TITLE: Discovery of RNA and DNA viruses using next-generation sequencing: Metagenomics
AUTHORS: Lily Tong, Katherine Smollett, Jenna Nichols, Kirsty Kwok, Kyriaki Nomikou, Ma. Jowina Galarion, Daniel Mair, Ana Filipe 

[DESCRIPTION]
Next-generation sequencing is a powerful tool for viral genomics. Viruses often constitute a very small proportion of any given sample meaning that methods that enable detection of viral nucleic acids are frequently needed for detection and characterisation. Improvement of sensitivity can be achieved by depletion of unwanted nucleic acid during sample pre-treatment or by enrichment such as PCR amplification with virus specific primers, or probe-based targeted enrichment. However, some methods for specific enrichment rely on prior knowledge of the viruses. Here we describe a method for next-generation sequencing to identify unknown viruses (see Figure 1). 
    Following simultaneous extraction of RNA and DNA samples are split into two and subjected to non-specific treatments that deplete host nucleic acids and improve chances of detecting RNA or DNA viruses, respectively. Illumina sequencing libraries are then prepared and these metagenomic libraries can directly sequenced. These sequencing libraries can then be subjected to targeted enrichment using a pan-viral probe set to achieve higher sensitivity (for details see accompanying protocol Discovery of RNA and DNA viruses using next-generation sequencing: Targeted enrichment).

We applied this approach to an outbreak of acute hepatitis of unknown aetiology in children, enabling the identification of adeno-associated virus 2 (AAV2) in all patients but not in samples from controls. This method also led to the identification of adenovirus and human herpesviruses.

This protocol describes the pre-treatment of nucleic acid extracts to improve detection of DNA and RNA viral reads followed by Illumina library preparation and metagenomic sequencing.  

[BEFORE_START]
This protocol starts with nucleic acid extracts.

[STEPS]
SECTION: Initial sample preparation
2. Split each nucleic acid extract into two subsamples for RNA and DNA virus detection.
SECTION: Initial sample preparation
2.1. If required, make up each sample to 50 µL with Nuclease-free water.
SECTION: Initial sample preparation
2.2. Prepare two 0.2 mL PCR tubes per sample labelled with R (for the RNA pre-treatment) or D (for DNA pre-treatment) along with the sample names.  
 

SECTION: Initial sample preparation
2.3. Add 25 µL to the tube R and 25 µL to tube D.  

Subsample R  → proceed to RNA virus detection - DNase I treatment (Step 3).
Subsample D → proceed to DNA virus detection - Microbiome enrichment (step 14).
 


SECTION: RNA virus detection - DNase I treatment
3. Prepare DNase I mix as follows (for multiple samples prepare a master mix with 10% excess): 

 

  


 


SECTION: RNA virus detection - DNase I treatment
3.1. Add 5 µL to 25 µL.
SECTION: RNA virus detection - DNase I treatment
3.2. Incubate as follows:

37 °C for 15 min then place on ice.  
SECTION: RNA virus detection - DNase I treatment
4. Perform clean-up with 2X volume of RNAclean XP magnetic beads.

  

 

SECTION: RNA virus detection - DNase I treatment
4.2. Incubate at Room temperature for 5 min. 

SECTION: RNA virus detection - DNase I treatment
4.3. Place on a magnetic rack until beads and solution have fully separated.
SECTION: RNA virus detection - DNase I treatment
4.4. Carefully remove supernatant being careful not to disturb the beads.
SECTION: RNA virus detection - DNase I treatment
4.5. Wash 2X with 200 µL. 
 


SECTION: RNA virus detection - DNase I treatment
4.6. Remove all traces of ethanol and air dry for up to 5 min.
 


SECTION: RNA virus detection - DNase I treatment
4.7. To elute DNase I treated RNA add 12 µL and incubate at Room temperature for at least 2 min
SECTION: RNA virus detection - DNase I treatment
4.1. Add 60 µL  to the 30 µL and mix by pipetting.
SECTION: RNA virus detection - Ribosomal depletion
5. Transfer 10 µL to fresh 0.2 mL tubes/plate for Ribo-zero probe hybridisation.


  

 

SECTION: RNA virus detection - Ribosomal depletion
5.1. Thaw DB1 and DP1 at Room temperature , vortex to mix and centrifuge briefly.

SECTION: RNA virus detection - Ribosomal depletion
5.2. Prepare the hybridisation probe mix on ice (for multiple samples prepare a master mix with 10% excess):    

 

SECTION: RNA virus detection - Ribosomal depletion
5.4. Add 4 µL to each 10 µL and fully mix by pipetting 10 times.

