number: V4221 ) (5 M NaCl 1 L bottle, DNase-, RNase-, protease-free; aliquoted in 1 ml aliquots and stored at -20 °C)
7. Saline magnesium (SM) buffer (Fisher Scientific, catalog number: 50-329-444 ) (SM buffer with gelatin pH 7.5, 100 ml)
8. 10% SDS (diluted from 20% SDS stock [Thermo Fisher Scientific, Ambion^TM, catalog number: AM9820 ] in RNase, DNase, Protease free H₂O; aliquoted and stored at -20 °C)
9. Lysozyme (10 mg/ml) (EMD Millipore, catalog number: 71412 ) – aliquoted and stored at -20 °C
10. Turbo DNase I (2 U/µl) (Thermo Fisher Scientific, Ambion^TM, catalog number: AM2238 )
11. BaseLine zero DNase (1 U/µl) (Epicentre, catalog number: DB0711K )
12. Chloroform (Fisher Scientific, catalog number: C298-500 )
13. Phenol:chloroform:isoamyl alcohol (25:24:1) pH 8.0 (Fisher Scientific, catalog number: BP1752I-100 )
14. QIAGEN DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69506 )
15. CTAB (Sigma-Aldrich, catalog number: 52365 )
16. CTAB/NaCl (see Recipes) [0.45 µm filtered + 0.22 µm filtered]
Library construction
1. GenomiPhi V2 DNA Amplification Kit (GE Healthcare, catalog number: 25-6600-31 )
2. Covaris microTUBE AFA fiber snap-cap 50 µl (Covaris, catalog number: 520045 )
3. NEBNext^® Ultra^TM DNA Library Prep Kit for Illumina (New England Biolabs, catalog number: E7370L , 96 rxns)
4. NEBNext^® Multiplex Oligos for Illumina^®
  Index Primers Set 1 (New England Biolabs, catalog number: E7335L )
  Index Primers Set 2 (New England Biolabs, catalog number: E7500L )
5. AMPure XP beads (Beckman Coulter, catalog number: A63881 )
6. TE low EDTA
7. D1K reagents (Agilent Technologies, catalog number: 5067-5362 )
8. D1K screen tape for TapeStation (Agilent Technologies, catalog number: 5067-5361 )

Equipment

Biosafety hood
6 mortar/pestles, 100 ml capacity
Beaker
Microcentrifuge
PCR hood
-80 °C freezer
Covaris E210
PCR Thermocycler (Eppendorf)
NanoDrop Micro-Volume UV-Vis spectrophotometer
Agilent 2200 TapeStation (Agilent Technologies, model: 2200 TapeStation)
Loading tips for TapeStation (Agilent Technologies, catalog number: 5067-5153 )
Dynamag^TM-spin magnet (Thermo Fisher Scientific, catalog number: 12320D )

