Jan 22, 2026
Version 1
Nanopore amplicon-based whole genome sequencing of human Monkeypox Virus V.1
This protocol is a draft, published without a DOI.
- Valentin Leducq1
- 1Sorbonne Université, INSERM, Institut Pierre Louis d'Epidémiologie et de Santé Publique, AP-HP, Hôpitaux Universitaires Pitié-Salpêtrière - Charles Foix, Laboratoire de Virologie, Paris, France.
Protocol Citation: Valentin Leducq 2026. Nanopore amplicon-based whole genome sequencing of human Monkeypox Virus. protocols.io https://protocols.io/view/nanopore-amplicon-based-whole-genome-sequencing-of-dbku2kww
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: April 02, 2024
Last Modified: January 22, 2026
Protocol Integer ID: 97652
Keywords: Next Generation Sequencing, Virology, Monkeypox virus, whole genome sequencing of human monkeypox virus, human monkeypox virus, monkeypox virus, yale hmpxv amplicon panel pcr primer, yale hmpxv amplicon panel pcr primers from idt, nanopore amplicon, pcr amplicon, based whole genome sequencing, gridion instrument from oxford nanopore, sequencing protocol, oxford nanopore, sequencing reaction, minion
Funders Acknowledgements:
Sorbonne University
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Abstract
This procedure is adapted from the Monkeypox virus multiplexed PCR amplicon sequencing protocol by Nicholas F.G. Chen et al. (DOI: dx.doi.org/10.17504/protocols.io.5qpvob1nbl4o/v4). It uses Yale hMPXV Amplicon Panel PCR primers from IDT in two pre-pooled formats at a concentration of 100µM. For this version of the protocol, libraries are prepared using the Rapid Barcoding kit (SQK-RBK110.96) and the sequencing reaction is carried out on a MinION or GridION instrument from Oxford Nanopore.
Guidelines
This protocol is intended for use with R9.4.1 flow cells (FLO-MIN106) and the Rapid Barcoding Kit 96 (SQK-RBK110.96) utilising V10 Oxford Nanopore chemistry. However, you can adapt this procedure to use R10.4.1 flow cells (FLO-MIN114) and V14 chemistry kit from Oxford Nanopore. The recommended replacement is Rapid Barcoding Kit 96 V14 (SQK-RBK114.96).
Materials
- Yale hMPXV Amplicon Panel PCR primers (pre-pooled 100µM)
Note: Yale hMPXV Amplicon Panel PCR primers can be ordered through Integrated DNA Technologies (IDT) with the following link https://go.idtdna.com/Yale-Mpox-Amplicon-Panel-Request-Consult.html.
- Q5 Hot Start High-Fidelity 2X Master Mix from New England Biolabs (M0494)
- Rapid Barcoding Kit 96 from Oxford Nanopore (SQK-RBK110.96)
AMPure XP reagent manufactured by Beckman Coulter, Inc.
- R9.4.1 flow cells from Oxford Nanopore (FLO-MIN106)
- Nuclease-free water
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)
- Qubit Assay Tubes (ThermoFisher Q32856)
- Eppendorf DNA LoBind tubes
- Eppendorf PCR plate 96 LoBind with PCR seals
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Centrifuge capable of taking 96-well plates
- Micro centrifuge
- Vortex mixer
- Thermal cycler
- Multichannel pipettes suitable for dispensing 0.5–10 μl, 2–20 μl and 20–200 μl, and tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Ice bucket with ice
- Timer
- Qubit fluorometer (or equivalent)
Troubleshooting
Safety warnings
Processing of any sample type which could potentially be positive for hMPXV should be conducted in BSL3 settings. Before starting work with these samples, please contact your local EHS (environment, health and safety) or biosafety office for proper guidance on how to work with these samples in your laboratory.
PCR amplification
Dilute primers to a working concentration of 10µM with nuclease-free water (i.e. 1:10 dilution)
*Safe Stopping Point: Pooled Primers Can Be Stored at -20°C*
According to your number of samples, prepare an appropriate volume of the following master mixes in Eppendorf DNA LoBind tubes and mix thoroughly.
Volume per sample:
| Reagent | Pool 1 | Pool 2 | |
| Nuclease-free water | 1.9 µl | 1.9 µl | |
| Yale hMPXV Amplicon pool 1 (10µM) | 1.85 µl | - | |
| Yale hMPXV Amplicon pool 2 (10µM) | - | 1.85 µl | |
| Q5 HS Master Mix 2X | 6.25 µl | 6.25 µl | |
| Total | 10 µl | 10 µl |
Using a stepper pipette or a multichannel pipette, aliquot 10 μl of Pool 1 and Pool 2 master mixes into a clean 96-well plate(s).