SECTION: RNA virus detection - Ribosomal depletion
5.5. Incubate samples as follows:  
95 °C for 2 min 
Decrease 0.1 °C  until temperature reaches 37 °C  then hold.

 


SECTION: RNA virus detection - Ribosomal depletion
6. Prepare rRNA Depletion.
SECTION: RNA virus detection - Ribosomal depletion
6.1. Thaw RDB and RDE at Room temperature , vortex or flick (RDE) to mix and centrifuge briefly.

SECTION: RNA virus detection - Ribosomal depletion
6.2. Prepare the rRNA Depletion mix (for multiple samples prepare a master mix with 10% excess):     

 

SECTION: RNA virus detection - Ribosomal depletion
6.4. Add 5 µL to each 14 µL and fully mix by pipetting 10 times.

SECTION: RNA virus detection - Ribosomal depletion
6.5. Incubate samples as follows:  
37 °C for 15 min 
4 °C 


SECTION: RNA virus detection - Ribosomal depletion
7. Prepare probe removal.
SECTION: RNA virus detection - Ribosomal depletion
7.1. Thaw PRB and PRE at Room temperature , vortex (PRB) or flick (PRE) to mix and centrifuge briefly.

SECTION: RNA virus detection - Ribosomal depletion
7.2. Prepare the Probe Removal mix on ice  (for multiple samples prepare a mastermix with 10% excess):   


 

  
SECTION: RNA virus detection - Ribosomal depletion
7.4. Add 10 µL  to each 19 µL and fully mix by pipetting 10 times.

SECTION: RNA virus detection - Ribosomal depletion
7.5. Incubate samples as follows:  
37 °C for 15 min 
70 °C for 15 min 
4 °C  


SECTION: RNA virus detection - Ribosomal depletion
8. Perform clean-up with 2X volume of RNAclean XP magnetic beads.

  

 

SECTION: RNA virus detection - Ribosomal depletion
8.1. Add 60 µL to the 30 µL , mix by pipetting.

SECTION: RNA virus detection - Ribosomal depletion
8.2. Incubate at Room temperature for 5 min
SECTION: RNA virus detection - Ribosomal depletion
8.3. Place on a magnetic rack until beads and solution have fully separated.
SECTION: RNA virus detection - Ribosomal depletion
8.4. Remove and discard supernatant.
SECTION: RNA virus detection - Ribosomal depletion
8.5. Wash 2X with 175 µL.

SECTION: RNA virus detection - Ribosomal depletion
8.6. Remove all traces of ethanol and air dry for up to 2 min 

 

SECTION: RNA virus detection - Ribosomal depletion
8.7. Elute in 12 µL by incubating at Room temperature for 2 min.
SECTION: RNA virus detection - Low input reverse transcription
9. Transfer 10 µL to fresh 0.2 mL tubes/plates.

 


SECTION: RNA virus detection - Low input reverse transcription
10. Prepare NTP/Hex mix (for multiple samples prepare a master mix with 10% excess):     

 
  
  

SECTION: RNA virus detection - Low input reverse transcription
10.1. Add 2 µL  to each 10 µL.

SECTION: RNA virus detection - Low input reverse transcription
10.2. Incubate as follows:  
65 °C for 5 min 
immediately place on ice 
SECTION: RNA virus detection - Low input reverse transcription
11. Prepare the SSIII master mix (for multiple samples prepare a master mix with 10% excess): 


 

  
  

SECTION: RNA virus detection - Low input reverse transcription
11.1. Add 8 µL  to each 12 µL.
SECTION: RNA virus detection - Low input reverse transcription
12. Prepare second strand mix (for multiple samples prepare a mastermix with 10% excess):    


 

  

SECTION: RNA virus detection - Low input reverse transcription
12.1. Add 60 µL to each 20 µL
SECTION: RNA virus detection - Low input reverse transcription
12.2. Incubate as follows on PCR machine  
16 °C for 150 min 
4 °C 
SECTION: RNA virus detection - Low input reverse transcription
11.2. Incubate samples as follows:  
25 °C  for 10 min 
55 °C  for 60 min 
70 °C  for 15 min 
4 °C 

SECTION: RNA virus detection - Low input reverse transcription
13. Perform clean up with 1X volume of AmPure XP beads.

  

 


SECTION: RNA virus detection - Low input reverse transcription
13.1. Add 80 µL  to each 80 µL (1:1 Ampure:sample ratio) and mix well.

SECTION: RNA virus detection - Low input reverse transcription
13.2. Incubate at Room temperature for 5 min.

