Jul 09, 2025
Version 2
Seasonal and zoonotic influenza WGS using ONT rapid barcoding technology V.2
- Richard Chalvignac1,
- Quentin Semanas1,
- Hadrien Regue1,
- antonin.bal BAL1,
- laurence.josset 1
- 1Hospices Civils de Lyon - Genepii Sequencing Platform
Protocol Citation: Richard Chalvignac, Quentin Semanas, Hadrien Regue, antonin.bal BAL, laurence.josset 2025. Seasonal and zoonotic influenza WGS using ONT rapid barcoding technology. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl41onjlo5/v2Version created by Richard Chalvignac
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 03, 2025
Last Modified: July 09, 2025
Protocol Integer ID: 221681
Keywords: zoonotic influenza wg, zoonotic rapid whole genome, ont rapid barcoding technology this protocol, using oxford nanopore technology, clade resolution, tierce bioinformatic tool, oxford nanopore technology, sequencing result, genome, using ont, rapid barcoding technology, oxford nanopore
Abstract
This protocol is suitable for seasonal and zoonotic rapid whole genome sequencing for influenza A and B using Oxford Nanopore Technologies (ONT) rapid chemistry (SQK-RBK114.24 or SQK-RBK114.96).
Using ONT and tierce bioinformatic tools, it enables sub-typing and clade resolution sequencing result in one working-day.
Troubleshooting
Nucleic acid extraction
1h
RNA extraction is performed using either Maxwell‱ CSC Pathogen Total Nucleic Acid Kit AS1860 or EMAG‱ Nucleic Acid Extraction System Generic program, allowing the management of up to 16 or 24 samples simustaneously depending on the extraction system.
1h
Primers
Primers scheme and PCR should be chosen based on flu-type as follows
For Influenza A (individual primers)
| Primer name | 5'-3' Sequences | |
| Uni12 Forward (Fw) | GGG-GGG-AGC-AAA-AGC-AGG | |
| Uni12b Forward (Fw) (Uni12_inf3) | GGG-GGG-AGC-GAA-AGC-AGG | |
| Uni13 Reverse (Rv) | CGG-GTT-ATT-AGT-AGA-AAC-AAG-G |
Table 1 : Influenza A Universal Primers
For Influenza B (Primer pool preparation prior to PCR setup)
| Primer name | 5'-3' Sequences | [Primer Stock] (µM) | Primer Volume (µL) | |
| B-PBs-UniF | GGGGGGAGCAGAAGCGGAGC | 100 | 10 | |
| B-PBs-UniR | CCGGGTTATTAGTAGAAACACGAGC | 100 | 10 | |
| B-PA-UniF | GGGGGGAGCAGAAGCGGTGC | 100 | 5 | |
| B-PA-UniR | CCGGGTTATTAGTAGAAACACGTGC | 100 | 5 | |
| HANA-UniF | GGGGGGAGCAGAAGCAGAGC | 100 | 10 | |
| HANA-UniR | CCGGGTTATTAGTAGTAACAAGAGC | 100 | 10 | |
| NP-UniF | GGGGGGAGCAGAAGCACAGC | 100 | 6 | |
| NP-UniR | CCGGGTTATTAGTAGAAACAACAGC | 100 | 6 | |
| M-Uni3F | GGGGGGAGCAGAAGCACGCACTT | 100 | 3 | |
| Mg-Uni3F | GGGGGGAGCAGAAGCAGGCACTT | 100 | 3 | |
| M-Uni3R | CCGGGTTATTAGTAGAAACAACGCACTT | 100 | 6 | |
| NS-Uni3F | GGGGGGAGCAGAAGCAGAGGATT | 100 | 5 | |
| NS-Uni3R | CCGGGTTATTAGTAGTAACAAGAGGATT | 100 | 5 | |
| H2O | - | - | 756 | |
| Total | - | - | 840 |
Table 2 : Influenza B Primers Pool preparation. Stock at -20°C and use when needed.