Depending on the number of samples, fill each well per column as follows:
| Plate location | x24 samples | x48 samples | x96 samples | |
| Columns | Pool 1: 1-3 Pool 2: 4-6 | Pool 1: 1-6 Pool 2: 7-12 | Pool 1: 1-12 Pool 2: 1-12 |
Note: For x96 samples, Pool 1 is a separate plate to Pool 2.
Transfer 2.5 μl of DNA to the corresponding well for both Pool 1 and Pool 2 in the PCR plate(s), taking care not to cross-contaminate different wells. Mix by pipetting the contents of each well up and down.
*There should be two PCR reactions per sample*
Seal the plate(s) and spin down briefly.
Incubate using the following program, with the heated lid set to 105°C:
| Step | Temperature | Time | Cycle | |
| Initial denaturation | 98°C | 3 min | 1 | |
| Denaturation | 98°C | 15 sec | 35 times | |
| Annealing and extension | 63°C | 5 min | ||
| Hold | 4°C | Hold | Hold |
*Safe Stopping Point: Amplified DNA may remain in the thermocycler at 4°C or stored at -20°C until ready to use*
Addition of rapid barcodes
Spin down the Rapid Barcode Plate and PCR reactions prior to opening to collect material in the bottom of the wells.
Using a multichannel pipette or stepper pipette, transfer 2.5 μl nuclease-free water to the wells of a fresh 96-well plate (Barcode Attachment Plate).
Depending on the number of samples, aliquot into each well of the columns as follows:
| Plate location | x24 samples | x48 samples | x96 samples | |
| Columns | 1-3 | 1-6 | 1-12 |
Using a multichannel pipette, transfer the entire contents of each well of PCR Pool 2 to the corresponding well of PCR Pool 1 and mix by pipetting.
Using a multichannel pipette, transfer 5 μl from each well of PCR Pool 1 (now containing pooled PCR products) to the corresponding well of the Barcode Attachment Plate and mix by pipetting.
Using a multichannel pipette, transfer 2.5 μl from the Rapid Barcode Plate to the corresponding well of the Barcode Attachment Plate, taking care not to cross-contaminate different wells.
Mix by pipetting.
Seal the Barcode Attachment Plate and spin down.
Incubate the plate in a thermal cycler at 30°C for 2 minutes and then at 80°C for 2 minutes.
Pooling samples and clean-up
Briefly spin down the Barcode Attachment Plate to collect the liquid at the bottom of the wells prior to opening.
Pool the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.
We expect to have about ~10 μl per sample.
Mix pooled samples by vortexing.
Transfer half of the barcoded pooled sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Per sample, we expect to take forward ~5 μl.
Resuspend the AMPure XP Beads (AXP, or SPRI) by vortexing.
To the pooled barcoded sample, add an equal volume of resuspended AMPure XP Beads (AXP, or SPRI) and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
Prepare at least 3 ml of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
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 30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet by pipetting in 15 μl Elution Buffer (EB). Incubate for 10 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colourless.
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 HS Assay Kit.
Take forward 800 ng of library and make up the volume to 11 μl with Elution Buffer (EB).
Add 1 μl of Rapid Adapter F (RAP F) to 11 μl of barcoded DNA.
Incubate at room temperature for 5 minutes.
The prepared library is used for loading into the MinION flow cell. Store the library on ice until ready to load.
Priming and loading the SpotON Flow Cell
Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), 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.
Note: For the one-bottle FB format, dilute 30 µl of FLT into 1.170 µl of FB.
Open the MinION / GridION device lid and slide the flow cell under the clip.
Press down firmly on the flow cell to ensure correct thermal and electrical contact.
Complete a flow cell check to assess the number of pores available before loading the library.
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 µl, to draw back 20-30 µl, 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, 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 | |
| Sequencing Buffer II (SBII) | 37.5 μl | |
| Loading Beads II (LBII) mixed immediately before use | 25.5 μl | |
| DNA library | 12 µl | |
| Total | 75 µl |
Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer II (SBII) and Loading Beads II (LBII) because the fuel in the buffer will start to be consumed by the adapter.
Complete the flow cell priming:
Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
Load 200 μl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
Note: Load the library as soon as possible after this step.
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 / GridION device lid.
Sequencing run settings in MinKnow
Barcoding parameters must be set up on MinKNOW prior to the sequencing.
During the run setup, in the Analysis tab, enable Barcoding. Select Edit options, enable Mid-read barcode filtering. Enable Override minium barcoding score and set the value to 60. Enable Override minimum mid-read barcoding score and set the value to 50.
You can now start the sequencing run.
Note: We recommend using superaccurate basecalling for downstream analyses.
Protocol references
DOI: dx.doi.org/10.17504/protocols.io.5qpvob1nbl4o/v4