Dec 12, 2025
Version 2
Severe fever with thrombocytopenia syndrome virus (SFTSV) Genome Sequencing Protocol for illumina 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
Protocol Citation: Tsubasa Narita, Tamaki Okabayashi 2025. Severe fever with thrombocytopenia syndrome virus (SFTSV) Genome Sequencing Protocol for illumina. 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 11, 2025
Last Modified: December 12, 2025
Protocol Integer ID: 234812
Keywords: SFTS, NGS, genome sequencing protocol for illumina, thrombocytopenia syndrome virus, whole genome analysis of sftsv, severe fever with thrombocytopenia syndrome virus, genome sequencing protocol, performing whole genome analysis, sequencing protocol, illumina iseq, severe fever, sftsv, virus, illumina
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.
The library was prepared at 1/4 scale according to the procedure of 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 Illumina iSeq 100.
Protocol materials
LunaScript RT SuperMix KitNew England BiolabsCatalog #E3010L
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 KitThermo Fisher ScientificCatalog #Q32854
QIAseq FX DNA Library KitQiagenCatalog #180475
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 the 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 outside of Japan
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 primers.
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;
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
11m 30s
Prepare the new 8-strip tube 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 00:02:00 .
11. The supernatant is transferred to a new 8-strip PCR tubes or 96-well PCR plate.
11m 30s
Quantification
Quantify the amplicon pools using fluorescent based method such as Qubit dsDNA HS kit (Invitrogen).
Qubit dsDNA HS Assay KitThermo Fisher 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.
Fragmentation & End-prep
37m
Note
This protocol uses 1/4 the amount of material used in the original QIAseq FX DNA Library Kit protocol.
Transfer 2 µL of pooled & purified PCR products to each well of the new 8-strip PCR tubes or 96-well PCR plate. Then, place the tube or plate at aluminum block On ice .
Set up the thermal cycler program in advance as follows and make it ready to start immediately. Keep the heat-lid at 80 °C .
Tempreture Time
4 °C 00:40:00 (*2)
32 °C 00:06:00
65 °C 00:30:00
Hold at 4 °C
Note
*2 The time is preset to 00:40:00 , but after adding the sample to the master mix below, set it in the thermal cycler, skip after 00:01:00 , and raise the temperature to 32 °C .
Prepare the master mix per one sample as below.
QIAseq FX DNA Library KitQiagenCatalog #180475
Component Volume/Sample
FX Buffer, 10x 1.25 µL
Nuclease-free Water 6.75 µL
FX Enzyme Mix 2.5 µL
Total 10.5 µL
Add 10.5 µL of master mix to 2 µL of pooled & purified PCR products On ice .
Note
Keep On ice during this procedure.
Place the library tube or plate into the thermal cycler posing at 4 °C and immediately skip to the next step (32 °C )(*2).
37m
Adaptor Ligation
35m
Remove the tube or plate from the thermal cycler after finishing the thermal program.
Take the adapter plate out of the kit box and thaw the content.
Note
Keep both end-prepped DNA mixture and thawed adapter solution On ice during this step.
QIAseq FX DNA Library KitQiagenCatalog #180475
Add 1 µL adapter solution to each end-prepped DNA mixture.
Prepare the master mix per sample as below.
Component Volume/Sample
DNA Ligase Buffer, 5x 5 µL
DNA Ligase 2.5 µL
Nuclease-free water 4 µL
Total 11.5 µL
Set up the thermal cycler program in advance as follows. Keep the heat-lid at 80 °C .
Temperature Time
20 °C 00:15:00
65 °C 00:20:00
Hold at 4 °C
35m
Library pooling & purfication
22m
Prepare the 2.0ml low-binding tube.
Take3 µL or 6 µL ligated mixture from each well and pool them into the 2.0 ml low-binding tube.
The amount of ligated mixture to be added is determined based on the amount of DNA measured by Qubit in the Quantification section.
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Note
ex) Regarding the addition of ligated mixture, if DNA amount is 10 ng/l or less, add 6 µL . If it is 10 ng/mµl or more, add 3 µL .
Measure the volume of pooled mixture (V)
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, and add v × 0.8 µl of the AMPureXP solution to the pooled library solution. Incubate 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 the 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 500 µL of the 80 % ethanol on the magnetic stand.
Note
Prepare 80% ethanol just before use.
4. Vortex and spin down.
5. Place the tube on a magnetic stand to collect the magnetic beads.
6. Remove the 80% ethanol from the tube on the magnetic stand.
7. Repeat steps 3-6.
8. 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.
9. Remove the tube from the magnetic stand, add 50 µL of Nuclease-free water, and mix thoroughly with the magnetic beads.
10. Leave the tube for 00:01:00 .
11. Spin the tube down and place the tube on a magnetic stand for 00:02:00 .
12. Transfer the supernatant to the new 2.0 ml low-binding tube.
Prepare Agencourt AMPure XP and vortex thoroughly to disperse any precipitated beadsand, and add 60 µl (1.2x) of AMPureXP solution. Leave at room temperature for 00:05:00 .
Note
When adding AmpureXP to samples, pipette thoroughly.
Further, re-purification is carried out according to the following procedure.
1. The tube 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 500 µL of the 80 % ethanol on the magnetic stand.
Note
Prepare 80% ethanol just before use.
4. Vortex and spin down.
5. Place the tube on a magnetic stand to collect the magnetic beads.
6. Remove the 80% ethanol on the magnetic stand.
7. Repeat steps 3-6.
8. 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.
9. Remove the tube from the magnetic stand, add 50 µL of Low-TE Buffer, and mix thoroughly with the magnetic beads.
10. Leave the tube for 00:01:00 .
11. Spin the tube down and place on a magnetic stand for 00:02:00 .
12. Transfer the supernatant to the new 2.0 ml low-binding tube.
22m
Quantification & Sequencing
Quantify the Library using fluorescent based method such as Qubit dsDNA HS kit (Invitrogen).
Perform sequencing according to the manual provided by Illumina.
Protocol references
nCoV-2019 sequencing protocol for illumina V.4 , Itokawa et al.