Procedure

Stool aliquoting/pulverization
Note: All steps were performed in a biosafety hood due to use of human feces and risk of viable infectious organisms.
1. Washing and preparation of mortars and pestles
2. Pulverization of stool
VLP enrichment protocol
1. Pulverize as above ~200 mg of stool into sterile 2 ml screw cap tube on dry ice to keep frozen.
2. Add 400 µl cold SM buffer per sample. Keep on ice. Vortex on high speed for 5 min.
  Note: It is possible to use a 2 ml tube adapter plate to hold all sample tubes at once if available. Avoid touching tip to tube when adding reagents. Change tips between samples to avoid cross-contamination. Low speed centrifugation is used to avoid lysing bacteria. Goal is to get everything into solution. More SM buffer can be added if needed.
3. Centrifuge for 10 min at 2,000 x g at 4 °C. Transfer supernatant to a clean 1.5 ml Eppendorf tube. Centrifuge a second time for 10 min at 2,000 x g at 4 °C.
  Note: Sometimes a third spin is needed to clarify supernatant. Final supernatant volume should be at least 200 µl because a fraction of the volume will be lost in the subsequent filtration. More SM buffer can be added if needed.
4. Filter supernatant once through a 0.45 µm filter. Then filter twice through 0.22 µm filters. All filtration steps use 1 ml Luer-lok syringes and 13 mm diameter filters.
  Note: Filtering once through 0.22 µm filter is sufficient, but 2 x 0.22 µm filtration steps were used in this manuscript. If the filter clogs: pull the plunger out to relieve pressure, take off the filter and put a new one on. If the initial sample volume was low, may need to use a pipette to recover remaining sample from the old, clogged filter.
5. Check final volume of filtrate. Bring to 200 µl with cold SM buffer.
6. Lysozyme/chloroform/DNase treatment
  1. Add 20 µl (10% volume) lysozyme (10 mg/ml stock) to each tube. Incubate at 37 °C 30 min.
  2. Add 44 µl (20% volume) chloroform, briefly vortex, and incubate 10 min at room temperature (RT; 15-25 °C).
  3. Centrifuge at 2,500 x g 5 min at RT.
  4. Collect aqueous phase and transfer to 2 ml screw cap tube.
    Note: Screw cap is preferable to prevent sample loss from the top popping open during the heating.
  5. Make DNase master mix.
7. Add 50 µl DNase master mix to each tube. Incubate at 37 °C for 1 h.
8. Heat-inactivate DNase at 65 °C for 15 min followed by a quick spin to pull down condensation.
9. SDS/CTAB cleanup
Note: All steps here below were performed in a PCR hood.
1. Clean DNA using QIAGEN DNeasy Blood and Tissue Kit. Elute in 200 μl elution buffer.
  Note: Start at the buffer AL step in the Purification of Total DNA from Animal Blood or Cells protocol. Other column clean-up kits may be used, but should remain consistent between samples used for comparison. We would recommend only clean-up kits that have a large size range of DNA retained.
Phi29 polymerase DNA amplification
Note: 2 μl of each sample is used as template in 4 independent MDA reactions using the GenomiPhi V2 DNA Amplification Kit to reduce amplification bias. The 4 replicates are then pooled after MDA and quantified by NanoDrop.
1. Add 8 µl of GenomiPhi V2 DNA Amplification Kit sample buffer + 2 μl of template to a clean 0.5 ml PCR tube.
2. Heat at 95 °C for 3 min then cool to 4 °C on ice. Keep cold.
3. Prepare master mix on ice:
4. Add 10 µl of the above master mix per tube and return to thermocycler.
5. Heat to 30 °C for 2 h. Then heat-kill enzyme at 65 °C for 10 min. Cool to 4 °C. Can store at -20 °C.
  Note: Incubation time should be optimized to the shortest time that allows sufficient amplification to minimize amplification bias.
6. Pool the 4 independent MDA reactions from the same sample. Adjust volume to 200 µl with DNase-free H₂O.
7. Purify MDA product using QIAGEN DNeasy Blood and Tissue Kit. Elute in 100 µl buffer AE, re-circulate through column once to increase yield.
Covaris fragmentation with Covaris E210
1. Dilute 200 ng DNA to 50 µl total volume dH₂O per sample in the Covaris snap-top tubes.
  Note: Increasing to 500 ng DNA at this step did not increase number of different viral sequences.
2. Pre-chill the Covaris.
3. Fragment nucleic acid using the 400 setting as per Covaris manual: Intensity 5, Duty cycle 5%, Cycles per burst 200, Treatment time 55 sec, Temp 7 °C, Water level 6, Sample volume 50 µl.
4. Remove samples and store on ice if proceeding immediately to the next step (recommended) or store at -20 °C for later use.
NEBNext DNA library construction
Note: Perform as per NEBNext^® Ultra^TM DNA Library Prep Kit for Illumina manual protocol.
1. End repair
  1. Bring DNA up to 55.5 µl with dH₂O.
  2. Add 6.5 µl End repair reaction buffer.
  3. Add 3 µl End Prep enzyme mix.
  4. In thermocycler:
    20 °C for 30 min
    65 °C for 30 min
    4 °C Hold
2. Adaptor ligation
3. Add 3 µl USER enzyme to ligation mix and heat in thermocycler at 37 °C for 15 min.
4. AmPure bead size selection for 400-500 bp library product (300-400 bp insert).
  1. Add 13.5 µl nuclease-free H₂O to the ligation reaction for a final volume of 100 µl.
  2. Add 40 µl AmPure beads and mix well by pipetting up and down.
  3. Incubate at RT for 5 min.
  4. Centrifuge tubes briefly. Place on magnet and incubate at RT for 5 min (until clear).
  5. Transfer supernatant to a clean tube.
  6. Add 20 µl AmPure beads and mix well. Incubate at RT for 5 min.
  7. Centrifuge tubes briefly. Place on magnet and incubate at RT for 5 min.
  8. Leaving tubes on magnet, remove and discard supernatant, careful to not dislodge beads.
  9. With tubes on magnet, add 200 µl freshly prepared 80% EtOH and incubate at RT for 30 sec.
  10. With tubes on magnet, remove EtOH wash.
  11. Repeat steps E4i and E4j two more times for a total of 3 washes.
  12. Air dry beads for 10 min at RT on the magnet with the tube top open.
  13. Remove tubes from magnet and add 28 µl TE low EDTA, pH 8 to beads and resuspend by vortexing or mixing well by pipetting.
  14. Centrifuge tubes briefly, replace tubes on magnet and incubate at RT for 5 min, or until clear.
  15. Collect 23 µl of DNA and transfer to a clean PCR tube. Be sure not to transfer any beads.
5. PCR amplification
6. PCR cleanup
7. Assay for library yield and quality on TapeStation HS DNA as per TapeStation HS DNA protocol.
8. Pool samples (~12 samples/run, equimolar) to a final concentration of 10 nM and verify concentration on TapeStation HS DNA.
9. Submit for Illumina Miseq as per facility protocol and recommendations (we used loading concentration 7 pM, 1% PhiX spike-in, Std flowcell, 2 x 250 bp run).
  Note: This protocol is also applicable for Illumina Hiseq.