SECTION: RNA virus detection - Low input reverse transcription
13.3. Place on a magnetic rack until beads and solution have fully separated.
SECTION: RNA virus detection - Low input reverse transcription
13.4. Remove supernatant.
SECTION: RNA virus detection - Low input reverse transcription
13.5. Wash 2X with 200 µL.

SECTION: RNA virus detection - Low input reverse transcription
13.6. Remove all traces of ethanol and air dry for 5 min.

SECTION: RNA virus detection - Low input reverse transcription
13.7. Elute in 27 µL by incubation at Room temperature for 2 min.


 


cDNA → proceed to Section – Library prep (step 22) or store until DNA samples ready so can process together.

SECTION: DNA virus detection - Microbiome enrichment
14. Pre-bind MBD2-Fc Protein to Magnetic Beads.

  

See following attachment for reagent calculations:
  
 

SECTION: DNA virus detection - Microbiome enrichment
14.1. Pipette 1 µL  (see column B of reagent calculation table) for every 6.25 ng  into a 1.5 mL DNA LoBind tube. 
 

 


SECTION: DNA virus detection - Microbiome enrichment
14.2. Add 0.1 V (see column C of reagent calculation table) to the Protein A magnetic beads.

SECTION: DNA virus detection - Microbiome enrichment
14.3. Mix the bead-protein mixture by placing the tube in a rotating mixer for 10 min.

SECTION: DNA virus detection - Microbiome enrichment
14.4. Prepare the 1X Bind/Wash buffer and keep it on ice:     

 

SECTION: DNA virus detection - Microbiome enrichment
14.5. After the incubation, briefly spin the tube and place on the magnetic rack for 5 min  until the beads have collected.

SECTION: DNA virus detection - Microbiome enrichment
14.6. Remove the supernatant with a pipette without disturbing the beads.
SECTION: DNA virus detection - Microbiome enrichment
14.7. Add 1 mL  to the tube to wash the beads. Pipet up and down a few times to mix.

SECTION: DNA virus detection - Microbiome enrichment
14.8. Mix the beads on a rotating mixer for 3 min at Room temperature.
SECTION: DNA virus detection - Microbiome enrichment
14.9. Briefly spin the tube and place on the magnetic rack for 5 min  until the beads have collected.
SECTION: DNA virus detection - Microbiome enrichment
14.10. Remove the supernatant with a pipette without disturbing the beads.
SECTION: DNA virus detection - Microbiome enrichment
14.11. Repeat wash step (2 washes in total).

SECTION: DNA virus detection - Microbiome enrichment
14.12. Resuspend the beads in the volume of 1X Bind/Wash buffer equal to the initial magnetic bead volume in step 13.1 (see sum of column B of reagent calculation table).
SECTION: DNA virus detection - Microbiome enrichment
15. Capture Methylated Host DNA.
SECTION: DNA virus detection - Microbiome enrichment
15.1. Add appropriate volume of 5X Bind/Wash buffer to fresh tubes for each sample to give a 1X solution (see column D of reagent calculation table).
SECTION: DNA virus detection - Microbiome enrichment
15.2. Add the volume of sample to give up to 1 µg (see column A of reagent calculation table).

 

 

SECTION: DNA virus detection - Microbiome enrichment
15.3. Add appropriate volume of washed Fc-bead/protein mix (see column B of reagent calculation table) to the DNA/buffer mix.
SECTION: DNA virus detection - Microbiome enrichment
15.4. Mix and incubate in a rotating mixer at RT for 15 min to 240 min depending on sample type .

 

 

SECTION: DNA virus detection - Microbiome enrichment
16. Elute Microbiome DNA.
SECTION: DNA virus detection - Microbiome enrichment
16.1. Briefly centrifuge and place on a magnet for at least 5 min to separate the bead-bound host DNA.

SECTION: DNA virus detection - Microbiome enrichment
16.2. Remove the supernatant containing host depleted/microbiome enriched DNA to fresh tubes.  

 


 

SECTION: DNA virus detection - sonication
17. If required make sample up to 55 µL.
SECTION: DNA virus detection - sonication
18. Prepare sonicator for use.

