Aug 19, 2025
SMART-9N amplification enabling rapid nanopore sequencing of RNA viruses
- Vanessa Smilansky1
- 1SecureBio
- SecureBio Detection
External link: https://naobservatory.org
Protocol Citation: Vanessa Smilansky 2025. SMART-9N amplification enabling rapid nanopore sequencing of RNA viruses . protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6yr8g5b/v1
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: July 15, 2025
Last Modified: August 19, 2025
Protocol Integer ID: 222535
Keywords: wastewater, wastewater sample, viromics, metagenomics, nanopore sequencing, untargeted sequencing, RNA, viral RNA, untargeted RNA, total rna extraction of virus, diverse range of rna virus, nucleic acid extraction of virus, rna virus, total rna extraction, enabling rapid nanopore, rapid oxford nanopore technology, viral rna extract, rapid nanopore, nucleic acid extraction, rna, nucleic acid observatory, nasal swab, high genome coverage, untargeted sequencing, virus, workflow for untargeted sequencing, wastewater
Funders Acknowledgements:
Open Philanthropy
Grant ID: NA
Abstract
This protocol describes a rapid Oxford Nanopore Technologies (ONT) workflow for untargeted sequencing of RNA viruses. It has been optimized for wastewater and nasal swabs processed using the Nucleic Acid Observatory (NAO) protocols Concentration and nucleic acid extraction of viruses from wastewater influent V.3 and Concentration and total RNA extraction of viruses from nasal swabs V.1, respectively. It assumes that viral RNA extracts have undergone background depletion and does not detail those steps. In brief, RNA is reverse transcribed using SMART-9N amplification and prepared for sequencing with ONT's Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24). In our experience, this approach consistently yields sufficient material for ONT’s recommended inputs and recovers a diverse range of RNA viruses from both wastewater and nasal swabs, with long reads, high genome coverage, and reduced background noise.
Guidelines
RNA processing and handling: Please review Protocol Note: Working with RNA Samples before handling RNA samples.
Materials
Consumables:
- 100 µM RLB RT 9N primer (IDT - Sequence: 5’-TTTTTCGTGCGCCGCTTCAACNNNNNNNNN-3’)
- 100 µM RLB TSOmG template-switching oligo (IDT - Sequence: 5’-GCTAATCATTGCTTTTTCGTGCGCCGCTTCAACATmGmGmG-3’)
- LunaScript RT Master Mix Kit (NEB, E3025)
- Sequenase Version 2.0 DNA Polymerase (ABI, 70775Y200UN)
- Rapid PCR Barcoding Kit 24 V14 (ONT, SQK-RPB114.24)
- 2X LongAmpTaq Master Mix (NEB, M0287)
- Agencourt AMPure XP beads (Beckman CoulterTM, A63881)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 100% ethanol
- 1.5 mL Eppendorf DNA LoBind tubes
- 0.2 mL thin-walled PCR tubes
- QubitTM dsDNA HS Assay Kit (ThermoFisher, Q32851)
- QubitTM Assay Tubes (Invitrogen, Q32856)
Equipment:
- Ice bucket with ice
- Microfuge
- Micropipettes (1000 uL, 200 uL, 20 uL, 2 uL) and holder
- Timer
- Vortex mixer
- Thermal cycler
- Magnetic separation racks: compatible with 0.2 mL tubes and 1.5 mL tubes
- Hula mixer (gentle rotator mixer)
- QubitTM fluorometer
Troubleshooting
Before start
Prepare a biosafety cabinet or dead air box for RNA processing and handling. Gather materials and reagents. Ensure proper PPE.
Stage 1. cDNA synthesis and clean-up
1h 16m
Prepare 12 µL of each viral RNA extract in a separate clean 0.2 mL tube.
1m
Add 2 µL of RLB RT 9N (100 µM) to each 0.2 mL tube containing viral RNA extract.
1m
Incubate the reaction at 65 °C for 00:05:00 , then transfer the samples to ice immediately. Keep the samples on ice for 00:02:00 .
7m
Add 2 µL of TSOmG (100 µM) followed by 4 µL of LunaScript RT Master Mix Kit (Primer-free) to each 0.2 mL tube for a total volume of 20 µL .
2m
Incubate in thermal cycler using the following protocol:
| Step | Temperature | Time | |
| Primer Annealing | 25°C | 2 minutes | |
| cDNA Synthesis | 55°C | 10 minutes | |
| Heat Inactivation | 95°C | 1 minute |
13m
| A | B | C | |
| Enter the number of samples: | |||
| Reagents | Volume (µL) per sample | 2 | |
| 5X Sequenase Buffer | 2 | 4.4 | |
| Sequenase Dilution Buffer | 0.9 | 1.98 | |
| Sequenase | 0.6 | 1.32 | |
| NFW | 7.7 | 16.94 | |
| TOTAL | 11.2 | 24.64 |
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
5m
Add 11.2 µL of Sequenase master mix to each sample.
