TITLE: Discovery of RNA and DNA viruses using next-generation sequencing: Targeted enrichment
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. The development of probe-capture panels targeting multiple viruses have enabled simultaneous sequencing of multiple viruses. Here we describe a highly sensitive and semi-agnostic sequencing method to identify unknown viruses using a pan-viral probe capture design (see Figure 1). 
    
Following simultaneous extraction of RNA and DNA, samples are first split into two and subjected to non-specific enrichment treatments that improve chances of detecting RNA or DNA viruses, respectively and generate untargeted Illumina sequencing libraries as described in the accompanying protocol Discovery of RNA and DNA viruses using next-generation sequencing: Metagenomics. The same sequencing libraries can be subjected to targeted enrichment using a pan-viral probe set to achieve higher sensitivity. 

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 how to perform targeted enrichment on metagenomic Illumina sequencing libraries.  We enrich for unknown viruses using VirCapSeq-VERT probes, a panel of ~2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates. 

[BEFORE_START]
This protocol starts with DNA and RNA metagenomic Illumina sequencing libraries prepared as described in protocol Discovery of RNA and DNA viruses using next-generation sequencing: Metagenomics.


[STEPS]
SECTION: Hybridisation
1. Prepare enrichment pools from the pre-prepared Illumina metagenomic sequencing libraries. Each pool should contain 8-16 libraries equal ng of each and a total of 1 μg DNA in a 1.5 mL DNA LoBind tube.     
 


SECTION: Hybridisation
3. To each pool add the following blocking reagents:    
 
 
  
  
   
SECTION: Hybridisation
4. Concentrate the pool using Ampure XP.
  

 

 

SECTION: Hybridisation
4.1. Add 2X total volume of the pool plus blocking reagent of AmpureXP.

SECTION: Hybridisation
4.2. Place on a magnetic rack until beads and solution have fully separated 5 min.
SECTION: Hybridisation
4.3. Remove supernatant being careful not to disturb the beads.
SECTION: Hybridisation
4.4. Add 800 µL and incubate Room temperature for 1 min.

SECTION: Hybridisation
4.5. Remove all traces of ethanol being careful not to disturb the beads.
SECTION: Hybridisation
4.6. Air-dry the beads for around 3 min taking care not to over dry the beads.

SECTION: Hybridisation
5. Prepare the hybridisation mix (for multiple samples prepare a master mix with 10% excess):  
 
 
  

SECTION: Hybridisation
6. Add 10.5 µL directly to the bead-bound DNA samples, remove from magnet and mix thoroughly.

SECTION: Hybridisation
7. Incubate at Room temperature for 2 min.

SECTION: Hybridisation
8. Place on magnetic rack and elute the entire 10.5 µL to a new 0.2 mL PCR tube tube containing 4.5 µL.

 

 

SECTION: Hybridisation
9. Mix thoroughly by pipetting.
SECTION: Hybridisation
10. Incubate as follows on a PCR machine with lid set to 105 °C :  
95 °C for 5 min 
cool to 47 °C

SECTION: Hybridisation
11. Quickly transfer to second PCR machine with lid set to 57 °C and incubate as follows:
47 °C for 4320 min 

 


SECTION: Capture and washing
12. Prepare the wash buffers per capture as follows:
 
  

SECTION: Capture and washing
13. Transfer tubes A and B 200 µL and tube C 100 µL to the PCR machine to equilabrate to 47 °C.

SECTION: Capture and washing
14. Prepare capture beads.
SECTION: Capture and washing
14.1. For each capture, place 100 µL in a 1.5 mL tube. 

