Severe fever with thrombocytopenia syndrome virus (SFTSV) Genome Sequencing Protocol for Nanopore MinION and GridION

Tsubasa Narita; Tamaki Okabayashi

Feb 16, 2026

Version 2

Severe fever with thrombocytopenia syndrome virus (SFTSV) Genome Sequencing Protocol for Nanopore MinION and GridION V.2

This protocol is a draft, published without a DOI.

Tsubasa Narita¹,
Tamaki Okabayashi²

¹Miyazaki Prefectural Institute for Public Health and Environment, Miyazaki, 889-2155, Japan;
²Center for Animal Disease Control, University of Miyazaki, Miyazaki, 889-2192, Japan

Tsubasa Narita

Miyazaki Prefectural Institute for Public Health and Environ...

Protocol Citation: Tsubasa Narita, Tamaki Okabayashi 2026. Severe fever with thrombocytopenia syndrome virus (SFTSV) Genome Sequencing Protocol for Nanopore MinION and GridION. protocols.io https://dx.doi.org/Version created by Tsubasa Narita

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: December 12, 2025

Last Modified: February 16, 2026

Protocol Integer ID: 234860

Keywords: SFTS, NGS, genome sequencing protocol for nanopore minion, whole genome analysis of sftsv, thrombocytopenia syndrome virus, severe fever with thrombocytopenia syndrome virus, genome sequencing protocol, nanopore minion, performing whole genome analysis, sequencing protocol, sftsv, severe fever, virus, gridion this protocol, nanopore

Funders Acknowledgements:

JSPS KAKENHI

Grant ID: JP23K21273

Abstract

This protocol is for performing whole genome analysis of SFTSV.
This protocol is based on the nCoV-2019 sequencing protocol for Illumina V.4 by Itokawa et al.
This protocol assumes that the S, M, and L segments will be analyzed separately.
This protocol is currently validated to work on the Nanopore MinION and GridION.

Protocol materials

LunaScript RT SuperMix KitNew England BiolabsCatalog #E3010L 
NEB Q5® Hot Start High-Fidelity 2X Master MixNew England BiolabsCatalog #M0494L 
Agencourt AMPure XP beadsBeckman CoulterCatalog #A63881 
1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA) 
Qubit dsDNA HS assay kitFisher ScientificCatalog #Q32854 
Native Barcoding Expansion 13-24 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD114 
NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767 
SFB expansionOxford Nanopore TechnologiesCatalog #EXP-SFB001 
NEB Blunt/TA Ligase Master MixNew England BiolabsCatalog #M0367 
NEBNext Ultra II End repair/dA-tailing ModuleNew England BiolabsCatalog #E7546 
Native Barcoding Expansion 1-12 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD104 
NEBNext Quick Ligation Module - 100 rxnsNew England BiolabsCatalog #E6056L 

Safety warnings

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the
Ethics Committee of Miyazaki prefectural Institute for Public Health and Environment (No. 6 and 2022-July.-21 as date of approval). 
If this protocol is to be carried out using human samples, prior approval by the user's Institutional Ethics Board or equivalent ethucs committee is required.

Ethics statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the
Ethics Committee of Miyazaki prefectural Institute for Public Health and Environment (No. 6 and 2022-July.-21 as date of approval). 

Before start

We use this protocol and it's working.
This protocol has now been verified to work for human and feline blood and tick samples.

cDNA Synthesis

23m

Mix the following components in an 0.2mL 8-strip tube or 96 well PCR plate;

LunaScript RT SuperMix KitNew England BiolabsCatalog #E3010L   

Component                                      Volume/sample

LunaScript RT SuperMix        2 µL   
Template RNA                         8 µL   
Total                                                   10 µL   

    Gently mix by pipetting and spin down.

Incubate the reaction as follows:

25 °C   for 00:02:00   
55 °C   for 00:20:00   (*1)
95 °C   for 00:01:00   
Hold at 4 °C   

Note
*1 Following the method of Itokawa et al., the time was set to 20 min.

23m

Multiplex PCR

3h 4m 15s

 According to the attached primer list, prepare primer sets, pool1 and pool2, for each segment by adding equal amounts of each primer diluted to 10 µM.

Primer list.xlsx12.9KB 

Note
These primer set are compatible with strains confirmed in Japan, but has not been verified with strains outside of Japan. If strains from outside japan are used, we recommend using Primal Scheme (https://primalscheme.com/) to design primer.

Note
We recommend adding twice the amount of each 5' end and 3' end primer pair. In addition, read depth can be improved by doubling the amount of primer pair added in areas with poor reaction.