1m
Incubate at 37 °C for 00:08:00 .
8m
Resuspend AMPure XP Beads (AXP) by vortexing. Add 25 µL (0.8X) of AXP beads to each sample and mix by flicking the tube.
2m
Incubate on a Hula mixer at Room temperature for 00:05:00 .
5m
Spin down and pellet on a magnet for 00:05:00 ; remove supernatant.
6m
Add 200 µL freshly prepared 80% ethanol (do not remove from magnet); incubate 00:00:30 , remove ethanol. Repeat.
2m
Dry spin and remove residual ethanol. Allow pellet to air dry for about 00:00:30 on the magnet, but do not dry the pellet to the point of cracking.
2m
Remove the tube from the magnet and resuspend pellet in 11 µL NFW.
1m
Incubate on a Hula mixer at Room temperature for 00:10:00 .
10m
Spin down, pellet on magnet, and transfer 10 µL of eluate to a clean 0.2 mL PCR tube.
5m
Use 2 µL of each sample to quantify with Qubit dsDNA HS assay. Use remaining 8 µL sample for Rapid Barcode PCR.
Note
Take your cDNA sample(s) forward to Stage 2 of the protocol.
Optional: if you are not using your sample(s) immediately, store at -20°C.
5m
Stage 2. Rapid Barcode PCR
2h 10m
Prepare 5 µL each of viral cDNA (diluted to a concentration of 1 ng/µL, for a total of 5 ng) in a separate clean 0.2 ml tube.
Note
If the concentration of viral cDNA is <1 ng/µL, then the volume can be increased to reach a total of 5 ng. Additional volume beyond 5 µL must be subtracted from the 20 µL of NFW added in step 19.
5m
To each tube, add 20 µL of NFW, 1 µL of Rapid Barcode Primer (RLB01-24, at 10 µM), and 25 µL 2X LongAmp Taq Master Mix. Mix by gently flicking the tube and spin down.
5m
Amplify using the following cycling conditions: 95 °C for 00:00:45 ; 25 cycles of 95 °C for
00:00:15 , 56 °C for 00:00:15 and 65 °C for 00:04:00 followed by a final extension step of 65 °C for 00:06:00 . Hold at 10 °C .
Note
Take your PCR product(s) forward to the clean-up, quantification, and adapter attachment steps.
Optional: PCR amplification may be performed and held at the final hold temperature overnight.
2h
Stage 3. Clean-up, quantification, and adapter attachment
47m
Quantify the sample tubes (PCR products from the previous step) using the Qubit dsDNA HS Assay Kit.
5m
In a new 1.5 mL Eppendorf DNA LoBind tube, pool all barcoded samples in equal ratios to a combined final concentration 800 ng.
Note
For example: if 10 barcodes were used, add 80 ng from each sample.
5m
Resuspend the AXP beads by vortexing. Add 0.6X volume of AXP beads to the pooled samples.
2m
Incubate on a Hula mixer at Room temperature for 00:05:00 .
5m
Spin down and pellet on a magnet for 00:05:00 ; remove supernatant.
6m
Add 500 µL freshly prepared 80% ethanol (do not remove from magnet); incubate 00:00:30 , remove ethanol. Repeat.
2m
Dry spin and remove residual ethanol. Allow pellet to air dry for about 00:00:30 on the magnet, but do not dry the pellet to the point of cracking.
2m
Remove the tube from the magnet and resuspend pellet in 14 µL Elution Buffer (EB).
1m
Incubate at Room temperature for 00:02:00 .
2m
Spin down, pellet on magnet, and transfer 13 µL of eluate to a clean 1.5 mL tube.
5m
Use 2 µL of eluate to quantify with Qubit dsDNA HS assay. Use remaining 11 µL for adapter attachment.
Expected result
The eluate should be approximately 10–40 ng/µL.
5m
In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the Rapid Adapter (RA) dilution as described below and mix by pipetting:
| Reagent | Volume | |
| Rapid Adapter (RA) | 1.5 µL | |
| Adapter Buffer (ADB) | 3.5 µL | |
| TOTAL | 5 µL |
1m
Add 1 µL of diluted RA to the library pool. Mix gently by flicking the tube and spin down.
1m
Incubate at Room temperature RT for 00:05:00 .
5m
The prepared library pool is used for loading into a MinION/GridION/PromethION Flow Cell. Keep the pool on ice or at 4 °C until ready to load.