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Next-generation Sequencing of the DNA Virome from Fecal Samples
粪便样本中DNA病毒组的下一代测序法   

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Abstract

Herein we describe a detailed protocol for DNA virome analysis of low input human stool samples (Monaco et al., 2016). This protocol is divided into four main steps: 1) stool samples are pulverized to evenly distribute microbial matter; 2) stool is enriched for virus-like particles and DNA is extracted by phenol-chloroform; 3) purified DNA is multiple-strand displacement amplified (MDA) and fragmented; and 4) libraries are constructed and sequenced using Illumina Miseq. Subsequent sequence analysis for viral sequence identification should be sensitive but stringent.

Keywords: Virome(病毒组), Viral microbiome(病毒微生物组), Virus-like particles(病毒样颗粒), MDA(MDA), Illumina Miseq(Illumina Miseq)

Background

The virome, a dynamic community of eukaryotic viruses, bacteriophages and endogenous retroviruses, represents a minimally characterized component of the human microbiome (Virgin, 2014). In fact, it is estimated that only 1% of the virome has been sequenced and annotated (Mokili et al., 2012). Next generation sequencing (NGS) enables examination of the entire virome, including unculturable viruses. Stool is a readily obtainable specimen type for study of the virome, and alterations in the fecal virome have been associated with a number of disease states (Handley et al., 2012; Norman et al., 2015; Monaco et al., 2016). The fecal virome is largely comprised of bacteriophages, which affect the gastrointestinal tract through alterations in bacterial functions and populations (Duerkop and Hooper, 2013; Reyes et al., 2013; Virgin, 2014). Enteric eukaryotic viruses, while less ubiquitous than bacteriophages, play a more direct role in gastrointestinal tract dysfunction by inducing gastroenteritis, enteritis and colitis. Despite the abundance of bacteriophages in fecal samples, only a few studies thus far have examined the contributions of fecal bacteriophages in human diseases. Inflammatory bowel disease has been associated with increased enteric bacteriophage richness (Norman et al., 2015). In contrast, profound immunosuppression from AIDS in a sub-Saharan cohort resulted in an expanded eukaryotic virome, but had minimal impact on bacteriophage populations (Monaco et al., 2016). More studies are needed to elucidate the role the fecal virome plays in disease states. A key roadblock to studying the stool virome is viral nucleic acid extraction and enrichment from fecal material. Several factors can contribute to difficulty in isolating viral sequences from fecal samples, chief among them the fact that viruses constitute a minority of fecal sample material. Additionally, dilution of feces in collection media (such as RNAlater RNA stabilization reagent) can further hamper the ability to find viral sequences. While many nucleic acid extraction protocols can be used for high input nucleic acid samples to enrich for viral nucleic acid, low input samples, such as those diluted in collection media, represent a challenge with virome studies. After comparison and optimization of several methods, the following protocol was identified as the most universally applicable for isolation of phage and DNA viral sequences from both low (Monaco et al., 2016) and high (Norman et al., 2015) input samples.

Materials and Reagents

  1. Stool aliquoting and pulverization
    1. Versi-dry sheets (Thermo Fisher Scientific, Thermo ScientificTM, 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

  2. 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, AmbionTM, 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, AmbionTM, 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]

  3. 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® UltraTM 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

  1. Biosafety hood
  2. 6 mortar/pestles, 100 ml capacity
  3. Beaker
  4. Microcentrifuge
  5. PCR hood
  6. -80 °C freezer
  7. Covaris E210
  8. PCR Thermocycler (Eppendorf)
  9. NanoDrop Micro-Volume UV-Vis spectrophotometer
  10. Agilent 2200 TapeStation (Agilent Technologies, model: 2200 TapeStation)
  11. Loading tips for TapeStation (Agilent Technologies, catalog number: 5067-5153 )
  12. DynamagTM-spin magnet (Thermo Fisher Scientific, catalog number: 12320D )