Mix the following components in the 0.2mL 8-strip tube or 96 well PCR plate;

NEB Q5® Hot Start High-Fidelity 2X Master MixNew England BiolabsCatalog #M0494L  

S Segment

Component                                      Pool1                        Pool2

Q5 HS Hi-Fi 2X Master Mix     12.5 µL       12.5 µL   
Primer pool1  (10µM)                0.3 µL          
primer pool2 (10µM)                                           0.3 µL   
Nuclease-free water                9.7 µL          9.7 µL   
Total                                                      22.5 µL       22.5 µL   

M Segment                                       

Component                                      Pool1                        Pool2

Q5 HS Hi-Fi 2X Master Mix     12.5 µL          12.5 µL     
Primer pool1  (10µM)                0.45 µL    
primer pool2 (10µM)                                              0.45 µL                                
Nuclease-free water                9.55 µL         9.55 µL   
Total                                           22.5 µL         22.5 µL   

L Segment

Component                                      Pool1                         Pool2

Q5 HS Hi-Fi 2X Master Mix     12.5 µL          12.5 µL   
Primer pool1  (10µM)                0.75 µL               
primer pool2 (10µM)                                             0.75 µL                             
Nuclease-free water                9.25 µL         9.25 µL  
Total                                          22.5 µL         22.5 µL   

Add 2.5 µL    of cDNA to each of the Pool 1 and Pool 2 reactions and mix well by pipetting.

Spin down pool1 and pool2 tubes.

Set-up the following program on the thermal cycler:  

Stage                         Temperature          　　　　　　　　      Time

Heat Activation    98 °C              　　　　　　　　    00:00:30   

35 cycles of...
  Denaturation        98 °C               　　　　　　　　 00:00:15   
  Annealing             60 °C  (L segment:61℃) 　　　  00:05:00    　　　

Hold at 4 °C   

3h 4m 15s

Purification

9m 30s

Prepare the new 8-strip tube or 96-well plate and add Pool 1 to Pool 2

Agencourt AMPure XP beadsBeckman CoulterCatalog #A63881  
1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)  

Prepare Agencourt AMPure XP and vortex thoroughly to disperse any precipitated beads. Add 50 µL    of AMPure XP solution to each sample, mix thoroughly, and leave at Room temperature    for  00:05:00     .

Note
When adding AmpureXP to samples, pipette thoroughly.

Follow the steps below to wash the beads and elute DNA.

1. The 8-strip PCR tubes or 96-well  PCR plate is placed on the magnetic stand and left to stand until the magnetic beads are attracted to the magnet and the liquid becomes transparent.

2. Remove the supernatant on the magnetic stand, being careful not to aspirate the magnetic beads.

Note
DNA is binded to the magnetic beads, so if the beads are aspirated, it will cause DNA loss.

3. Add 150 µL    of the 80 % ethanol on the magnetic stand.

Note
Prepare 80% ethanol just before use.

4. Leave the tube or plate on the magnetic stand for 00:00:30   .

5. Remove the 80% ethanol from the tube or plate on the magnetic stand.

6. Repeat steps 3-5.

7. Leave the tube or plate on the magnetic stand for about 00:03:00    until the remaining ethanol evaporates. It is not necessary to dry it completely.

8. Remove the tube or plate from the magnetic stand and add 20 µL    of Low TE buffer to each sample and mix thoroughly with the magnetic beads.

9. Leave the tube or plate for 00:01:00   .

10. Place the tube or plate on the magnetic stand for 2 minutes.

11. The supernatant is transferred to the new  8-strip PCR tubes or 96-well  PCR plate.

9m 30s

Quantification

Quantify the amplicon pools using fluorescent based method such as Qubit dsDNA HS kit (Invitrogen).  

Qubit dsDNA HS assay kitFisher ScientificCatalog #Q32854  

Note
If you perform analysis with both Illumina sequencer and Nanopore sequencer, you can reuse the samples up to this point if you stock them.

End-prep & Barcoding

1h 2m 30s

Prepare  the new 8-strip PCR tubes or 96-well PCR plate.

Based on the quantification results, the purified PCR product of each sample was diluted with Nuclease-free water to a total DNA volume of 50 ng/12.5 µL.

Note
Samples with DNA concentrations below 4ng/µl may not be able to be analyzed. If you absolutely must analyze samples with DNA concentration below 4ng/µl, we recommend that you use the sample undiluted.

Prepare the End-Prep Master Mix as follows:

NEBNext Ultra II End repair/dA-tailing ModuleNew England BiolabsCatalog #E7546  

Component                                                                                         Volume/Sample

NEBNext Ultra II End Prep Reaction Buffer                    1.75 µL   
NEBNext Ultra II End Prep Enzyme Mix                         0.75 µL   
Total                                                                                 2.5 µL   

Add 2.5 µL    of the above master mix to each well of the 12.5 µL    diluted DNA solution.

Set up the thermal cycler program in advance as follows. Keep the heat-lid at 80 °C   .

Temprature               Time

20 °C                00:10:00   
65 °C                00:10:00   
Hold at 4 °C   

Prepare the new 8-strip PCR tubes or 96-well PCR plate and prepare the Reaction Mix as follows:

Native Barcoding Expansion 1-12 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD104  

Native Barcoding Expansion 13-24 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD114  

Note
As of July 2025, the above products are no longer available for sale. Please use the following kit as an alternative.

Native Barcoding Kit 24 V14 (SQK-NBD114.24)Oxford Nanopore TechnologiesCatalog #SQK-NBD114.24  

NEB Blunt/TA Ligase Master MixNew England BiolabsCatalog #M0367  

Component                                           Volume/Sample

Nuclease-free water                   6 µL   
End-preped DNA                         1.5 µL   
Native Barcode                            2.5 µL   
Blunt/TA Ligase Master Mix        10 µL   
Total                                                          20 µL   

Note
Dispense with care as the reagent is highly viscous.