Data analysis

Sequence analysis methods are rapidly evolving due to advances in both hardware speed and software coding. Many sequence processing software tools are open-source (such as BBTools, http://jgi.doe.gov/data-and-tools/bbtools/), as are statistical analysis and graphing packages in R (https://www.r-project.org/). We used VirusSeeker (Zhao et al., 2017), a customized automated bioinformatics pipeline based on VirusHunter (Zhao et al., 2013), to detect sequences sharing nucleotide and amino acid sequence similarity to known viruses (Figure 1 below). We recommend using a stringent protocol for viral sequence identification, such as VirusSeeker, that removes low-quality sequences, repeat sequences, and non-specific viral ‘hits.’ Similarly stringent methods have identified novel viral sequences (Zhao et al., 2013). Basic steps in the analysis protocol are shown in Figure 1. Custom viral databases can be generated after downloading sequences corresponding to all viral genomes from the NCBI database (make note of the date of download as new sequences are frequently added). Deduplication is recommended to minimize amplification bias, and taxon-assigned sequences should be normalized to account for variations in sequencing depth between samples. Novel viral sequences identified or viral sequences of interest should be validated by real-time qPCR using primers specific to the viral sequence. Additionally, sequences can be de novo assembled into longer contigs and compared to the NCBI nr/nt databases to better identify phylogeny of viral sequences of interest (Monaco et al., 2016). Phylogenetic trees comparing sequences of interest to known related viral sequences can be made using free software such as FigTree (http://tree.bio.ed.ac.uk/software/figtree/).

Figure 1. Sequence analysis schematic

Notes

Stool pulverization is performed in order to evenly distribute microbial matter in the sample. The use of SM buffer containing gelatin stabilizes bacteriophage populations after freezing for further characterization of bacteriophage of interest, including culturing. Due to risk of contamination, reagents should be used only for fecal microbiome studies. Use the same reagents for all Illumina Miseq runs (i.e., same bottle of sterile water, kits, etc.) as many reagents may be contaminated by microbial DNA, and this ensures even contamination across runs.