Procedure

  1. 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
      1. Thoroughly sterilize working area with 10% bleach followed by 75% EtOH and UV exposure for 10 min to minimize contamination.
      2. Prepare 10% bleach beaker for scrapers.
      3. Bleach mortar/pestles for 10 min in 10% bleach solution.
      4. Place versi-dry sheets in hood.
      5. Place samples on dry ice in hood.
      6. With remaining dry ice, place open microtube freezer box stably on top of dry ice with empty pre-labeled tubes and place in hood.
      7. Tear heavy duty aluminum foil into pieces around 10 inches wide. Then using a flat surface, tear this into 3 approximately equal squares. Do this twice (6 squares). Place these in the hood.
      8. Once the 10 min bleach is finished, rinse all 6 mortars and pestles with dH2O and set on another versi-dry to dry slightly, and then set upright in hood on versi-dry. UV for 10 min along with all other equipment to be used.
      9. When done, place pestles in mortars, and set all but one to side, stack aluminum foil.
      10. Add large mound of gloves to hood.
      11. Add liquid N2 to Dewar.
    2. Pulverization of stool
      1. Double glove.
        Note: This is not needed if not using human stool.
      2. Place large Kim-wipe on top of the versi-dry. Place mortar and pestle on it.
      3. Open a sterile package of stool scrapers.
      4. Place a piece of aluminum foil into mortar and mold to inner shape of mortar.
      5. Select sample to use, bring out of dry ice and set on Kim-wipe.
      6. Identify corresponding pre-labelled screw-cap tubes, and bring them to front of the box.
      7. Fill Styrofoam cup 3/4 full with liquid N2 from Dewar. Pour some liquid N2 into the box of screw-top tubes, and pour enough into the mortar to fill it ½ way full. Place cup to side of mortar (recommend having some liquid N2 remaining).
      8. Take a stool scraper out of package.
      9. Open sample tube.
      10. Use stool scraper to carefully get sample out of tube into the mortar. Try to get the whole sample out at once.
        Note: May need to warm sample tube in hands to slightly thaw around edges. Do not thaw longer than necessary to remove sample.
      11. Once out, place now empty sample tube into the box of tubes to re-freeze, consider adding small amount of liquid N2 near the tube to aid with re-freezing.
      12. For large chunks of stool, use pestle to break apart. When all large chunks have been crushed to small pieces, then pulverize with pestle. Intermittently add more liquid N2 to mortar as needed to keep sample from thawing out until pulverized completely.
      13. Let liquid N2 boil off. Sample now has the texture of a powder.
      14. Immediately take a fresh tube and scrape approximately 200 mg sample into the tube, tap the tube onto clean side of cold mortar to settle contents to bottom, and place it back into microtube box. Repeat with the remaining 3 tubes. If any sample remains after aliquoting 200 mg to new tubes, scrape remaining sample into the original tube, tapping tube onto a clean cold portion of the mortar to settle contents to the bottom of the tube. If needed, remove aluminum foil from the mortar to get any remaining bits of sample.
        Notes:
        a. Sample left in aluminum foil thaws VERY quickly once out of the mortar.
        b. Do not screw on caps tightly as it is likely they still have some liquid N2 present.
      15. Place stool scraper into bleach beaker, aluminum foil into autoclave bag, pestle into mortar, deglove, take Styrofoam cup out of hood. Place Kim-wipe into an autoclave bag. If versi-dry is dirty, remove versi-dry and place it into the autoclave bag.
      16. Carefully place mortar and pestle in bleach bath.
      17. Repeat process for total of 6 samples.
      18. Store samples at -80 °C.
      19. Wash as above and repeat as needed.

  2. 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.
      1. Take 200 µl to a clean 1.5 ml tube on ice.
      2. Store any remainder for later use at -80 °C.
    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
      1. Add 10 µl SDS (10%) + 1 µl proteinase K (10 mg/ml stock) to each tube and incubate at 56 °C for 20 min. At this point, pre-incubate CTAB/NaCl solution at 65 °C.
      2. Add 35 µl NaCl (5 M soln) + 28.1 µl CTAB/NaCl (2.5% soln). Pulse vortex. Incubate at 65 °C for 10 min then perform a quick spin.
        Note: Sample will turn cloudy after CTAB/NaCl is added.
      3. Add 200 µl phenol:chloroform:isoamyl alcohol (25:24:1) pH 8.0. Pulse vortex. Centrifuge 8,000 x g for 5 min at RT.
      4. Collect the aqueous fraction from step B9c. Add 200 µl chloroform. Pulse vortex for 3-5 sec. Centrifuge 8,000 x g for 5 min at RT.
      5. Collect the aqueous fraction from step B9d. This is final Virus Nucleic Acid. 
      6. Place in dry ice for transport and store at -80 °C.
    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.
      1. Add 200 μl buffer AL. Mix thoroughly by vortexing.
      2. Add 200 μl ethanol (96-100%). Mix thoroughly by vortexing.
      3. Pipet 650 µl of mixture into a DNeasy mini spin column placed in a 2 ml collection tube. Centrifuge at ≥ 6,000 x g for 1 min. Discard the flow-through. Repeat as necessary until all of sample is used.
      4. Add 500 μl buffer AW1. Centrifuge for 1 min at ≥ 6,000 x g. Discard the flow-through.
      5. Add 500 μl buffer AW2, and centrifuge for 2 min at 20,000 x g. Discard the flow-through and collection tube.
      6. Centrifuge for 1 min at 20,000 x g in a new collection tube.
      7. Transfer the DNeasy spin column to a new 1.5 ml or 2 ml microcentrifuge tube.
      8. Elute the DNA by adding 200 μl buffer AE to the center of the DNeasy spin column membrane. Incubate for 1 min at RT. Centrifuge for 1 min at ≥ 6,000 x g.
      9. Recirculate the eluate through the column once (Add eluate to the center of the DNeasy spin column membrane. Incubate for 1 min at RT. Centrifuge for 1 min at ≥ 6,000 x g). 