 

 

SECTION: DNA virus detection - sonication
18.1. Fill the tank with water to FILL level -2.
SECTION: DNA virus detection - sonication
18.2. Switch on the chiller and ensure set to 7 °C.
SECTION: DNA virus detection - sonication
18.3. Switch on the water conditioning system, the Covaris and the computer.
SECTION: DNA virus detection - sonication
18.4. Open the SonoLab software.
SECTION: DNA virus detection - sonication
18.5. Select Home and the transducer will get submerged, the degas pump should start automatically.
SECTION: DNA virus detection - sonication
18.6. Degas the water bath for ~45 min.
SECTION: DNA virus detection - sonication
19. Set up the sonication conditions as follows:

 

 


SECTION: DNA virus detection - sonication
20. Sonicate samples.
SECTION: DNA virus detection - sonication
20.1. Add 55 µL to either the 8 microTUBE-50 AFA Fiber Strip V2 or 96 microTUBE AFA Fiber Plate Thin Foil.

SECTION: DNA virus detection - sonication
20.2. Place the strip/plate in the appropriate holder and screw into place (ensure that it is set up the same as the program conditions).
SECTION: DNA virus detection - sonication
20.3. Select Load position to move the support arm forward.
SECTION: DNA virus detection - sonication
20.4. Press the green button and open the door to put the holder into the support arm, ensure in the correct orientation and close the door.
SECTION: DNA virus detection - sonication
20.5. Press start position to submerge the rack and confrim the correct volume of water has been added (samples should NOT be fully submerged).
SECTION: DNA virus detection - sonication
20.6. Press Run to start the sonication.
SECTION: DNA virus detection - sonication
20.7. Once completed press Load Position to remove the plate.
SECTION: DNA virus detection - sonication
20.8. Transfer 50 µL to PCR tubes. 

 
  
  

 

SECTION: DNA virus detection - sonication
21. 1.4X Ampure clean up.
  

SECTION: DNA virus detection - sonication
21.1. Add 70 µL  to the samples (ratio 1.4:1).

SECTION: DNA virus detection - sonication
21.2. Incubate at Room temperature for 15 min.
SECTION: DNA virus detection - sonication
21.3. Place on a magnetic rack until beads and solution have fully separated.
SECTION: DNA virus detection - sonication
21.4. Remove supernatant.
SECTION: DNA virus detection - sonication
21.5. Wash 2X with 200 µL.
SECTION: DNA virus detection - sonication
21.6. Remove all traces of Ethanol. Air dry for 5 min.

SECTION: DNA virus detection - sonication
21.7. Elute samples in 25 µL.
SECTION: DNA virus detection - sonication
21.8. Transfer 25 µL to new tubes. 

 
Sheared DNA → proceed to Section – Library prep (step 22) can process alongside the prepared cDNA samples.
SECTION: Illumina sequencing library preparation
22. Prepare the End Repair mix (for multiple samples prepare a master mix with 10% excess):     

 


 

 
  

SECTION: Illumina sequencing library preparation
22.1. Add 5 µL  to each 25 µL.
SECTION: Illumina sequencing library preparation
22.2. Incubate as follows (if using a PCR machine do not use hot lid):  
20 °C for 30 min 
SECTION: Illumina sequencing library preparation
23. 1.4X Ampure XP clean up.
  

SECTION: Illumina sequencing library preparation
23.1. Add 20 µL increase volume of each sample to a total of 50 µL.
SECTION: Illumina sequencing library preparation
23.2. Add 70 µL  (1.4:1 Ampure:sample ratio). Pipette up and down to mix.

SECTION: Illumina sequencing library preparation
23.3. Incubate at Room temperature for 5 min. 
SECTION: Illumina sequencing library preparation
23.4. Place samples on a magnetic rack until beads and solution have fully separated.
SECTION: Illumina sequencing library preparation
23.5. Remove supernatant.
SECTION: Illumina sequencing library preparation
23.6. Wash 2X with 200 µL.
SECTION: Illumina sequencing library preparation
23.7. Remove all traces of ethanol and air dry for 5 min.

SECTION: Illumina sequencing library preparation
23.8. Elute in 21 µL  leaving beads in solution.

SECTION: Illumina sequencing library preparation
24. 13.  Prepare the A-Tail mix (for multiple samples prepare a master mix with 10% excess):     


 
  

SECTION: Illumina sequencing library preparation
24.1. Add 4 µL  to each 21 µL.

SECTION: Illumina sequencing library preparation
24.2. Incubate as follows:
30 °C   for  60 min
SECTION: Illumina sequencing library preparation
25. 1.4X SPRI clean up.

 

SECTION: Illumina sequencing library preparation
25.1. Add 25 µL  to increase volume of each sample to a total of 50 µL.

SECTION: Illumina sequencing library preparation
25.2. Add 70 µL (1.4:1 SPRI:sample ratio). Pipette up and down to mix.  

