Discovery of RNA and DNA viruses using next-generation sequencing: Metagenomics
Katherine Smollett, Lily Tong, Jenna Nichols, Kirsty Kwok, Kyriaki Nomikou, Ma. Jowina Galarion, Daniel Mair, Ana Filipe
Viral genomics
Next-generation sequencing
viral discovery
Illumina
Metagenomics
RNA
DNA
Untargeted detection of viruses
Disclaimer
Abstract
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
Initial sample preparation
Quantify the DNA and RNA concentration of your samples using Qubit HS reagents.
Equipment
| Value | Label |
|---|---|
| Qubit | NAME |
| Flurometer | TYPE |
| Invitrogen | BRAND |
| Q33228 | SKU |
Split each nucleic acid extract into two subsamples for RNA and DNA virus detection.
If required, make up each sample to 50µL with Nuclease-free water.
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.
Add 25µL to the tube R and 25µL 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).
RNA virus detection - DNase I treatment
Prepare DNase I mix as follows (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| 10X DNase I buffer | 3 |
| DNase I | 2 |
| Total | 5 |
Add 5µL to 25µL.
Incubate as follows:
37°C for 0h 15m 0s then place On ice.
Perform clean-up with 2X volume of RNAclean XP magnetic beads.
Add 60µL to the 30µL and mix by pipetting.
Incubate at Room temperature for 0h 5m 0s.
Place on a magnetic rack until beads and solution have fully separated.
Carefully remove supernatant being careful not to disturb the beads.
Wash 2X with 200µL.
Remove all traces of ethanol and air dry for up to 0h 5m 0s.
To elute DNase I treated RNA add 12µL and incubate at Room temperature for at least 0h 2m 0s
RNA virus detection - Ribosomal depletion
Transfer 10µL to fresh 0.2 mL tubes/plate for Ribo-zero probe hybridisation.
Thaw DB1 and DP1 at Room temperature , vortex to mix and centrifuge briefly.
Prepare the hybridisation probe mix on ice (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| DB1 (Depletion Probe Buffer) | 3 |
| DP1 (Depletion Probe Pool) | 1 |
| Total | 4 |
Thoroughly pipette mix.
Add 4µL to each 10µL and fully mix by pipetting 10 times.
Incubate samples as follows:
95°C for 0h 2m 0s
Decrease 0.1°C until temperature reaches 37°C then hold.
Prepare rRNA Depletion.
Thaw RDB and RDE at Room temperature , vortex or flick (RDE) to mix and centrifuge briefly.
Prepare the rRNA Depletion mix (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| RDB (RNA Depletion Buffer) | 4 |
| RDE (RNA Depletion Enzyme) | 1 |
| Total | 5 |
Thoroughly pipette mix.
Add 5µL to each 14µL and fully mix by pipetting 10 times.
Incubate samples as follows:
37°C for 0h 15m 0s
4°C
Prepare probe removal.
Thaw PRB and PRE at Room temperature , vortex (PRB) or flick (PRE) to mix and centrifuge briefly.
Prepare the Probe Removal mix On ice (for multiple samples prepare a mastermix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| PRB (Probe Removal Buffer) | 7 |
| PRE (Probe Removal Enzyme) | 3 |
| Total | 10 |
Thoroughly pipette mix.
Add 10µL to each 19µL and fully mix by pipetting 10 times.
Incubate samples as follows:
37°C for 0h 15m 0s
70°C for 0h 15m 0s
4°C
Perform clean-up with 2X volume of RNAclean XP magnetic beads.
Add 60µL to the 30µL , mix by pipetting.
Incubate at Room temperature for 0h 5m 0s
Place on a magnetic rack until beads and solution have fully separated.
Remove and discard supernatant.
Wash 2X with 175µL.
Remove all traces of ethanol and air dry for up to 0h 2m 0s
Elute in 12µL by incubating at Room temperature for 0h 2m 0s.
RNA virus detection - Low input reverse transcription
Transfer 10µL to fresh 0.2 mL tubes/plates.
Prepare NTP/Hex mix (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| 10 mM dNTP | 1 |
| Random Hexamers | 1 |
| Total | 2 |
Add 2µL to each 10µL.