  3. 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.
      1. Add 200 μl buffer AL. Mix thoroughly by vortexing.
      2. Add 200 μl ethanol (96-100%). Mix thoroughly by vortexing.
      3. Pipet the mixture into a DNeasy mini spin column placed in a 2 ml collection tube. Centrifuge at ≥ 6,000 x g for 1 min. Discard the flow-through.
      4. Add 500 μl buffer AW1. Centrifuge for 1 min at ≥ 6,000 x g. Discard the flow-through.
      5. Add 500 μl buffer AW2, and centrifuge for 2 min at 20,000 x g. Discard the flow-through and collection tube.
      6. Centrifuge for 1 min at 20,000 x g.
      7. Transfer the spin column to a new 1.5 ml or 2 ml microcentrifuge tube.
      8. Elute the DNA by adding 100 μl buffer AE to the center of the spin column membrane. Incubate for 1 min at RT. Centrifuge for 1 min at ≥ 6,000 x g.
      9. Recirculate the eluate through the column once.
      10. Check the concentration by NanoDrop.

  4. 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.

  5. NEBNext DNA library construction
    Note: Perform as per NEBNext® UltraTM 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
      1. Add 15 µl Blunt/TA ligase master mix.
      2. Add 2.5 µl undiluted NEBNext adaptor.
      3. Add 1 µl ligation enhancer.
      4. In thermocycler, incubate at 20 °C for 15 min.
    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
      1. Add the following to each tube:


      2. In a thermocycler:

    6. PCR cleanup
      1. Add 50 µl AmPure beads and mix well.
      2. Incubate at RT x 5 min.
      3. Centrifuge tubes briefly, place on magnet and incubate at RT for 3-5 min.
      4. Leaving tubes on magnet, remove supernatant, careful to not dislodge beads.
      5. With tubes on magnet, add 200 µl freshly prepared 80% EtOH and incubate at RT for 30 sec.
      6. With tubes on magnet, remove EtOH wash.
      7. Repeat steps E6e and E6f once.
      8. Air dry beads for 10 min at RT.
      9. Remove tubes from magnet and add 33 µl TE low EDTA to beads and resuspend. Incubate at RT for 2 min.
      10. Replace tubes on magnet and incubate at RT for 4 min.
      11. Collect 30 µl of DNA and transfer to a clean PCR tube.
    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

  1. 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

  1. Duerkop, B. A. and Hooper, L. V. (2013). Resident viruses and their interactions with the immune system. Nat Immunol 14(7): 654-659.
  2. 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.
  3. Mokili, J. L., Rohwer, F. and Dutilh, B. E. (2012). Metagenomics and future perspectives in virus discovery. Curr Opin Virol 2(1): 63-77.
  4. 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.
  5. 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.
  6. 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.
  7. Virgin, H. W. (2014). The virome in mammalian physiology and disease. Cell 157(1): 142-150.
  8. 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.
  9. 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.

简介

在这里,我们描述了低输入人粪便样品的DNA病毒分析的详细方案(摩纳哥等人,2016)。该方案分为四个主要步骤:1)粪便样品粉碎均匀分布微生物; 2)粪便富集病毒样颗粒,DNA由苯酚 - 氯仿提取; 3)纯化的DNA是多链置换扩增(MDA)并分裂的;和4)使用Illumina Miseq构建和排序库。病毒序列鉴定的后续序列分析应该是敏感但严格的。

背景 真菌病毒,噬菌体和内源性逆转录病毒的动态社区是维生素组织,代表人类微生物组织的最低限度特征(维珍,2014年)。事实上,估计只有1%的病毒已被排序和注释(Mokili等人,2012)。下一代测序(NGS)可以检测整个病毒,包括不可培养的病毒。粪便是易于获得的用于研究病原体的样本类型,并且粪便病毒的改变已经与许多疾病状态相关联(Handley等人,2012; Norman等人,2015;摩纳哥等人,2016)。粪便病毒主要由噬菌体组成,通过细菌功能和群体的改变影响胃肠道(Duerkop和Hooper,2013; Reyes等人,2013; Virgin,2014)。肠道真核病毒虽然比噬菌体普遍存在,但通过诱导胃肠炎,肠炎和结肠炎在胃肠道功能障碍中起直接作用。尽管粪便样本中噬菌体丰富,但迄今为止只有少数研究已经检查了粪便噬菌体对人类疾病的贡献。炎症性肠病与肠道噬菌体丰富度增加有关(Norman等,2015)。相比之下,撒哈拉沙漠以南的队列中艾滋病的深度免疫抑制导致扩增的真核病毒,但对噬菌体群体的影响最小(摩纳哥等,2016)。需要更多的研究来阐明粪便病毒在疾病状态中的作用。研究粪便病毒的一个重要障碍是病毒核酸提取和粪便物质的富集。病毒序列与粪便样本的分离有几个因素,其中主要是病毒构成少数粪便样本物质。此外,收集介质(如RNAlater RNA稳定剂)中的粪便稀释可能进一步阻碍寻找病毒序列的能力。虽然许多核酸提取方案可以用于高输入核酸样品以富集病毒核酸,但是低输入样品(例如在收集介质中稀释的那些样品)代表了病毒学研究的挑战。在比较和优化几种方法之后,以下方案被确定为最普遍适用于从低分离(摩纳哥等等,2016)和高(Norman)的噬菌体和DNA病毒序列>等等,2015)输入样本。

