Jan 12, 2026

Public workspaceNuclease-MDA for Enriching Monkeypox Virus Genomes for NGS

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l22524l1y/v1
  • Masayasu Misu1,
  • Takeshi Kurosu1,
  • Tomoki Yoshikawa1,
  • Madoka Kawahara1,
  • Kohei Oishi1,
  • Masayuki Shimojima1,
  • Hideki Ebihara1
  • 1Department of Virology I, National Institute of Infectious Diseases
Tomoki Yoshikawa
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l22524l1y/v1
Protocol Citation: Masayasu Misu, Takeshi Kurosu, Tomoki Yoshikawa, Madoka Kawahara, Kohei Oishi, Masayuki Shimojima, Hideki Ebihara 2026. Nuclease-MDA for Enriching Monkeypox Virus Genomes for NGS. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22524l1y/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: June 20, 2023
Last Modified: January 12, 2026
Protocol Integer ID: 83667
Keywords: Oxford Nanopore Technology, RNA virus, Sequence method, MinION, Nanopore sequencing, RCA-NGS, mda for enriching monkeypox virus, enrichment of the monkeypox virus, enriching monkeypox virus, viral genome, genome sequencing of mpxv, monkeypox virus, intact viral genome, idt for illumina dna, unwanted host dna via micrococcal nuclease treatment, illumina dna prep, high proportion of viral read, illumina dna, genome sequencing, viral read, genome amplification, cowpox virus, virus concentration step, micrococcal nuclease treatment, unwanted host dna, other poxvirus, rna, employing phi29 polymerase, genome, phi29 polymerase, nuclease, sequencing, oxford nanopore technology, vaccinia virus, iseq100 system
Funders Acknowledgements:
Ministry of Health, Labor and Welfare of Japan
Grant ID: 20HA2005 and 23HA2004
Japan Agency for Medical Research and Development
Grant ID: 21fk0108426j0001, 25fk0108660h0003, 22fk0108502j0101 and 23fk0108581j0701
Japan Society for the Promotion of Science
Grant ID: JP22K085
Abstract
This protocol describes the enrichment of the monkeypox virus (MPXV) genome using the Nuclease-MDA method, optimized for Oxford Nanopore Technologies (ONT) sequencing with V14 chemistry.

This protocol can be applied using the ONT Native Barcoding Kit(SQK-NBD114.24, SQK-NBD114.96), Rapid Barcoding Kit V14 (SQK-RBK114.24 or SQK-RBK114.96) or Illumina DNA Prep, (M) Tagmentation (24 Samples, IPB) (Cat#20060060), IDT for Illumina DNA/RNA UD Indexes Set A (Cat#20027213), and the iSeq100 system.

This approach eliminates unwanted host DNA via micrococcal nuclease treatment, preserving the intact viral genome. The viral genome is then amplified using multiple displacement amplification (MDA), an isothermal whole-genome amplification (WGA) technique employing Phi29 polymerase. This method provides a high proportion of viral reads (~90%) without requiring specific PCR or virus concentration steps.

This method enables high-depth whole-genome sequencing of MPXV and can be readily adapted to other poxviruses, such as vaccinia virus and cowpox virus, with appropriate primer modifications.
Materials
  • ReagentMicrococcal Nuclease - 320,000 gel unitsNew England BiolabsCatalog #M0247S
  • ReagentHigh Pure Viral Nucleic Acid KitRocheCatalog #11858874001
  • Dr.GenTLE Precipitation Carrier - Takara Catalog #9094
  • ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
  • GenomiPhi V3 Ready-To-Go DNA Amplification Kit - Cytiva Catalog #25-6601-24
  • ReagentT7 Endonuclease I - 250 unitsNew England BiolabsCatalog #M0302S
  • ReagentNEBNext FFPE DNA Repair Mix - 24 rxnsNew England BiolabsCatalog #M6630S
  • ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
  • ReagentBlunt/TA Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0367S
  • ReagentNEBNext Quick Ligation Module - 20 rxnsNew England BiolabsCatalog #E6056S
  • Native Barcoding Kit 24 V14 - Oxford Nanopore Technologies Catalog #SQK-NBD114.24
  • ReagentQubit 4 FluorometerThermo Fisher ScientificCatalog #Q33238
  • ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230
  • ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021
  • Reagent0.2 ml PCR Tube stripsEppendorfCatalog #0030124359
  • 100 % ethanol
  • 70 % ethanol
  • TE(pH8.0)
  • nuclease-free H2O