Incubate as follows:
65°C for 0h 5m 0s
immediately place On ice
Prepare the SSIII master mix (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| 5X Reverse transcription buffer | 4 |
| SuperScript III | 2 |
| RNaseOUT | 1 |
| DTT | 1 |
| Total | 8 |
Add 8µL to each 12µL.
Incubate samples as follows:
25°C for 0h 10m 0s
55°C for 1h 0m 0s
70°C for 0h 15m 0s
4°C
Prepare second strand mix (for multiple samples prepare a mastermix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| 10X Second strand synthesis buffer | 8 |
| Second strand synthesis enzyme | 4 |
| Nuclease-free water | 48 |
| Total | 60 |
Add 60µL to each 20µL
Incubate as follows on PCR machine
16°C for 2h 30m 0s
4°C
Perform clean up with 1X volume of AmPure XP beads.
Add 80µL to each 80µL (1:1 Ampure:sample ratio) and mix well.
Incubate at Room temperature for 0h 5m 0s.
Place on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200µL.
Remove all traces of ethanol and air dry for 0h 5m 0s.
Elute in 27µL by incubation at Room temperature for 0h 2m 0s.
cDNA → proceed to Section – Library prep (step 22) or store until DNA samples ready so can process together.
DNA virus detection - Microbiome enrichment
Pre-bind MBD2-Fc Protein to Magnetic Beads.
See following attachment for reagent calculations:
Pipette 1µL (see column B of reagent calculation table) for every 6.25ng into a 1.5 mL DNA LoBind tube.
Remove the supernatant with a pipette without disturbing the beads.
Repeat wash step (2 washes in total).
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).
Add 0.1V (see column C of reagent calculation table) to the Protein A magnetic beads.
Mix the bead-protein mixture by placing the tube in a rotating mixer for 0h 10m 0s.
Prepare the 1X Bind/Wash buffer and keep it On ice:
| A | B |
|---|---|
| Component | Volume (μl) |
| 5X NEBNext Bind/Wash buffer | 800 |
| Nuclease-free water | 3200 |
| Total | 4000 |
After the incubation, briefly spin the tube and place on the magnetic rack for 0h 5m 0s until the beads have collected.
Remove the supernatant with a pipette without disturbing the beads.
Add 1mL to the tube to wash the beads. Pipet up and down a few times to mix.
Mix the beads on a rotating mixer for 0h 3m 0s at Room temperature.
Briefly spin the tube and place on the magnetic rack for 0h 5m 0s until the beads have collected.
Capture Methylated Host DNA.
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).
Add the volume of sample to give up to 1µg (see column A of reagent calculation table).
Add appropriate volume of washed Fc-bead/protein mix (see column B of reagent calculation table) to the DNA/buffer mix.
Mix and incubate in a rotating mixer at RT for 0h 15m 0s to 4h 0m 0s depending on sample type .
Elute Microbiome DNA.
Briefly centrifuge and place on a magnet for at least 0h 5m 0s to separate the bead-bound host DNA.
Remove the supernatant containing host depleted/microbiome enriched DNA to fresh tubes.
DNA virus detection - sonication
If required make sample up to 55µL.
Prepare sonicator for use.
Equipment
| Value | Label |
|---|---|
| LE220 | NAME |
| High-throughput focused ultrasonicators | TYPE |
| Covaris | BRAND |
| 500569 | SKU |
Fill the tank with water to FILL level -2.
Switch on the chiller and ensure set to 7°C.
Switch on the water conditioning system, the Covaris and the computer.
Open the SonoLab software.
Select Home and the transducer will get submerged, the degas pump should start automatically.
Degas the water bath for ~0h 45m 0s.
Set up the sonication conditions as follows:
| A | B |
|---|---|
| Peak power | 450 |
| Duty factor | 10 |
| Cycles/burst | 1000 |
| Treatment time (s) | 89 |
| Dithering | on |
Example sonication conditions to achieve ~350 bp fragments.
Sonicate samples.
Add 55µL to either the 8 microTUBE-50 AFA Fiber Strip V2 or 96 microTUBE AFA Fiber Plate Thin Foil.
Place the strip/plate in the appropriate holder and screw into place (ensure that it is set up the same as the program conditions).
Select Load position to move the support arm forward.
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.