关键字:病毒组, 病毒微生物组, 病毒样颗粒, MDA, Illumina Miseq

材料和试剂

  1. 粪便等分和粉碎
    1. Versi-dry sheet(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:74018-00)
    2. 大金箔(KCWW,Kimberly-Clark,目录号:34721)
    3. 超重型铝箔(VWR,目录号:89107-734)
    4. 干冰
    5. 液氮和杜瓦以及泡沫塑料杯
    6. 冷冻箱中预先标记的螺旋管(STARSTEDT)(每个样品4个预先标记的管)
    7. 粪便刮刀(以6组高压灭菌)(Fisher Scientific,目录号:21-401-25B)
    8. 灭菌袋(小)
    9. 高压灭菌袋
    10. 漂白剂(5.25%次氯酸钠溶液)
    11. 75%EtOH

  2. 病毒样颗粒(vlp)制剂
    1. 无菌1.5毫升和2.0毫升螺旋盖管
    2. 1 ml luer-lok注射器(BD,目录号:309628)
    3. 直径为13 mm的0.45μm过滤器(EMD Millipore,目录号:SLHV013SL)
    4. 直径为13 mm的0.22μm过滤器(EMD Millipore,目录号:SLGV013SL)
    5. 1 M Tris,pH 7.5(Fisher Scientific,目录号:MT-46-030-CM)(Tris 1M pH 7.5,DNase-,RNase-,不含蛋白酶的[6×1L瓶])
    6. 5 M NaCl(Promega,目录号:V4221)(5M NaCl 1L瓶,DNase-,RNase-,不含蛋白酶;等分在1ml等份中并储存在-20℃)
    7. 盐水镁(SM)缓冲液(Fisher Scientific,目录号:50-329-444)(SM缓冲液,明胶pH 7.5,100ml)
    8. 10%SDS(从20%SDS储备[Thermo Fisher Scientific,Ambion TM,目录号:AM9820]在RNase,DNase,不含蛋白酶的H 2 O中稀释;等分和储存于-20°C)
    9. 溶解酶(10 mg/ml)(EMD Millipore,目录号:71412) - 等分并储存于-20°C
    10. Turbo DNase I(2U /μl)(Thermo Fisher Scientific,Ambion TM ,目录号:AM2238)
    11. 碱基零DNase(1U /μl)(Epicentre,目录号:DB0711K)
    12. 氯仿(Fisher Scientific,目录号:C298-500)
    13. 苯酚:氯仿:异戊醇(25:24:1)pH8.0(Fisher Scientific,目录号:BP1752I-100)
    14. QIAGEN DNeasy血液和组织试剂盒(QIAGEN,目录号:69506)
    15. CTAB(Sigma-Aldrich,目录号:52365)
    16. CTAB/NaCl(参见食谱)[0.45μm过滤+0.22μm过滤]

  3. 图书馆建设
    1. GenomiPhi V2 DNA扩增试剂盒(GE Healthcare,目录号:25-6600-31)
    2. Covaris microTUBE AFA纤维卡帽50μl(Covaris,目录号:520045)
    3. NEBNext ® Ultra TM Illumina的DNA文库准备工具包(New England Biolabs,目录号:E7370L,96 rxns)
    4. NEBNext ® Illumina的复用Oligos ®
      Index Primers Set 1(New England Biolabs,目录号:E7335L)
      Index Primers Set 2(New England Biolabs,目录号:E7500L)
    5. AMPure XP珠(Beckman Coulter,目录号:A63881)
    6. TE低EDTA
    7. D1K试剂(Agilent Technologies,目录号:5067-5362)
    8. 用于TapeStation的D1K屏幕胶带(Agilent Technologies,目录号:5067-5361)

设备

  1. 生物安全罩
  2. 6支砂浆/杵,100毫升容量
  3. 烧杯
  4. 微量离心机
  5. PCR罩
  6. -80°C冰箱
  7. Covaris E210
  8. PCR热循环仪(Eppendorf)
  9. NanoDrop微量紫外 - 可见分光光度计
  10. Agilent 2200 TapeStation(Agilent Technologies,型号:2200 TapeStation)
  11. 载入TapeStation的技巧(Agilent Technologies,目录号:5067-5153)
  12. Dynamag TM 旋转磁铁(Thermo Fisher Scientific,目录号:12320D)