 
SECTION: Illumina sequencing library preparation
25.3. Incubate at Room temperature for 5 min.
SECTION: Illumina sequencing library preparation
25.4. Place the samples on a magnetic rack until beads and solution have fully separated.
SECTION: Illumina sequencing library preparation
25.5. Remove supernatant.
SECTION: Illumina sequencing library preparation
25.6. Wash 2X with 200 µL.
SECTION: Illumina sequencing library preparation
25.7. Remove all traces of ethanol and air dry for 5 min.
SECTION: Illumina sequencing library preparation
25.8. Elute in 15 µL  leaving beads in solution.

SECTION: Illumina sequencing library preparation
26. Quantify 1 µL  using Qubit high sensitivity dsDNA.

  

 

SECTION: Illumina sequencing library preparation
26.1. Calculate the amount of pmol per in each 14 µL as follows (alternatively use the calculation in the attached sheet):

  
 

  

SECTION: Illumina sequencing library preparation
27. Prepare the Ligation mix (for multiple samples prepare a master mix with 10% excess):   

 
    

SECTION: Illumina sequencing library preparation
27.1. Add 7.5 µL  to 14 µL (ensure remains on the beads).

SECTION: Illumina sequencing library preparation
27.2. Add 5 µL.

SECTION: Illumina sequencing library preparation
27.3. Incubate as follows (if using a PCR machine ensure the hot lid is not turned on):  
20 °C for 60 min. 
SECTION: Illumina sequencing library preparation
28. Add 0.75 µL  to each tube.


 
 
  

SECTION: Illumina sequencing library preparation
29. 0.9X SPRI clean up.
SECTION: Illumina sequencing library preparation
29.1. 
Add 25 µL to make volume up to 50 µL.

SECTION: Illumina sequencing library preparation
29.3. Incubate at Room temperature for 5 min.

SECTION: Illumina sequencing library preparation
29.4. Place on a magnetic rack until beads and solution have fully separated.
SECTION: Illumina sequencing library preparation
29.5. Remove supernatant.
SECTION: Illumina sequencing library preparation
29.6. Wash 2X with 200 µL.
SECTION: Illumina sequencing library preparation
29.7. Remove all traces of ethanol and air dry for up to 5 min.
 

SECTION: Illumina sequencing library preparation
29.8. Elute samples in 22 µL.
SECTION: Illumina sequencing library preparation
29.9. Transfer 20 µL  to new freah 0.2 mL PCR tubes.

SECTION: Illumina sequencing library preparation
30. Add 5 µL each sample.

 
 
  

SECTION: Illumina sequencing library preparation
30.1. Add 25 µL  to each sample.

 
    

SECTION: Illumina sequencing library preparation
30.2. Incubate as follows:  
98 °C for 45 s 
4-20* cycles of  
98 °C for 15 s 
65 °C for 30 s   
72 °C for 30 s
 Final cycle of  
72 °C for 1 min 
4 °C 4oC hold  
 

 


 



SECTION: Illumina sequencing library preparation
26.2. Calculate the amount of adapter required (aim for 20:1 ratio adapter to sample - see calculation sheet).

 

  
  

SECTION: Illumina sequencing library preparation
26.3. Dilute adapter in water to achieve appropriate concentration in a total volume of 5 µL  per reaction.

 

SECTION: Illumina sequencing library preparation
28.1. Step 15 Incubate as follows:  
37 °C for 15 min 
4 °C 
SECTION: Illumina sequencing library preparation
29.2. Add 45 µL to the samples (ratio 0.9:1).
SECTION: Illumina sequencing library preparation
31. Clean up and QC the libraries as in protocol Library clean up and quality control for Illumina sequencing.
SECTION: Metagenomic sequencing
32. Metagenomic sequencing can speed up identification of any viruses in the samples and, depending on the virus and viral load, may be suficient to generate full genomes.
SECTION: Metagenomic sequencing
34. Pool the libraries by equal molarity and QC the pools as described in the protocol Library pooling and quality control for Illumina sequencing.

 

SECTION: Metagenomic sequencing
35. Sequence the pools on an Illumina sequencer following the manufacturer's guidelines.

 

SECTION: Metagenomic sequencing
33. Using the bp size and ng/μl concentration calculate the nM concentration for each library as follows:

  

SECTION: RNA virus detection - Ribosomal depletion
5.3. Thoroughly pipette mix.
SECTION: RNA virus detection - Ribosomal depletion
6.3. Thoroughly pipette mix.
SECTION: RNA virus detection - Ribosomal depletion
7.3. Thoroughly pipette mix.
SECTION: Initial sample preparation
1. Quantify the DNA and RNA concentration of your samples using Qubit HS reagents.