Protocol materials
ReagentMicrococcal Nuclease - 320,000 gel unitsNew England BiolabsCatalog #M0247S
ReagentT4 RNA Ligase 2, truncated KQ - 2,000 unitsNew England BiolabsCatalog #M0373S
ReagentSuperscript IVThermo Fisher ScientificCatalog #18090050
Reagent0.2 ml PCR Tube stripsEppendorfCatalog #0030124359
ReagentNEBNext FFPE DNA Repair Mix - 24 rxnsNew England BiolabsCatalog #M6630S
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
ReagentHigh Pure Viral Nucleic Acid KitRocheCatalog #11858874001
ReagentQubit 4 FluorometerThermo Fisher ScientificCatalog #Q33238
ReagentQubit 1X dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q33230
ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021
ReagentT7 Endonuclease I - 250 unitsNew England BiolabsCatalog #M0302S
ReagentBlunt/TA Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0367S
ReagentNEBNext Quick Ligation Module - 20 rxnsNew England BiolabsCatalog #E6056S
ReagentQubit dsDNA BR (Broad Range) assayThermo Fisher ScientificCatalog #Q32850
Troubleshooting
Safety warnings
Follow your facility's regulations and biosafety practices.
Before start
It is recommended to check for the absence of bacterial contamination (e.g., Mycoplasma spp.).
Preparation for virus supernatant
Centrifuge the working stock virus to remove debris.
Centrifigation6000 x g, Room temperature, 00:10:00

10m
Transfer Amount180 µL virus supernatant to a 1.5ml screw cap tube.
Unwanted DNA and RNA mainly originating from the virus-infected cells are digested usingReagentMicrococcal Nuclease - 320,000 gel unitsNew England BiolabsCatalog #M0247S .
Total 200 μl reaction

  • Amount179 µL virus supernatant
  • Amount20 µL 10X Micrococcal Nuclease Reaction Buffer
  • Amount1 µL Micrococcal nuclease

Mix by pipetting and spin down.
Temperature37 °C water bath Duration01:00:00


1h
The viral genomic DNA extraction
The viral genomic DNA extraction is performed using ReagentHigh Pure Viral Nucleic Acid KitRocheCatalog #11858874001 .

Add Amount200 µL of binding buffer, Amount4 µL PolyA carrier RNA and Amount50 µL Proteinase K.

Mix well by pipetting and inverting the tube thoroughly, and spin down.
Temperature72 °C Duration00:10:00
10m
Add Amount100 µL of binding buffer and mix well by pipetting or inverting the tube thoroughly, and spin down.
Transfer the whole sample to a High Pure Filter Tube.
Centrifigation8000 x g, Room temperature, 00:01:00

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube.
1m
Add Amount500 µL of inhibitor removal buffer.
Centrifigation8000 x g, Room temperature, 00:01:00

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube.
1m
Add Amount450 µL of wash buffer.
Centrifigation8000 x g, Room temperature, 00:01:00

Discard the flow-through liquid and Collection Tube, and insert the Filter Tube into a new Collection Tube.
1m
Add Amount450 µL of wash buffer.
Centrifigation13000 x g, Room temperature, 00:01:00 and discard the flow-through liquid.

Discard the Collection Tube and insert the Filter Tube into a 1.5 ml tube -ReagentDNA LoBind Tube 1.5ml EppendorfCatalog #022431021 .

1m
Add Amount50 µL elution Buffer.
Centrifigation13000 x g, Room temperature, 00:01:00

Note
The purified Amount50 µL viral genomic DNA can be stored at -80℃.


1m
Amplification of DNA by multiple displacement amplification (MDA)
DNA is amplified by MDA using GenomiPhi V3 Ready-To-Go DNA Amplification Kit - Cytiva Catalog #25-6601-24.