程序

  1. 粪便等分/粉碎
    注意:所有步骤都是在生物安全罩中进行的,因为使用人类粪便和存在感染性生物体的风险。
    1. 砂浆和杵的洗涤和制备
      1. 用10%漂白剂,75%乙醇彻底灭菌工作区域,并进行紫外线曝光10分钟,以尽量减少污染。
      2. 准备10%的漂白烧杯用于刮刀。
      3. 漂白砂浆/杵在10%漂白溶液中10分钟。
      4. 将干燥的床单放在敞篷中。
      5. 将样品放在防冰罩上的干冰上。
      6. 使用剩余的干冰,将空的预先标记的管子放置在干冰的顶部,并放置在发动机罩内,将开放的微管冷冻箱放置在干冰的顶部。
      7. 将重型铝箔撕成10英寸宽的碎片。然后使用平坦的表面,将其撕成3个近似相等的正方形。做这两次(6个方块)。把它们放在罩子里。
      8. 一旦10分钟的漂白剂完成,用dH <2> O冲洗所有6个迫击炮和杵,另一个干燥,稍微干燥,然后直立在罩上。紫外线10分钟以及所有其他使用的设备。
      9. 完成后,将杵放在迫击炮中,将所有物品放在一边,堆叠铝箔。
      10. 将大号手套加入罩中。
      11. 向Dewar加入液体N 2 。
    2. 粪便粉碎
      1. 双手套
        注意:如果不使用人体粪便,则不需要。
      2. 将大金箔抹在干燥的上面。将砂浆和杵放在上面。
      3. 打开无菌包装的粪便刮刀。
      4. 将一块铝箔放入砂浆和模具中至砂浆的内部形状。
      5. 选择要使用的样品,取出干冰,并放在Kim-wipe上。
      6. 识别相应的预先标记的螺旋盖管,并将其带到箱体前面。
      7. 从Dewar填充泡沫塑料杯3/4,充满液体N 2。将一些液体N 2倒入螺旋顶管的盒子中,并倒入足够的泥浆中以充满半径。将杯子放在砂浆的侧面(推荐留下一些液体N 2 )
      8. 把一个粪便刮刀从包装中取出。
      9. 打开样品管。
      10. 使用粪便刮刀将样品从管中小心地取入砂浆。尝试一次取出整个样品。
        注意:可能需要在手中加热样品管以在边缘稍微解冻。不要解冻更长的时间来除去样品。
      11. 一旦出现,现在将空样品管放入管子内重新冻结,考虑在管附近加入少量液体N 2,以帮助重新冷冻。
      12. 对于大块大便,用杵分开。当所有大块被粉碎成小块时,用研杵粉碎。根据需要间歇性地向砂浆中加入更多的液体N 2,以保持样品解冻直至完全粉碎。
      13. 使液体N 2沸腾。样品现在具有粉末的质感。
      14. 立即取出新鲜的管子,将约200毫克的样品刮到管中,将管子抽到冷灰浆的干净的一侧,将内容物放到底部,放回微管箱中。与其余3根管重复。如果任何样品在等分200毫克新管后保留,将残留的样品刮到原始管中,将管子轻轻地放在干净的冷却部分的研钵上,以将内容物沉淀到管的底部。如果需要,从砂浆中取出铝箔,以获得任何剩余的样品。
        注意:
        a。铝箔中的样品离开砂浆非常快速地一次。
        b。不要拧紧盖子,因为它们很可能还有一些液体N <2> 。
      15. 将粪便刮刀放入漂白烧杯中,将铝箔放入高压灭菌袋中,将杵杵倒入砂浆中,脱泡,将发泡胶杯从罩中取出。将Kim-wipe放入高压灭菌袋中。如果干燥不洁,请清洁干燥,并将其放入高压灭菌器袋中。
      16. 砂浆和杵仔细地在漂白浴中。
      17. 共6个样本重复过程。
      18. 将样品储存在-80°C。
      19. 如上所述进行洗涤,并根据需要重复。

  2. VLP浓缩方案
    1. 将上述粉碎至200mg的粪便放入干冰中的无菌2ml螺旋盖管中以保持冷冻
    2. 每个样品加入400μl冷SM缓冲液。保持在冰上高速涡旋5分钟。
      注意:如有可能,可以使用2 ml管接头板将所有样品管保持在一起。加入试剂时,避免接触尖端至管。更改样品之间的提示,以避免交叉污染。低速离心法用于避免细菌裂解。目标是让一切都变成解决方案。如果需要,可以添加更多SM缓冲区。
    3. 在4℃下以2,000 x g离心10分钟。将上清液转移至干净的1.5 ml Eppendorf管中。在4℃下以2,000×g离心分离10分钟。
      注意:有时需要第三次旋转以澄清上清液。最后的上清液体积应至少为200μl,因为在随后的过滤中体积的一部分将会丢失。如果需要,可以添加更多的SM缓冲区。
    4. 过滤上清液一次通过0.45μm过滤器。然后通过0.22μm过滤器过滤两次。所有过滤步骤均使用1毫升鲁尔 - 洛克注射器和13毫米直径的过滤器。
      注意:通过0.22μm过滤器过滤一次是足够的,但在该手稿中使用2×0.22μm的过滤步骤。如果过滤器堵塞:拉出柱塞以释放压力,取下过滤器并重新打开。如果初始样品体积较低,可能需要使用移液管从旧的堵塞过滤器中回收剩余的样品。
    5. 检查滤液的最终体积。用冷SM缓冲液加至200μl。
      1. 将200μl的冰块放入干净的1.5 ml管中
      2. 储存任何剩余物以供以后使用-80°C。
    6. 溶菌酶/氯仿/DNA酶处理
      1. 向每个管中加入20μl(10%体积)溶菌酶(10mg/ml储备液)。在37°C孵育30分钟。
      2. 加入44μl(20%体积)氯仿,短暂涡旋,并在室温(RT; 15-25℃)下孵育10分钟。
      3. 在室温下以2,500 x g离心5分钟。
      4. 收集水相并转移至2 ml螺旋盖管 注意:螺旋盖最好用于防止加热过程中顶部出现的样品损失。
      5. 使DNase主机混合。 