Pre-PCR and amplification
4h
Influenza A Pre-PCR is prepared as follows :
- ezDNase 11766051 (Thermofisher Scientific) is used for nucleic-acid extracts DNase treatment
| Reagent | Volume / Sample (µL) | |
| nuclease-free water | 0.625 | |
| ezDNase buffer (10X) | 1.25 | |
| ezDNase enzyme | 0.625 | |
| nucleic acid extract | 10 | |
| Total | 12.5 |
Table 2 : Influenza A nucleic-acid extract DNase treatment
DNase treatment is performed by incubating the prepared mix at 37 °C for 00:10:00
SuperScript III One Step RT-PCR System 12574035 (Thermofisher Scientific) combined with RNasin N2511 (Promega) is then used as follows :
| Reagent | Volume / Sample (µL) | |
| 2X Reaction Mix | 12.5 | |
| ezDNase - nucleic acid extract mix | 5 | |
| nuclease-free water | 4.75 | |
| Primer Uni12 forward (10µM) | 0.5 | |
| Primer Uni12b forward (10µM) | 0.5 | |
| Primer Uni13 reverse (10µM) | 1 | |
| RNasin | 0.25 | |
| SuperScript III RT/ Platinum Taq High Fidelity Enzyme Mix | 0.5 | |
| Total | 20 |
Table 3 : Influenza A mix preparation for one-step RT-PCR
One-step RT-PCR is performed on a thermocycler as follows 03:30:00 :
| Temp (C°) | Time | Cycle(s) | |
| 45 | 01:00:00 | 1 | |
| 94 | 00:02:00 | ||
| 94 | 00:00:30 | 5 | |
| 45 | 00:00:30 | ||
| 68 | 00:03:00 | ||
| 94 | 00:0030 | 31 | |
| 57 | 00:00:30 | ||
| 68 | 00:03:00 |
Table 4 : Influenza A thermocycler one-step RT-PCR program
Influenza B Pre-PCR is prepared as follows :
- ezDNase 11766051 (Thermofisher Scientific) is used for nucleic-acid extracts DNase treatment
| Reagent | Volume / Sample (µL) | |
| nuclease-free water | 0.75 | |
| ezDNase buffer (10X) | 1.5 | |
| ezDNase enzyme | 0.75 | |
| nucleic acid extract | 12 | |
| Total | 15 |
Table 2 : Influenza B nucleic-acid extract DNase treatment
DNase treatment is performed by incubating the prepared mix at 37 °C for 00:10:00
SuperScript III One Step RT-PCR System 12574035 (Thermofisher Scientific) combined with RNasin N2511 (Promega) is then used as follows :
| Reagent | Volume / Sample (µL) | |
| 2X Reaction Mix | 12.5 | |
| ezDNase - nucleic acid extract mix | 10 | |
| Influenza B Primer Pool (10µM) | 2 | |
| RNasin | 0.2 | |
| SuperScript III RT/ Platinum Taq High Fidelity Enzyme Mix | 0.75 | |
| Total | 25 |
Table 3 : Influenza B mix preparation for one-step RT-PCR
One-step RT-PCR is performed on a thermocycler as follows 04:00:00 :
| Temp (C°) | Time | Cycle(s) | |
| 45 | 01:00:00 | 1 | |
| 55 | 00:30:00 | 1 | |
| 94 | 00:02:00 | 1 | |
| 94 | 00:00:20 | 4 | |
| 40 | 00:00:30 | ||
| 68 | 00:03:30 | ||
| 94 | 00:00:20 | 44 | |
| 58 | 00:00:30 | ||
| 68 | 00:03:30 | ||
| 68 | 00:10:00 | 1 | |
| 4 | Hold |
Table 4 : Influenza B thermocycler one-step RT-PCR program
Library preparation
The library construction is performed according to manufacturer's instruction for the Rapid Barcoding Kit (SQK-RBK114.24/96).
Before starting this step it's possible to perform a flow cell check on the ONT device to gain some time on the whole experiment.
Briefly, each sample is quantified using QuBIt fluorometer and normalised to obtain 50 ng of amplified DNA in a 9 µL volume.
1 µL of rapid barcode are added to the reaction volume and the mix is then incubated as follows :
30 °C 00:02:00
80 °C 00:02:00
Once the incubation time is over, each sample is pooled in a single 1.5mL LoBind Eppendorf tube and a 1:1 AMPure XP beads purification is performed. Two 80% ethanol washes are performed on a magnetic rack. Elution is done in 11 µL of Elution Buffer (EB)
Rapid Adapter mix is performed as follows :
| Reagent | Volume (µL) | |
| Adapter Buffer (ADB) | 3.5 | |
| Rapid Adapter (RA) | 1.5 | |
| Total | 5 |
Table 5 : Rapid Adapter Mix (unused mix can be stored at -20°C for further use)
1 µL of Rapid Adapter mix is added to the 11 µL of previously barcoded and purified samples.
The resulting mix is incubated 00:05:00 at room temperature and then immediatly used to load on the flow cell.
It's possible to keep the remaining 4 µL of RA + ADB mix at -20 °C for further use
Sequencing and analysis
1h 30m
Sequencing is performed on any ONT sequencing device. Preferentially use one with computing capacity allowing for real-time High Accuracy basecalling mode (HAC).
R10.4.1 flow cell is loaded according to manufacturer's instruction and sequencing run is launched for at least 01:00:00 for small batches of samples (<8). Once the short run is over, it can be re-launched for a longer time to use as supplementary data if needed.
The time required for successful sequencing is depending on samples multiplexing but also individual samples overall quality and viral load thus should be tested to ensure good results. For higher throughput (eg : 96 samples), running the sequencing experiment over-night (>12h) is more suited.
1h
The RBK kit usage must be specified while setting-up the analysis run.
The wf-flu is not suited for zoonotic influenza type so the samples will always be Undetermined in the subtyping results of the report. If there is a suspicion for zoonotic influenza, an alternative solution can be used as described after.
Using intermediate files generated in the analysis directory, it's still possible to obtain the consensus for the samples of interest (wf-flu_ID\output\SampleID\consensus\draft.consensus.fasta).
The consensus can then be uploaded on Nextclade with the automatically suggested reference dataset, allowing for clade determination based on the HA sequence
30m