Recipes

CTAB/NaCl
3.5 ml 5 M NaCl (final conc. 0.7 M)
2.5 g CTAB (final conc. 10%)
12 ml nuclease-free H₂O
Final volume: 25ml
Heat to 55 °C to dissolve
Add water to final volume of 25 ml. Then 0.45 µm followed by 0.22 µm filter

Acknowledgments

CLM was supported by NIH training grant 5T32AI007172-34, and the work was funded by R24 ODO19793, R01 OD011170, R01 AI111918, and R01 DK101354. DSK is supported by the Burroughs Wellcome Fund. The stool pulverization protocol is adapted from a protocol generously provided by the Jeffrey Gordon laboratory at Washington University in St. Louis. The VLP protocol was adapted from a protocol for phage isolation (Reyes et al., 2013). We would like to thank Brian Keller, M.D., Ph. D for critical review of this manuscript.

References

Duerkop, B. A. and Hooper, L. V. (2013). Resident viruses and their interactions with the immune system. Nat Immunol 14(7): 654-659.
Handley, S. A., Thackray, L. B., Zhao, G., Presti, R., Miller, A. D., Droit, L., Abbink, P., Maxfield, L. F., Kambal, A., Duan, E., Stanley, K., Kramer, J., Macri, S. C., Permar, S. R., Schmitz, J. E., Mansfield, K., Brenchley, J. M., Veazey, R. S., Stappenbeck, T. S., Wang, D., Barouch, D. H. and Virgin, H. W. (2012). Pathogenic simian immunodeficiency virus infection is associated with expansion of the enteric virome. Cell 151(2): 253-266.
Mokili, J. L., Rohwer, F. and Dutilh, B. E. (2012). Metagenomics and future perspectives in virus discovery. Curr Opin Virol 2(1): 63-77.
Monaco, C. L., Gootenberg, D. B., Zhao, G., Handley, S. A., Ghebremichael, M. S., Lim, E. S., Lankowski, A., Baldridge, M. T., Wilen, C. B., Flagg, M., Norman, J. M., Keller, B. C., Luevano, J. M., Wang, D., Boum, Y., Martin, J. N., Hunt, P. W., Bangsberg, D. R., Siedner, M. J., Kwon, D. S. and Virgin, H. W. (2016). Altered virome and bacterial microbiome in human immunodeficiency virus-associated acquired immunodeficiency syndrome. Cell Host Microbe 19(3): 311-322.
Norman, J. M., Handley, S. A., Baldridge, M. T., Droit, L., Liu, C. Y., Keller, B. C., Kambal, A., Monaco, C. L., Zhao, G., Fleshner, P., Stappenbeck, T. S., McGovern, D. P., Keshavarzian, A., Mutlu, E. A., Sauk, J., Gevers, D., Xavier, R. J., Wang, D., Parkes, M. and Virgin, H. W. (2015). Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell 160(3): 447-460.
Reyes, A., Wu, M., McNulty, N. P., Rohwer, F. L. and Gordon, J. I. (2013). Gnotobiotic mouse model of phage-bacterial host dynamics in the human gut. Proc Natl Acad Sci U S A 110(50): 20236-20241.
Virgin, H. W. (2014). The virome in mammalian physiology and disease. Cell 157(1): 142-150.
Zhao, G., Krishnamurthy, S., Cai, Z., Popov, V. L., Travassos da Rosa, A. P., Guzman, H., Cao, S., Virgin, H. W., Tesh, R. B. and Wang, D. (2013). Identification of novel viruses using VirusHunter--an automated data analysis pipeline. PLoS One 8(10): e78470.
Zhao, G., Wu, G., Lim, E.S., Droit, L., Krishnamurthy, S., Barouch, D.H., Virgin, H.W., and Wang, D. (2017). VirusSeeker, a computational pipeline for virus discovery and virome composition analysis. Virology 503: 21-30.