    7. 在每个管中加入50μlDNase主混合物。在37°C孵育1 h。
    8. 在65℃下热灭活DNase 15分钟,然后快速旋转以拉下冷凝。
    9. SDS/CTAB清理
      1. 向每个管中加入10μlSDS(10%)+1μl蛋白酶K(10mg/ml储备液),并在56℃温育20分钟。此时,在65℃预孵育CTAB/NaCl溶液
      2. 加入35μlNaCl(5 M soln)+ 28.1μlCTAB/NaCl(2.5%soln)。脉冲涡流在65℃孵育10分钟,然后快速旋转。
        注意:添加CTAB/NaCl后,样品将变得阴天。
      3. 加入200μl苯酚:氯仿:异戊醇(25:24:1),pH8.0。脉冲涡流在室温下将8,000 x g离心5分钟。
      4. 收集步骤B9c的含水级分。加入200μl氯仿。脉冲涡流3-5秒在室温下将8,000 x g离心5分钟。
      5. 从步骤B9d收集含水部分。这是最终的病毒核酸。
      6. 放在干冰中运输并储存在-80°C。
    注意:以下所有步骤均在PCR引擎盖中进行。
    1. 使用QIAGEN DNeasy血液和组织试剂盒清洁DNA。在200μl洗脱缓冲液中洗脱。
      注意:从动物血液或细胞方案的总DNA纯化中的缓冲液AL步骤开始。可以使用其他柱清洁试剂盒,但在比较用样品之间应保持一致。我们建议只保留大范围DNA的清理工具。
      1. 加入200μl缓冲液AL。通过涡旋充分混合。
      2. 加入200μl乙醇(96-100%)。通过涡旋彻底混合。
      3. 将650μl混合物吸入放置在2ml收集管中的DNeasy微型旋转柱中。离心机≥6,000x g 1分钟。丢弃流通。必要时重复,直到使用所有样品。
      4. 加入500μl缓冲液AW1。以≥6,000x g离心1分钟。放弃流通。
      5. 加入500μl缓冲液AW2,并以20,000×g离心2分钟。丢弃流通和收集管。
      6. 在新收集管中以20,000 x g离心1分钟。
      7. 将DNeasy旋转柱转移到新的1.5 ml或2 ml微量离心管中
      8. 通过在DNeasy旋转柱膜的中心加入200μl缓冲液AE来洗脱DNA。在室温下孵育1分钟。离心1分钟,≥6,000x g
      9. 将洗脱液再循环通过柱子(将洗脱液加入到DNeasy旋转柱膜的中心,在室温下孵育1分钟,以≥6,000x g 离心1分钟) 

  3. Phi29聚合酶扩增
    注意:使用GenomiPhi V2 DNA扩增试剂盒将4μl独立MDA反应中的每个样品2μl用作模板,以减少扩增偏差。然后在MDA之后合并4个重复,并由NanoDrop定量
    1. 将8μlGenomiPhi V2 DNA扩增试剂盒样品缓冲液+ 2μl模板加入干净的0.5 ml PCR管中。
    2. 在95℃下加热3分钟,然后在冰上冷却至4℃。保持冷。
    3. 准备冰上的主人混合:


    4. 每管加入10μl上述主混合物,并返回热循环仪。
    5. 加热至30°C 2 h。然后在65℃下热灭菌10分钟。冷却至4°C。可在-20°C储存。
      注意:孵化时间应优化到允许充分放大以最小化放大偏差的最短时间。
    6. 从同一个样品中汇集4个独立的MDA反应。将体积调节至200μl,不含DNase的H 2 O。
    7. 使用QIAGEN DNeasy血液和组织试剂盒纯化MDA产品。在100μl缓冲液AE中洗脱,通过柱一次循环以提高产量。
      1. 加入200μl缓冲液AL。通过涡旋充分混合。
      2. 加入200μl乙醇(96-100%)。通过涡旋彻底混合。
      3. 将混合物吸入放置在2ml收集管中的DNeasy微型旋转柱中。离心机≥6,000x g 1分钟。放弃流通。
      4. 加入500μl缓冲液AW1。以≥6,000x g离心1分钟。放弃流通。
      5. 加入500μl缓冲液AW2,并以20,000×g离心2分钟。丢弃流通和收集管。
      6. 以20,000 x g离心1分钟。
      7. 将旋转柱转移到新的1.5 ml或2 ml微量离心管中
      8. 通过向旋转柱膜的中心加入100μl缓冲液AE来洗脱DNA。在室温下孵育1分钟。离心1分钟,≥6,000x g
      9. 将洗脱液再次循环通过色谱柱一次。
      10. 检查NanoDrop的浓度。