 
  

SECTION: Capture and washing
14.2. Place tube on a magnet, remove liquid being careful not to disturb the beads.
SECTION: Capture and washing
14.3. Add 2x the initial volume of beads of bead wash buffer (tube G).
SECTION: Capture and washing
14.4. Remove from magnet, vortex for 10 s then centrifuge briefly.
SECTION: Capture and washing
14.5. Place tube on a magnet, remove liquid.
SECTION: Capture and washing
14.6. Repeat bead wash one more time (2 washes in total).
SECTION: Capture and washing
14.7. Re-suspend beads in 1x original volume of bead wash buffer (tube G) by vortexing.
SECTION: Capture and washing
15. Transfer 100 µL per capture to a fresh 0.2 mL PCR tube. 
SECTION: Capture and washing
16. Place tube on a magnet, remove liquid and proceed immediately to next so that the beads do not dry out.
SECTION: Capture and washing
17. Immediately add the 15 µL to the prepared capture beads. Mix by pipetting ten times.

SECTION: Capture and washing
18. Incubate in a PCR machine for 45 min at 47 °C, with the heated lid set to 57 °C.

 

SECTION: Capture and washing
19. Add 100 µL pre-heated to 47 °C  (tube C).

SECTION: Capture and washing
20. Mix by vortexing for 10 s.

SECTION: Capture and washing
21. Place tube on a magnet, remove liquid.

 

SECTION: Capture and washing
22. Add 200 µL pre-heated to 47 °C (tube A). Pipette 10X to mix.

SECTION: Capture and washing
23. Incubate at 47 °C for 5 min.

SECTION: Capture and washing
24. Repeat stringent wash one more time (tube B, total 2 washes).
SECTION: Capture and washing
25. Transfer mixture to a fresh 1.5 mL DNA LoBind tube.

 

SECTION: Capture and washing
26. Place tube on a magnet, remove liquid.
SECTION: Capture and washing
27. Add 200 µL at Room temperature (tube D) and vortex for 2 min.
SECTION: Capture and washing
28. Place tube on a magnet, remove liquid.
SECTION: Capture and washing
29. Add 200 µL at Room temperature (tube E) and vortex for 1 min.

SECTION: Capture and washing
30. Place tube on a magnet, remove liquid.
SECTION: Capture and washing
31. Add 200 µL at Room temperature  (tube F) and vortex for 30 s.
SECTION: Capture and washing
32. Place tube on a magnet, remove liquid.
SECTION: Capture and washing
33. Remove from magnet, resuspend the beads in 20 µL and mix well by pipetting.
 
  
SECTION: Amplification
34. Prepare the PCR mix (for multiple samples prepare a master mix with 10% excess):   
 
  

SECTION: Amplification
35. Set up two PCR tubes per capture and add 15 µL to each tube.

SECTION: Amplification
36. Briefly vortex bead-bound captured DNA from step 33 and spin down.

SECTION: Amplification
37. Add 10 µL to each PCR reaction tube.  

 

SECTION: Amplification
38. Incubate on a PCR machine as follows:
95 °C for 3 min 
14 cycles of
98 °C for 20 s 
65 °C for 15 s 
72 °C for 30 s 
Final cycle of
72 °C for 2 min 
4 °C .

 

SECTION: Amplification
39. Briefly centrifuge PCR reactions and place on magnetic rack until the beads and solution have fully separated. 
SECTION: Amplification
40. Transfer the 25 µL into fresh tubes, combining the 2 reactions from each pool to make a total volume of 50 µL.

SECTION: Amplification
41. Pools can be cleaned up and undergo quality control as described for single libraries in protocol Library clean up and  quality control for Illumina sequencing. 
SECTION: Hybridisation
2. Enrichment is performed with VirCapSeq-VERT probes and Roche SeqCap reagents.
    
  
 
  

SECTION: Pooling and sequencing
42. Using the bp size and ng/μl concentration calculate the nM concentration for each pool as follows:

  

SECTION: Pooling and sequencing
43. If multiple pools are to be combined in the same sequencing run then pool by equal molarity with each pool weighted by the number of sequencing libraries contained within it as described in the protocol Library pooling and  quality control for Illumina sequencing.
SECTION: Pooling and sequencing
44. Sequence the pools on an Illumina sequencer following the manufacturer's guidelines.