Sep 05, 2025
Viral Metagenomics utilising SMART-9N Amplification
- Ingra Claro Morales1,
- Josh Quick2,
- Mia Weaver2
- 1Universidade de São Paulo;
- 2University of Birmingham
- ARTIC
Protocol Citation: Ingra Claro Morales, Josh Quick, Mia Weaver 2025. Viral Metagenomics utilising SMART-9N Amplification. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9xn5gx1/v1
Manuscript citation:
Claro, I.M., Ramundo, M.S., Coletti, T.M., Da Silva, C.A., Valenca, I.N., Candido, D.S., Sales, F.C., Manuli, E.R., de Jesus, J.G., de Paula, A. and Felix, A.C., 2023. Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing. Wellcome open research, 6, p.241. DOI: https://doi.org/10.12688/wellcomeopenres.17170.2
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 14, 2025
Last Modified: September 05, 2025
Protocol Integer ID: 224674
Keywords: Viral Metagenomics, RNA Template, RNA, cDNA, ONT, Viral Reference Material, Random 9N Priming, SMART, SMART-9N, cDNA Synthesis, Sequencing, Virus, Strand Switching, PCR, DNA, ideal for viral metagenomic, viral metagenomic, genome representation in diverse microbial community, rna template, viral pathogen, diverse microbial community, transcribe rna, rna present, genome coverage, allowing dna virus, dna virus, tail of rna, rna, genome, genome representation, viruses present in the sample, using random 9n, oxford nanopore technology, unmodified pcr primer, random 9n, removing extracellular dna present, total nucleic acid extraction, fungal, virus
Abstract
Following on from the original SMART-9N (Switch Mechanism at the 5′ End of RNA Templates) protocol utilised for viral metagenomics, this protocol offers an optimised approach to improve genome representation in diverse microbial communities and also incorporate DNA viruses.
This protocol is also adapted from the "Rapid metagenomic sequencing for surveillance of bacterial, fungal and viral pathogens using SQK-RPB114.24" protocol published by Oxford Nanopore Technologies (https://nanoporetech.com/document/rapid-sequencing-metagenomics-sqk-rpb114-2).
The protocol utilises a shotgun approach using random 9N priming to reverse transcribe RNA and PCR-amplify RNA present. Furthermore, as the random priming is independent of the poly(A) tail of RNA, DNA is also annealed and amplified (similarly to as described in https://doi.org/10.1016/j.meegid.2015.03.018). Therefore, it is ideal for viral metagenomics to enrich for the viruses present in the sample often obscured by high host backgrounds.
Optimisation Implemented:
- Host depletion has been moved before the total nucleic acid extraction, removing extracellular DNA present in the sample and allowing DNA viruses to be processed through the extraction.
- Total nucleic acid extraction utilising column purification has been replaced by a magnetic bead extraction.
- Primer concentrations have been adjusted to reflect the conditions providing the greatest yield and genome coverage.
- An unmodified PCR primer is utilised in place of the ONT RLB barcoding primers to ensure sufficient amplification for library preparation that the barcoding primers do not provide. We have found that utilising the barcoding primers result in 10x less yield post-PCR in comparison to the unmodified primer.