Press start position to submerge the rack and confrim the correct volume of water has been added (samples should NOT be fully submerged).
Press Run to start the sonication.
Once completed press Load Position to remove the plate.
Transfer 50µL to PCR tubes.
Equipment
| Value | Label |
|---|---|
| 4200 TapeStation System | NAME |
| Electrophoresis tool for DNA and RNA sample quality control. | TYPE |
| TapeStation Instruments | BRAND |
| G2991AA | SKU |
1.4X Ampure clean up.
Add 70µL to the samples (ratio 1.4:1).
Incubate at Room temperature for 0h 15m 0s.
Place on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200µL.
Remove all traces of Ethanol. Air dry for 0h 5m 0s.
Elute samples in 25µL.
Transfer 25µL to new tubes.
Illumina sequencing library preparation
Prepare the End Repair mix (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| 10X End repair buffer | 3 |
| End repair enzyme | 2 |
| Total | 5 |
Add 5µL to each 25µL.
Incubate as follows (if using a PCR machine do not use hot lid):
20°C for 0h 30m 0s
1.4X Ampure XP clean up.
Add 20µL increase volume of each sample to a total of 50µL.
Add 70µL (1.4:1 Ampure:sample ratio). Pipette up and down to mix.
Incubate at Room temperature for 0h 5m 0s.
Place samples on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200µL.
Remove all traces of ethanol and air dry for 0h 5m 0s.
Elute in 21µL leaving beads in solution.
- Prepare the A-Tail mix (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| 10X A-Tail buffer | 2.5 |
| A-Tail enzyme | 1.5 |
| Total | 4 |
Add 4µL to each 21µL.
Incubate as follows:
30°C for 1h 0m 0s
1.4X SPRI clean up.
Add 25µL to increase volume of each sample to a total of 50µL.
Add 70µL (1.4:1 SPRI:sample ratio). Pipette up and down to mix.
Incubate at Room temperature for 0h 5m 0s.
Place the samples on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200µL.
Remove all traces of ethanol and air dry for 0h 5m 0s.
Elute in 15µL leaving beads in solution.
Quantify 1µL using Qubit high sensitivity dsDNA.
Equipment
| Value | Label |
|---|---|
| Qubit | NAME |
| Flurometer | TYPE |
| Invitrogen | BRAND |
| Q33228 | SKU |
Calculate the amount of pmol per in each 14µL as follows (alternatively use the calculation in the attached sheet):
Calculate the amount of adapter required (aim for 20:1 ratio adapter to sample - see calculation sheet).
Dilute adapter in water to achieve appropriate concentration in a total volume of 5µL per reaction.
Prepare the Ligation mix (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| 5X ligation buffer | 5 |
| DNA ligase | 2.5 |
| Total | 7.5 |
Add 7.5µL to 14µL (ensure remains on the beads).
Add 5µL.
Incubate as follows (if using a PCR machine ensure the hot lid is not turned on):
20°C for 1h 0m 0s.
Add 0.75µL to each tube.
Step 15 Incubate as follows:
37°C for 0h 15m 0s
4°C
0.9X SPRI clean up.
Add 25µL to make volume up to 50µL.
Add 45µL to the samples (ratio 0.9:1).
Incubate at Room temperature for 0h 5m 0s.
Place on a magnetic rack until beads and solution have fully separated.
Remove supernatant.
Wash 2X with 200µL.
Remove all traces of ethanol and air dry for up to 0h 5m 0s.
Elute samples in 22µL.
Transfer 20µL to new freah 0.2 mL PCR tubes.
Add 5µL each sample.
Add 25µL to each sample.
Incubate as follows:
98°C for 0h 0m 45s
4-20* cycles of
98°C for 0h 0m 15s
65°C for 0h 0m 30s
72°C for 0h 0m 30s
Final cycle of
72°C for 0h 1m 0s
4°C 4oC hold
Clean up and QC the libraries as in protocol Library clean up and quality control for Illumina sequencing.
Metagenomic sequencing
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.
Using the bp size and ng/μl concentration calculate the nM concentration for each library as follows:
Pool the libraries by equal molarity and QC the pools as described in the protocol Library pooling and quality control for Illumina sequencing.
Sequence the pools on an Illumina sequencer following the manufacturer's guidelines.