Total 20 μl reaction

  • Amount10 µL purified viral genomic DNA
  • Amount10 µL 2X denaturation buffer

Mix by pipetting and spin down.
Temperature95 °C Duration00:03:00
Temperature4 °C on ice
3m
Add Amount20 µL denatured sample to Ready to go GenomiPhi cake.
Temperature30 °C Duration04:00:00
Temperature65 °C Duration00:10:00
Note
The amplified Amount20 µL viral genomic DNA can be stored at -80℃.



4h 10m
DNA purification by AMpure XP
The DNA is purified using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
Add Amount10 µL (X0.5 volume) AMPure beads
Incubate on rotor mixer.
Duration00:05:00 TemperatureRoom temperature

5m
Spin down and pellet on a magnet.
Wait forDuration00:01:00 and pipette off the supernatant.

1m
Wash twice by Amount100 µL 70 % ethanol, remove the ethanol using a pipette, and discard.

Spin down and pipette off any residual ethanol.
Resuspend pellet inAmount40 µL nuclease-free H2O.
Incubate on a rotor mixer.
Duration00:05:00 TemperatureRoom temperature
5m
Spin down and pellet the beads on the magnet until the elute is clear and colorless.
Remove and retainAmount40 µL elute into a new tube.
DNA concentration is measured using a Qubit 4 Fluorometer with ReagentQubit dsDNA BR (Broad Range) assayThermo Fisher ScientificCatalog #Q32850 .

  • Amount199 µL 1X working solution
  • Amount1 µL DNA

Mix by vortexing.

Incubate Duration00:02:00 TemperatureRoom temperature and measure.

Note
Confirm the total amplified DNA to be over 1500 ng.
The DNA can be stored at Temperature-30 °C for at least two months.


Note
At this step, whole-genome sequencing using an Illumina next-generation sequencer is also possible. In this case, the T7 endonuclease treatment and any subsequent steps in this protocol are not required. Instead, you should follow Illumina’s library preparation protocol. We have confirmed that this works using the Illumina DNA Prep, (M) Tagmentation (Cat#20060060) and the IDT for Illumina DNA/RNA UD Indexes Set A (Cat#20027213).

Similarly, the Oxford Nanopore Rapid Barcoding Kit 24 V14 (SQK-RBK114.24) can also be used. Here, the T7 endonuclease treatment is also not required. From this point, you should proceed by following the Rapid Barcoding Kit protocol, which significantly reduces library preparation time.

2m
T7 endonuclease treatment
The amplified DNA by MDA is digested using ReagentT7 Endonuclease I - 250 unitsNew England BiolabsCatalog #M0302S .

The following protocol is modified based on the Native barcoding amplicons (with EXP-NBD104, EXPNBD114, and SQK-LSK109) protocol (NBA_9093_v109_revA_12Nov2019) provided by the Oxford Nanopore Technologies website.
Total 30 μl reaction

  • Amountx µL (1.0 μg) DNA
  • Amount3 µL NEBuffer 2
  • Amount1.5 µL T7 endonuclease I
  • Amount25.5-x µL nuclease-free H2O

Mix by pipetting and spin down.
Temperature37 °C Duration00:30:00
30m
The DNA is purified using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
Add Amount15 µL (X0.5 volume) AMPure beads
Go to
Resuspend pellet in Amount13 µL nuclease-free H2O.
DNA repair and end-prep
The purified DNA is end-prepped using
ReagentNEBNext FFPE DNA Repair Mix - 24 rxnsNew England BiolabsCatalog #M6630S and
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
Total 15 μl reaction

  • Amount12 µL DNA
  • Amount0.875 µL NEB Next FFPE DNA repair buffer
  • Amount0.5 µL NEB Next FFPE DNA repair Mix
  • Amount0.875 µL Ultra II end-prep reaction buffer
  • Amount0.75 µL Ultra II end-prep reaction Mix

Mix by pipetting and spin down.
Temperature20 °C Duration00:30:00
Temperature65 °C Duration00:05:00
35m
The DNA is purified using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880 .
Add Amount15 µL (X1.0 volume) AMPure beads
Go to
Resuspend pellet in Amount10 µL nuclease-free H2O.
Note
The DNA can be stored at 4℃ overnight.