  4. Covaris分裂与Covaris E210
    1. 在Covaris弹簧管中将200 ng DNA稀释至每个样品50μl总体积dH 2 O。
      注意:在此步骤中增加到500ng DNA不会增加不同病毒序列的数量。
    2. 预冷Covaris。
    3. 片段核酸使用400设置根据Covaris手册:强度5,占空比5%,每爆发200次循环,处理时间55秒,温度7℃,水位6,样品体积50微升。
    4. 如果立即进行下一步(推荐)或在-20°C保存供以后使用,请取出样品并存放在冰上。

  5. NEBNext DNA文库构建
    注意:根据NEBNext ®执行Ultra TM 用于Illumina手册协议的DNA文库准备工具。
    1. 结束修复
      1. 将DNA加入到具有dH O的55.5μl。
      2. 加入6.5μl终止修复反应缓冲液
      3. 加入3μlEnd Prep酶混合物
      4. 在热循环仪中:
        20°C 30分钟
        65°C 30分钟
        4℃保持
    2. 适配器结扎
      1. 添加15μlBlunt/TA连接酶主混合物。
      2. 加入2.5μl未稀释的NEBNext适配器。
      3. 加入1μl结扎增强子
      4. 在热循环仪中,在20℃孵育15分钟
    3. 将3μlUSER酶加入到连接混合物中,并在37℃的热循环仪中加热15分钟
    4. 适用于400-500bp文库产品(300-400bp插入片段)的AmPure小珠大小选择
      1. 向连接反应物中加入13.5μl无核酸酶的H 2 O,终体积为100μl。
      2. 加入40μlAmPure珠,并通过上下移液充分混合。
      3. 在室温下孵育5分钟
      4. 离心管短暂。放在磁铁上并在室温下孵育5分钟(直到清除)
      5. 将上清液转移到干净的管中。
      6. 加入20μlAmPure珠,并充分混匀。在室温下孵育5分钟
      7. 离心管短暂。放在磁铁上,在室温下孵育5分钟
      8. 将管放在磁铁上,取出并弃去上清液,小心不要脱落珠粒。
      9. 用磁铁管,加入200μl新鲜制备的80%EtOH,并在室温下孵育30秒
      10. 用磁铁上的管子,去除EtOH洗涤液。
      11. 重复步骤E4i和E4j两次,共3次洗涤。
      12. 空气干燥珠在RT上10分钟,磁头上的管顶部打开。
      13. 从磁铁中取出管,加入28μlTE低浓度EDTA,pH8至珠子,并通过旋转或通过移液进行良好的混合重新悬浮。
      14. 短暂离心管,更换磁铁上的管,并在室温下孵育5分钟,或直到清除。
      15. 收集23μlDNA并转移到干净的PCR管中。确保不要转移任何珠子。
    5. PCR扩增
      1. 将以下内容添加到每个管中:


      2. 在热循环仪中:

    6. PCR清除
      1. 加入50μlAmPure珠,混匀。
      2. 以RT×5分钟孵育
      3. 将离心管短暂放置在磁铁上,并在室温下孵育3-5分钟
      4. 将管放在磁铁上,除去上清液,小心不要脱落珠子。
      5. 用磁铁管,加入200μl新鲜制备的80%EtOH,并在室温下孵育30秒
      6. 用磁铁上的管子,去除EtOH洗涤液。
      7. 重复步骤E6e和E6f一次。
      8. 空气干珠在室温下10分钟。
      9. 从磁铁中取出管,并加入33μlTE低EDTA至珠粒并重新悬浮。在室温下孵育2分钟。
      10. 更换磁铁上的管,并在室温下孵育4分钟
      11. 收集30μlDNA,并转移到干净的PCR管中
    7. 根据TapeStation HS DNA方案测定TapeStation HS DNA上的文库产量和质量。
    8. 池样品(〜12个样品/运行,等摩尔)至最终浓度为10 nM,并验证TapeStation HS DNA上的浓度。
    9. 根据设施协议和建议提交Illumina Miseq(我们使用负载浓度7 pM,1%PhiX spike-in,Std流通池,2 x 250 bp运行)。
      注意:此协议也适用于Illumina Hiseq。

数据分析

序列分析方法由于硬件速度和软件编码的进步而迅速发展。许多序列处理软件工具是开源的(例如BBTools, http://jgi.doe.gov/data-and-tools/bbtools/),以及R中的统计分析和图表包( https://www.r-project.org/)。我们使用VirusSeeker(Zhao等人,2017年),一种基于VirusHunter(Zhao等人,2013)的定制自动化生物信息学管道,以检测共享核苷酸和氨基酸的序列酸序列与已知病毒的相似性(图1)。我们建议使用严格的病毒序列鉴定方案,例如VirusSeeker,可以去除低质量序列,重复序列和非特异性病毒"命中"。同样严格的方法也确定了新型病毒序列(Zhao等,/em>。,2013)。分析协议中的基本步骤如图1所示。在从NCBI数据库中下载与所有病毒基因组相对应的序列后,可以生成自定义病毒数据库(记录下载的日期,因为频繁添加新序列)。推荐使用重复数据删除来最大限度地减少放大偏差,并且分类群分配的序列应归一化以考虑采样间序列深度的变化。应使用特异于病毒序列的引物,通过实时qPCR来验证新的病毒序列或感兴趣的病毒序列。另外,序列可以从头开始组装成更长的重叠群,并与NCBI nr/nt数据库进行比较,以更好地鉴定感兴趣的病毒序列的系统发育(Monaco等人,2016年) )。将感兴趣的序列与已知的相关病毒序列进行比较的系统发生树可以使用诸如FigTree的免费软件( http://tree.bio.ed.ac。 uk/software/figtree/)。


