Jul 19, 2023
Nanopore Influenza A Virus WGS using one step RT/PCR with RBK barcoding
- Tom Williams1
- 1Guy's and St Thomas' NHS Foundation Trust
Protocol Citation: Tom Williams 2023. Nanopore Influenza A Virus WGS using one step RT/PCR with RBK barcoding. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj655wlk5/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: August 24, 2022
Last Modified: July 19, 2023
Protocol Integer ID: 69115
Keywords: reaction genomic amplification accelerates sequencing, swine origin human influenza, workflow for influenza, rapid multiplex minion nanopore, influenza, nanopore, rna extracts from sample, vaccine production, rna, rna extract, virus, sequencing, vaccine production for classical, virus wg
Abstract
A protocol to generate Influenza A Virus sequences from RNA extracts in under 24 hours with less than two hours of hands on time.
Based on:
- King, J., Harder, T., Beer, M., & Pohlmann, A. (2020). Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses.BMC Infectious Diseases,20(1). https://doi.org/10.1186/s12879-020-05367-y
- Zhou, B., Donnelly, M. E., Scholes, D. T., st. George, K., Hatta, M., Kawaoka, Y., & Wentworth, D. E. (2009). Single-Reaction Genomic Amplification Accelerates Sequencing and Vaccine Production for Classical and Swine Origin Human Influenza A Viruses.Journal of Virology,83(19), 10309–10313. https://doi.org/10.1128/jvi.01109-09
Tested with up to 20 RNA extracts from samples per run, alongside:
- Negative control of nuclease-free water
- Positive control of H1
- Positive control of H3
- Positive control of lambda phage, introduced at barcoding stage (i.e. not amplified), as an assessment of barcode crosstalk
Materials
Up to 20 RNA extracts from clinical samples
Consumables
- Zeptometrix H1N1 positive control (A/Puerto Rico/8/1934)
- Zeptometrix H3N2 positive control (A/Hong Kong/2671/19)
- 2X Reaction Mix (a buffer containing 0.4 mM of each dNTP, 3.2 mM MgSO4)
- TUNI 12.4: 5' ACG CGT GAT CAG CAA AAG CAG G 3'
- TUNI 12: 5' ACG CGT GAT CAG CAA AAG CAG G 3'
- TUNI 13: 5' ACG CGT GAT CAG CAA AAG CAG G 3'
- SQK-RBK110.96 (https://store.nanoporetech.com/uk/rapid-barcoding-kit-1.html), containing Rapid Barcode plates, AMPure XP Beads, Sequencing Buffer II, Rapid Adapter F, Elution Buffer, Loading Beads II, Loading Solution, Flush Tether, Flush Buffer
- 0.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water
- Freshly-prepared 80% ethanol in nuclease-free water
Equipment
- Cold block
- Microplate centrifuge
- PCR stip/Eppendorf centrifuge
- Timer
- Thermal cycler
- Magnetic rack
- Hula mixer (gentle rotator mixer)
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Multichannel pipette
- Qubit fluorometer (or equivalent for QC check)
- Flow Cell (R9.4.1)
- GridION Mk1
Troubleshooting
Combined RT/PCR
4h
Prepare up to 20 RNA extracts from samples plus:
- Negative control of nuclease-free water
- Positive control of H1N1
- Positive control of H3N2
If samples previously frozen, mix by briefly vortexing and pulse spin to collect liquid. Keep samples on cool block at all times.
In the template-free pre-PCR hood, prepare the following master mixes in Eppendorf DNA LoBind tubes, mix thoroughly by pipetting, and pulse spin to collect liquid at the bottom of the tube.
Pool A
| Component | Volume | x(n+2) | LOT | |
| Superscript III | 0.5 µL | |||
| TUNI 12.4/13 (10uM) | 1 uL | |||
| Nuclease Free Water | 1.75 µL | |||
| Reaction Mix x2 | 6.25 µL | |||
| Total | 9.5 µL |
Pool B
| Component | Volume | x(x+2) | LOT | |
| Superscript III | 0.5 µL | |||
| Nuclease Free Water | 1.75 µL | |||
| Reaction Mix x2 | 6.25 µL | |||
| TUNI 12/13 (10uM) | 1 uL | |||
| Total | 9.5 µL |
In the template-free pre-PCR hood, aliquot 9.5 µL of Pool A master mix into in 0.2 mL 8 well PCR strip-tubes, and 9.5 µL of Pool B into a seperate 0.2 mL 8 well PCR strip tubes.
