Discovery of RNA and DNA viruses using next-generation sequencing: Targeted enrichment
Katherine Smollett, Lily Tong, Jenna Nichols, Kirsty Kwok, Kyriaki Nomikou, Ma. Jowina Galarion, Daniel Mair, Ana Filipe
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. 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
Hybridisation
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.
Enrichment is performed with VirCapSeq-VERT probes and Roche SeqCap reagents.
To each pool add the following blocking reagents:
| A | B |
|---|---|
| Component | Volume (μl) |
| COT DNA | 5 |
| Salmon sperm DNA (1 mg/ml) | 5 |
| xGen Universal blockers | 2 |
| Total | 12 |
Concentrate the pool using Ampure XP.
Add 2X total volume of the pool plus blocking reagent of AmpureXP.
Place on a magnetic rack until beads and solution have fully separated 0h 5m 0s.
Remove supernatant being careful not to disturb the beads.
Add 800µL and incubate Room temperature for 0h 1m 0s.
Remove all traces of ethanol being careful not to disturb the beads.
Air-dry the beads for around 0h 3m 0s taking care not to over dry the beads.
Prepare the hybridisation mix (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| 2X Hybridisation buffer | 7.5 |
| Hybridisation component A | 3 |
| Total | 10.5 |
Add 10.5µL directly to the bead-bound DNA samples, remove from magnet and mix thoroughly.
Incubate at Room temperature for 0h 2m 0s.
Place on magnetic rack and elute the entire 10.5µL to a new 0.2 mL PCR tube tube containing 4.5µL.
Mix thoroughly by pipetting.
Incubate as follows on a PCR machine with lid set to 105°C :
95°C for 0h 5m 0s
cool to 47°C
Quickly transfer to second PCR machine with lid set to 57°C and incubate as follows:
47°C for 72h 0m 0s
Capture and washing
Prepare the wash buffers per capture as follows:
| A | B | C | D | E |
|---|---|---|---|---|
| Component | Tube label | Tube type | Reagent volume (μl) | Water volume (μl) |
| 10x stringent wash buffer | A | PCR | 20 | 180 |
| 10x stringent wash buffer | B | PCR | 20 | 180 |
| 10x wash buffer 1 | C | PCR | 10 | 90 |
| 10x wash buffer 1 | D | PCR | 20 | 180 |
| 10x wash buffer 2 | E | PCR | 20 | 180 |
| 10x wash buffer 3 | F | PCR | 20 | 180 |
| 2.5x bead wash buffer | G | 1.5ml | 200 | 300 |
Transfer tubes A and B 200µL and tube C 100µL to the PCR machine to equilabrate to 47°C.
Prepare capture beads.
For each capture, place 100µL in a 1.5 mL tube.
Place tube on a magnet, remove liquid being careful not to disturb the beads.
Add 2x the initial volume of beads of bead wash buffer (tube G ).
Remove from magnet, vortex for 0h 0m 10s then centrifuge briefly.
Place tube on a magnet, remove liquid.
Repeat bead wash one more time (2 washes in total).
Re-suspend beads in 1x original volume of bead wash buffer (tube G ) by vortexing.
Transfer 100µL per capture to a fresh 0.2 mL PCR tube.
Place tube on a magnet, remove liquid and proceed immediately to next so that the beads do not dry out.
Immediately add the 15µL to the prepared capture beads. Mix by pipetting ten times.
Incubate in a PCR machine for 0h 45m 0s at 47°C, with the heated lid set to 57°C.
Add 100µL pre-heated to 47°C (tube C ).
Mix by vortexing for 0h 0m 10s.
Place tube on a magnet, remove liquid.
Add 200µL pre-heated to 47°C (tube A ). Pipette 10X to mix.
Incubate at 47°C for 0h 5m 0s.
Repeat stringent wash one more time (tube B , total 2 washes).
Transfer mixture to a fresh 1.5 mL DNA LoBind tube.
Place tube on a magnet, remove liquid.
Add 200µL at Room temperature (tube D ) and vortex for 0h 2m 0s.
Place tube on a magnet, remove liquid.
Add 200µL at Room temperature (tube E ) and vortex for 0h 1m 0s.
Place tube on a magnet, remove liquid.
Add 200µL at Room temperature (tube F ) and vortex for 0h 0m 30s.
Place tube on a magnet, remove liquid.
Remove from magnet, resuspend the beads in 20µL and mix well by pipetting.
Amplification
Prepare the PCR mix (for multiple samples prepare a master mix with 10% excess):
| A | B |
|---|---|
| Component | Volume (μl) |
| 2X KAPA HiFi ready mix | 25 |
| Post-LM PCR oligos | 5 |
| Total | 30 |
Set up two PCR tubes per capture and add 15µL to each tube.
Briefly vortex bead-bound captured DNA from step 33 and spin down.
Add 10µL to each PCR reaction tube.
Incubate on a PCR machine as follows:
95°C for 0h 3m 0s
14 cycles of
98°C for 0h 0m 20s
65°C for 0h 0m 15s
72°C for 0h 0m 30s
Final cycle of
72°C for 0h 2m 0s
4°C .
Briefly centrifuge PCR reactions and place on magnetic rack until the beads and solution have fully separated.
Transfer the 25µL into fresh tubes, combining the 2 reactions from each pool to make a total volume of 50µL.
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.
Pooling and sequencing
Using the bp size and ng/μl concentration calculate the nM concentration for each pool as follows:
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.
Sequence the pools on an Illumina sequencer following the manufacturer's guidelines.