Materials
Reagents and Consumables:
| Reagent / Consumable | Supplier | Catalogue Number | |
| HL-SAN Triton Free | ArcticZymes | 70911-202 | |
| MagMAX Viral/Pathogen Nucleic Acid Isolation Kit | ThermoFisher Scientific | A42352 | |
| 10 mM dNTPs | ThermoFisher Scientific | R0192 | |
| SuperScript™ IV Reverse Transcriptase | ThermoFisher Scientific | 18090050 | |
| RNaseOUT™ Recombinant Ribonuclease Inhibitor | ThermoFisher Scientific | 10777019 | |
| Q5® High-Fidelity 2X Master Mix | New England Biolabs | M0492S | |
| AMPure XP Beads for DNA Cleanup | Beckman Coulter | A63880 | |
| Qubit™ dsDNA Quantification Assay | ThermoFisher Scientific | Q32851/Q32854 | |
| Qubit™ Assay Tubes | ThermoFisher Scientific | Q32856 | |
| 1.5 mL Eppendorf DNA LoBind tubes | Eppendorf | 0030108051 | |
| 0.2 mL PCR tubes | |||
| Nuclease-Free Water | |||
| Molecular Grade 100% Ethanol |
Equipment:
| Equipment | Supplier | Catalogue Number | |
| Qubit™ 4 Fluorometer | ThermoFisher Scientific | Q33226 | |
| Vortex | |||
| Microcentrifuge | |||
| Thermomixer | |||
| HulaMixer | |||
| Magnetic Rack (To fit 1.5 mL Eppendorf tubes) | |||
| Thermocycler |
Oligonucleotides:
| Oligonucleotide | Sequence | Type | |
| RLB RT 9N | TTTTTCGTGCGCCGCTTCAACNNNNNNNNN | DNA | |
| RLB SSP | GCTAATCATTGCTTTTTCGTGCGCCGCTTCAACATrGrGrG | RNA | |
| RLB PCR | TTTTTCGTGCGCCGCTTCA | DNA |
Troubleshooting
Host Depletion and Extraction
Transfer 500 µL of sample into a 1.5 mL Eppendorf tube.
Centrifuge at 10000 x g for 00:05:00 .
For each sample, transfer 300 µL of supernatant into a separate 1.5 mL Eppendorf tube.
To each sample, add 10 µL of HLSAN enzyme and vortex for 00:00:03 .
Incubate the reaction on a thermomixer at 37 °C for 00:10:00 at 1000 rpm .
Add the following reagents to the reaction and mix by inversion:
| Component | Volume | |
| Binding Buffer from the MagMAX kit | 520 µL | |
| Magnetic beads from the MagMAX kit | 20 µL | |
| Proteinase K from the MagMAX kit | 10 µL | |
| Total | 860 µL |
Transfer the samples into a thermomixer and incubate at 65 °C for 00:05:00 at 1000 rpm .
Transfer the sample tubes to a HulaMixerTM and incubate/mix at room temperature for 00:05:00 using the HulaMixerTM.
Prepare 2 mL of fresh 80% ethanol per sample in nuclease-free water.
Briefly spin down the tubes and pellet on a magnetic rack until the supernatant is clear and colourless for at least 00:05:00 .
Keep the tube on the magnetic rack and pipette off the supernatant.
Remove the tube from the magnetic rack and add 1 mL of Wash Buffer from the MagMAX kit and gently mix by inverting the tube until fully resuspended.
Briefly spin down the tubes and pellet on the magnetic rack until the supernatant is clear and colourless for at least 00:02:00 .
Keep the tube on the magnetic rack and pipette off the supernatant.
Remove the tube from the magnetic rack and add 1 mL of 80% ethanol and gently mix by inverting the tube until fully resuspended.
Briefly spin down the tubes and pellet on the magnetic rack until the supernatant is clear and colourless for at least 00:02:00 .
Keep the tube on the magnetic rack and pipette off the supernatant.
Remove the tube from the magnetic rack and add 500 µL of 80% ethanol and gently mix by inverting the tube until fully resuspended.
Briefly spin down the tubes and pellet on the magnetic rack until the supernatant is clear and colourless for at least 00:02:00 .
Keep the tube on the magnetic rack and pipette off the supernatant, allow the pellet to dry for 00:02:00 but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet by adding 20 µL of nuclease-free water and ensure the pellet is fully resuspended by pipette mixing.
To aid with sample elution, transfer the tubes to a thermomixer and incubate at 65 °C for 00:05:00 at 1000 rpm .
Pellet the beads on a magnet until the eluate is clear and colourless for at least 00:01:00 .
Remove and retain the eluate into a clean 1.5 mL Eppendorf tube.