Native barcode ligation
The end-prepped DNA is ligated with native barcode using Native Barcoding Kit V14 - Oxford Nanopore Technologies Catalog #SQK-NBD114.24 or #SQK-NBD114.24 with ReagentBlunt/TA Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0367S .
Total 20 μl reaction

  • Amount8.5 µL DNA
  • Amount1.5 µL native barcode
  • Amount10 µL Blunt/TA ligase master mix

Mix by pipetting and spin down.
TemperatureRoom temperature Duration00:20:00
20m
Add the following volume of EDTA to each well and mix thoroughly by pipetting and spin down briefly.
Note
Ensure you follow the instructions for the cap colour of your EDTA tube.
EDTA cap colourVolume per well
For clear cap EDTA2 µl
For blue cap EDTA 4 µ
EDTA is added at this step to stop the reaction.

Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.
The DNA is purified using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880
Add Amount8.8 or 9.6 µL (X0.4 volume) AMPure beads multiply by the original number of tubes.
Incubate on rotor mixer.
Duration00:05:00 TemperatureRoom temperature
5m
Spin down and pellet on a magnet.
Wait forDuration00:01:00 and pipette off the supernatant.
1m
Wash twice by Amount700 µL 70 % ethanol, remove the ethanol using a pipette, and discard.
Spin down and pipette off any residual ethanol.
Resuspend the pellet in 15 µL of nuclease-free H2O.
If the pooled sample volume exceeds 100 µL (i.e., more than 5 samples), resuspend the pellet in x0.2 volume of the pooled sample (e.g., 20 µL when 5 samples are pooled) of nuclease-free H2O.
Incubate on a rotor mixer.
Duration00:10:00 Temperature37 °C
Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
10m
Spin down and pellet the beads on the magnet until the elute is clear and colorless.
Remove and retain the elute into a new tube.
Adaptor ligation and clean-up
20m
Adaptor Ligation with pooled samples is performed using
Ligation Sequencing Kit - Oxford Nanopore Technologies Catalog #SQK-NBD114.24 or #SQK-NBD114.96 with ReagentNEBNext Quick Ligation Module - 20 rxnsNew England BiolabsCatalog #E6056S
Total 20 μl reaction

  • Amount12 µL DNA
  • Amount2 µL Native Adapter (NA)
  • Amount4 µL NEB Next Quick Ligation Reaction Buffer(5X)
  • Amount2 µL Quick T4 DNA ligase

Mix gently and incubate.
TemperatureRoom temperature Duration00:20:00
20m
The adaptor-ligated DNA is purified using ReagentAgencourt AMPure XPBeckman CoulterCatalog #A63880 .
Prepare AMpure XP beads for use; resuspend by vortexing.
Transfer amplified DNA sample to 1.5ml low binding tube.
Add Amount10 µL (X0.5 volume) AMPure XP reagent and mix by pipetting.
Incubate on a rotor mixer.
Duration00:05:00 TemperatureRoom temperature
Spin down and pellet on a magnet. Wait for Duration00:01:00 and pipette off the supernatant.
  • Wash twice by Amount100 µL Short Fragment Buffer(SFB) and remove the SFB using a pipette and discard.
Spin down and pipette off any residual SFB.
  • Resuspend pellet in Amount15 µL Elution Buffer (EB)
Duration00:05:00 TemperatureRoom temperature and tapping occasionally.

5m
Spin down and pellet the beads on the magnet until the elute is clear and colourless.
Remove retain Amount15 µL elute into a new tube.
DNA concentration is measured using a Qubit 4 Fluorometer with ReagentQubit dsDNA BR (Broad Range) assayThermo Fisher ScientificCatalog #Q32850 .

  • Amount199 µL 1X working solution
  • Amount1 µL DNA

Mix by vortexing.

Incubate Duration00:02:00 TemperatureRoom temperature and measure.

Note
The molar concentration of the DNA sample can be converted based on the length of the major band con¦rmed by electrophoresis after T7 endonuclease treatment. Typically, the fragment lengths are around 2000 base pairs.

2m
Make up the library to Amount12 µL at 10-20 fmol

Sequencing by MinION
Sequencing according to the manufacturer's instructions.