图1.序列分析原理图

笔记

进行粪便粉碎以均匀分布样品中的微生物物质。使用含有明胶的SM缓冲液在冷冻后稳定噬菌体群体,以进一步表征感兴趣的噬菌体,包括培养。由于污染风险,试剂应仅用于粪便微生物组研究。对于所有Illumina Miseq运行使用相同的试剂( ie ,同一瓶无菌水,试剂盒,等等),许多试剂可能被微生物DNA污染,确保跨运行均匀的污染。

食谱

  1. CTAB/NaCl
    3.5ml 5M NaCl(最终浓度0.7M)
    2.5g CTAB(最终浓度为10%)
    12毫升无核酸酶的H 2 O
    最终体积:25ml
    加热至55°C溶解
    加水至最终体积为25 ml。然后是0.45μm,然后是0.22μm的滤色片

致谢

CLM由NIH培训授权5T32AI007172-34支持,该工作由R24 ODO19793,R01 OD011170,R01 AI111918和R01 DK101354资助。伯克维尔康康基金支持DSK。粪便粉碎方案适用于圣路易斯华盛顿大学的Jeffrey Gordon实验室慷慨提供的方案。 VLP方案从用于噬菌体分离的方案(Reyes等人,2013)中改编。我们要感谢布莱恩·凯勒博士,博士D对本手稿的批评性评论。

参考文献

  1. Duerkop,BA和Hooper,LV(2013)。居民病毒及其与免疫系统的相互作用。 Nat Immunol 14(7):654-659。
  2. Handley,SA,Thackray,LB,Zhao,G.,Presti,R.,Miller,AD,Droit,L.,Abbink,P.,Maxfield,LF,Kambal,A.,Duan,E.,Stanley, ,Kramer,J.,Macri,SC,Permar,SR,Schmitz,JE,Mansfield,K.,Brenchley,JM,Veazey,RS,Stappenbeck,TS,Wang,D.,Barouch,DH和Virgin,HW(2012) 。病原性猿猴免疫缺陷病毒感染与肠道病毒。 细胞 151(2):253-266。
  3. Mokili,JL,Rohwer,F.和Dutilh,BE(2012)。  Metagenomics和病毒发现中的未来前景。 Curr Opin Virol 2(1):63-77。
  4. 摩纳哥,CL,Gootenberg,DB,Zhao,G.,Handley,SA,Ghebremichael,MS,Lim,ES,Lankowski,A.,Baldridge,MT,Wilen,CB,Flagg,M.,Norman,JM,Keller,BC ,Luevano,JM,Wang,D.,Boum,Y.,Martin,JN,Hunt,PW,Bangsberg,DR,Siedner,MJ,Kwon,DS and Virgin,HW(2016)。< a class = -insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/26962942"target ="_ blank">在人类免疫缺陷病毒相关获得性免疫缺陷综合征中改变的病毒和细菌微生物组合 < em> Cell Host Microbe 19(3):311-322。
  5. Norman,JM,Handley,SA,Baldridge,MT,Droit,L.,Liu,CY,Keller,BC,Kambal,A.,Monaco,CL,Zhao,G.,Fleshner,P.,Stappenbeck,TS,McGovern, DP,Keshavarzian,A.,Mutlu,EA,Sauk,J.,Gevers,D.,Xavier,RJ,Wang,D.,Parkes,M.and Virgin,HW(2015)。< a class = -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25619688"target ="_ blank">炎症性肠病肠道病毒中的疾病特异性改变 单元格 160(3):447-460。
  6. Reyes,A.,Wu,M.,McNulty,NP,Rohwer,FL和Gordon,JI(2013)。  哺乳动物生理学中的病毒和疾病。 细胞 157(1):142-150。
  7. Zhao,G.,Krishnamurthy,S.,Cai,Z.,Popov,VL,Travassos da Rosa,AP,Guzman,H.,Cao,S.,Virgin,HW,Tesh,RB and Wang,D。(2013) 。使用VirusHunter识别新病毒 - 一种自动数据 分析流水线。 PLoS One 8(10):e78470。
  8. Zhao,G.,Wu,G.,Lim,ES,Droit,L.,Krishnamurthy,S.,Barouch,DH,Virgin,HW和Wang,D。(2017)。< a class = insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/28110145"target ="_ blank"> VirusSeeker,用于病毒发现和病毒组成分析的计算流程。 病毒学 503:21-30。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Monaco, C. L. and Kwon, D. S. (2017). Next-generation Sequencing of the DNA Virome from Fecal Samples. Bio-protocol 7(5): e2159. DOI: 10.21769/BioProtoc.2159.
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