SMART-9N Annealing, cDNA Synthesis and Amplification
Per reaction, combine and mix thoroughly the following in a 0.2 mL PCR tube:
| Component | Volume | |
| dNTP mix (10 mM) | 1 µL | |
| RLB RT 9N (12 µM) | 1 µL | |
| RNA | 10 µL | |
| Total | 12 µL |
Incubate at 65 °C for 00:05:00 and then snap cool on ice.
Per reaction, combine the following to create a master mix:
| Component | Volume | |
| SSIV Buffer (5X) | 4 µL | |
| DTT (100 µM) | 1 µL | |
| RNase OUT | 1 µL | |
| SSIV RT (200 units/µL) | 1 µL | |
| RLB SSP (12 µM) | 1 µL | |
| Total | 8 µL |
Add 8 µL of the master mix to 12 µL of the annealed RNA.
Incubate on the thermocycler as follows:
| Temperature | Time | |
| 42 °C | 90 minutes | |
| 70 °C | 10 minutes | |
| 4 °C | Hold |
Combine the following per reaction to create the amplification master mix:
| Component | Volume | |
| Q5 High-Fidelity 2X Master Mix | 12.5 µL | |
| RLB PCR (10 µM) | 1 µL | |
| Nuclease-Free Water | 9 µL | |
| cDNA | 2.5 µL | |
| Total | 25 µL |
Incubate the reaction as follows on the thermocycler:
| Temperature | Time | Cycles | |
| 98 °C | 45 seconds | 1 | |
| 98 °C | 15 seconds | 30 | |
| 62 °C | 15 seconds | ||
| 65 °C | 5 minutes | ||
| 65 °C | 10 minutes | 1 | |
| 4 °C | Hold | 1 |
Quality Control
Quantify the PCR products utilising the QubitTM dsDNA HS (High Sensitivity) Assay Kit on the Qubit Fluorometer.
Note
If the QubitTM fluorometer is not available, alternative fluorometers can be utilised including:
- Promega QuantusTM Fluorometer and the QuantiFluor‱ dsDNA System (Catalogue Number: E6150 and E2671).
- “DIYNAFLUOR” (DIY Nucleic Acid FLUORometer) - a portable, open-source, <$40 USD Nucleic Acid fluorometer compatible with QubitTM reagents (https://doi.org/10.1101/2024.12.16.626200).
1X Clean-up
Add a ratio of 1X AMPure XP beads to the sample and mix gently.
Incubate the samples at room temperature on the HulaMixerTM for 00:05:00 .
Place the sample on the magnet and allow the beads to pellet before removing the supernatant.
On the magnetic rack, wash the beads with 150 µL of 80% Ethanol, being careful not to disturb the pellet. Remove the ethanol and discard.
Repeat the previous step.
Spin down the pellet and remove any excess ethanol remaining and allow the pellet to lose its shine.
Resuspend the pellet in 20 µL of Nuclease-free water by flicking the tube and incubate at room temperature for 00:05:00 .
Place the sample on the magnet and transfer the clear supernatant into a fresh 1.5 mL Eppendorf tube.
Sequencing
The samples can now be sequenced. We recommend utilising the ONT Native Barcoding Kit (SQK-NBD114.24 / SQK-NBD114.96) for numerous samples or the ONT Ligation Sequencing kit (SQK-LSK114) for a single sample, following the manufacturer's protocol. For metagenomics, it is recommended to utilise the PromethION to generate sufficient data, however, the MinION or GridION can be used for a smaller number of samples or lower input libraries.
Protocol references
Claro, I.M., Ramundo, M.S., Coletti, T.M., Da Silva, C.A., Valenca, I.N., Candido, D.S., Sales, F.C., Manuli, E.R., de Jesus, J.G., de Paula, A. and Felix, A.C., 2023. Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing. Wellcome open research, 6, p.241. DOI: https://doi.org/10.12688/wellcomeopenres.17170.2
"Rapid metagenomic sequencing for surveillance of bacterial, fungal and viral pathogens using SQK-RPB114.24" protocol published by Oxford Nanopore Technologies (https://nanoporetech.com/document/rapid-sequencing-metagenomics-sqk-rpb114-24)