Mix thoroughly with gentle vortexing and react in the thermal cycler as follows. Keep the heat-lid at 80 °C    .

Temperature              Time
20 °C                  00:20:00   
65 °C                  00:10:00   
Hold at 4 °C   

Prepare the new 2.0 mL low-binding tube and pool all the barcoding reaction samples. After pooling, measure the volume of the solution (V µl).

Mix the NEBNext Sample Purification Beads solution thoroughly by vortexing, add 0.4 volume of the NEBNext Sample Purification Beads solution to the pooled reactions, mix thoroughly, and let sit for 00:05:00   .

NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767  
SFB expansionOxford Nanopore TechnologiesCatalog #EXP-SFB001  

Follow the steps below to wash the beads and elute DNA.

1. The tubes is placed on the magnetic stand and left to stand until the magnetic beads are attracted to the magnet and the liquid becomes transparent.

2. Remove the supernatant on the magnetic stand, being careful not to aspirate the magnetic beads.

Note
DNA is binded to the magnetic beads, so if the beads are aspirated, it will cause DNA loss.

3. Add 250 µL    of SFB to the aggregated beads on a magnetic stand, and vortex.

4. After leaving the tube for 00:01:00   , spin it down and return it to the magnetic stand.

5. Remove the supernatant on the magnetic stand, taking care not to aspirate the beads.

6. Repeat SFB washing (steps 3-5) once more.

7. Add 200 µL    of the the 80% ethanol to the tube while it is still on the stand. Leave it for 00:00:30   .

Note
Prepare the 80% ethanol just before use.

8. Remove the supernatant on the magnetic stand, taking care not to aspirate the beads.

9. Leave the tube on the magnetic stand for about  00:03:00    until the remaining ethanol evaporates. It is not necessary to dry it completely.

10. Remove the tube from the magnetic stand and add 32 µL of Nuclease-free water and mix thoroughly with the magnetic beads.

11. Leave the tube for 00:01:00   .

12. Spin the tube down and place on the magnetic stand for 00:02:00   .

13. Transfer the supernatant to a new 2.0 ml low-binding tube.

1h 2m 30s

Quantification

Quantify the amplicon pools using fluorescent based method such as Qubit dsDNA HS kit (Invitrogen). 

Qubit dsDNA HS assay kitFisher ScientificCatalog #Q32854  

Adaptor Ligation & Purification

29m

In the new 2.0 mL low-binding tube, dilute the purified barcoded DNA to 1 ng/µL with Nuclease-free water.
If the amount of DNA is less than 1ng/µl, proceed to the next step without dilution.

Prepare Adapter Ligation Reaction Mix as follows:

NEBNext Quick Ligation Module - 100 rxnsNew England BiolabsCatalog #E6056L  

Component                                                                        Volume
Barcoded DNA (Diluted)                                     30 µL   
Adapter Mix II                                                      5 µL   
NEBNext Quick Ligation Reaction Buffer           10 µL   
NEBNext Quick T4 Ligase                                   5 µL   
Total                                                                                         50 µL   

Incubate at Room temperature    for 00:20:00   

Mix the NEBNext Sample Purification Beads solution thoroughly by vortexing, add 0.4 volume of the NEBNext Sample Purification Beads solution to the pooled reactions, mix thoroughly, and let sit for 00:05:00    .

NEBNext Sample Purification BeadsNew England BiolabsCatalog #E7767  

Follow the steps below to wash the beads and elute the DNA.

SFB expansionOxford Nanopore TechnologiesCatalog #EXP-SFB001  

Note
This is similar to the previous elution process, but be careful not to use the ethanol. If you do use the ethanol, you must restart over from Barcoding or Adapter Ligation.

1. The tubes is placed on the magnetic stand and left to stand until the magnetic beads are attracted to the magnet and the liquid becomes transparent.

2. Remove the supernatant on the magnetic stand, being careful not to aspirate the magnetic beads.

Note
DNA is binded to the magnetic beads, so if the beads are aspirated, it will cause DNA loss.

3. Add 250 µL     of SFB to the aggregated beads on the magnetic stand, and vortex.

4. After leaving the tube for 00:01:00    , spin it down and return it to the magnetic stand.

5. Removed the supernatant on the magnetic stand, taking care not to aspirate the beads.

6. Repeat SFB washing (steps 3-5) once more.

7. Remove the tube from the magnetic stand and add 15 µL    of Elution Buffer (EB) and mix thoroughly with the magnetic beads.

11. Leave the tube for 00:01:00    .

12. Spin the tube down and place on a magnetic stand for 00:02:00    .

13. Transfer the supernatant to a new 2.0 ml low-binding tube.

29m

Priming & Loading

Perform Priming & Loading according to the manual provided by Nanopore.

Protocol references

nCoV-2019 sequencing protocol for illumina V.4 ,Kentaro Itokawa
nCoV-2019 sequencing protocol V.1 , Josh Quick