In the template pre-PCR hood, transfer 3 µL of sample into the corresponding well of both Pool A and Pool B. Mix by pipetting each well up and down. Spin down.
In the post-PCR lab, incubate the reaction as follows:
| Step | Temperature | Time | Cycles | |
| RT step | 55 | 30 minutes | 1 | |
| Denaturation | 94 | 2 minutes | 1 | |
| Denaturation | 94 | 30 seconds | 5 | |
| Annealing | 45 | 30 seconds | 5 | |
| Extension | 68 | 3 minutes | 5 | |
| Denaturation | 94 | 30 seconds | 31 | |
| Annealing | 57 | 30 seconds | 31 | |
| Extension | 68 | 3 minutes | 31 | |
| Final Extension | 68 | 3 minutes | 1 | |
| Hold | 4 | - | - |
Rapid Barcoding
10m
Add 5 µL of pool A and 2.5 µL of pool B of each sample to a new strip tube.
In another well of the new 0.2ml PCR strip tube, add0.1 µL of lambda phage to 7.4 µL nuclease free water
Transfer 2.5 µL from the Rapid Barcode Plate to the corresponding well of 7.5 µL containing pooled PCR product. Mix by pipetting. Spin down.
Incubate the plate:
- 30 °C for 00:02:00
- 80 °C for 00:02:00
- 4 °C hold
4m
Pooling and clean-up
30m
Briefly spin down the Barcode reaction product to collect the liquid at the bottom of the wells prior to opening.
Pool 10 µL from each sample into a 2ml Eppendorf
Vortex pooled samples
Resuspend the AMPure XP Beads (AXP, or SPRI) by vortexing
To the pooled barcoded sample, add an 0.4 x volume of resuspended AMPure XP Beads (AXP, or SPRI) (e.g. 40 µL of beads to 100 µL of pooled barcode reaction) and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for00:05:00 at room temperature.
5m
Prepare at least3 mL of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet for 00:03:00 . Keep the tube on the magnet, and pipette off the supernatant.
3m
Keep the tube on the magnet and wash the beads with 1 mL of freshly-prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step.
Briefly spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend the pellet by pipetting in 15 µL Nuclease Free water. Incubate for 00:10:00 at room temperature.
10m
Pellet the beads on a magnet for 00:03:00 until the eluate is clear and colourless.
3m
Remove and retain 15 µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify DNA concentration by using the Qubit dsDNA BS Assay Kit.
Take forward 1000 ng of library and make up the volume to11.25 µL Nuclease Free water.
Add0.75 µL of Rapid Adapter F (RAP F) to 11.25 µL of barcoded DNA.
Incubate at room temperature on Hula mixer (rotator mixer) for 00:05:00 . Then store on ice until ready to load
5m
Priming and loading flow cell
15m
Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII), Flush Tether (FLT) and Flush Buffer (FB) at room temperature before mixing the reagents by vortexing, and spin down the SBII and FLT at room temperature.
To prepare the flow cell priming mix, add 30 µL of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.
On the GridION Mk1, place flow cell and perform flow cell check. Proceed if more than >800 pores.
Slide the priming port cover clockwise to open the priming port.
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl):
- Set a P1000 pipette to 200 μl
- Insert the tip into the priming port
- Turn the wheel until the dial shows 220-230 ul, to draw back 20-30 ul, or until you can see a small volume of buffer entering the pipette tip
- Note:Visually check that there is continuous buffer from the priming port across the sensor array.
Load 800 µL of the priming mix into the flow cell via the priming port, again by turning wheel of pipette and not by pressing on button, avoiding the introduction of air bubbles. Wait for 5 minutes. During this time, prepare the library for loading by following the steps below.
Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting
In a new tube, prepare the library for loading as follows:
| Reagent | Volume | |
| SBII | 37.5 uL | |
| LBII | 25.5 uL | |
| DNA library | 12 uL | |
| Total | 75 uL |
Complete the flow cell priming:
1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
2. Load200 µL of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
Mix the prepared library gently by pipetting up and down just prior to loading.
Add 75 µL of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION device lid.
MinKNOW settings
When starting the sequencing run, ensure the following settings are selected
Rapid Barcoding Kit 96 (SQK-RBK110.96)
High accuracy base calling
Enable barcoding
Mid-read barcoding filtering on
Over-ride minimum barcode score 60
Fastq size set to 250 reads
Sequencing performed on MinKNOW version 22.10.7 with guppy_basecaller 6.3.9