May 23, 2025

West Nile Virus (Orthoflavivirus nilense, (Lineage 2) amplicon sequencing for Illumina(LowCost) V.2

This  protocol  is a draft, published without a DOI.
West Nile Virus (Orthoflavivirus nilense, (Lineage 2) amplicon sequencing for Illumina(LowCost)
  • 1Bernhard Nocht Institute for Tropical Medicine
Gábor óth
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Gábor óth, Marike Petersen, Alexandra Bialonski, Heike Baum, Marcy A. Sikora, Alex Tomazatos, Balázs orváth, Jonas Schmidt-Chanasit, Dániel adar 2025. West Nile Virus (Orthoflavivirus nilense, (Lineage 2) amplicon sequencing for Illumina(LowCost). protocols.io https://dx.doi.org/Version created by Gábor óth
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: In development
We are still developing and optimizing this protocol
Created: February 05, 2025
Last Modified: May 23, 2025
Protocol  Integer ID: 119628
Keywords: WNV, West Nile virus, Targeted Sequencing, Illumina, iSeq100, Lineage-2, west nile virus, orthoflavivirus nilense, many human pathogenic virus, widespread arbovirus, important virus, zika virus, sequencing method, ebola virus, methods for wnv, humans via mosquito bite, illumina iseq, virus, wnv, mosquito bite, iseq100, natural circulation of wnv, illumina platform, shorter amplicon length, molecular epidemiology, illumina platform on the market, illumina, sequencing
Abstract
Amplicon based targeted sequencing methods proved their importance in case of many human pathogenic viruses (e.g. Zika virus, Ebola virus, SARS-Cov 2). Application and development of this methods for other medically important viruses could be also beneficial as it ensure the possibility of molecular epidemiology.
West Nile virus (WNV, Orthoflavivirus nilense) is widespread arbovirus which is mainly transmitted to humans via mosquito bites. In the natural circulation of WNV multiple hosts are involved which raise the need for sufficient sequencing method.
There are already available, established targeted sequencing methods for WNV lineage-2, but we wanted to develop an additional one which uses shorter fragment length and optimised for Illumina iSeq 100. Shorter amplicon length works better on low quality samples and iSeq100 is the cheapest (most accessible) Illumina platform on the market.
Materials

ABC
Reagents
ComponentSupplierCatalog number
QIAamp Viral RNA Mini KitQiagen52904
RealStar® WNV RT-PCR Kit 2.0altona Diagnostics GmbH322013
SuperScript IV Reverse TranscriptaseInvitrogen18090010
Random Hexamers (50 μM)InvitrogenN8080127
RNaseOUT™ Recombinant Ribonuclease InhibitorInvitrogen10777019
Q5® High-Fidelity DNA PolymeraseNew England Biolabs M0491S
dNTP Mix (10 mM each)Thermo Scientific™R0192
Individual primersIDT or Eurofins etc.
NEBNext® Ultra™ II End Repair/dA-Tailing ModuleNew England Biolabs E7546S
QIAseq FX DNA Library UDI Kit (24)Qiagen180477
Agencourt® AMPure® XPBeckman CoulterA63880
Qubit™ 1X dsDNA High Sensitivity (HS)InvitrogenQ33230
Absolute Ethanol (Molecular Biology Grade)any-
Nuclease free waterany-
SeaKem® LE AgaroseLonza50004
iSeq 100 i1 Reagent v2 (300-cycle)Illumina20031371
Consumables
Qubit™ Assay TubesInvitrogenQ32856
PCR tubes 0,2 mlany-
1,5 ml tubesany-
Pipette tips 0,1-1000 μl
Equipments
Magnetic stand or rackany-
Pipettes 0,1-1000 μlany-
Qubit™ 4 FluorometerInvitrogenQ33238
End-point PCR machinesany-
Gel electrophoresis systemany-
iSeq 100 SystemIllumina

Safety warnings
Multiplex PCR based amplification of a targeted pathogen genome could be highly effective, so it could serve as potential contamination source! This and other amplicon based method should be separated from diagnostic units to avoid cross-contamination. The implementation of negative controls are necessary to check the reagents purity.
Nucleic Acid Extraction
RNA extraction with QIAamp Viral RNA kit based on manufacturers recommendation. Other RNA extraction kits can be used as well e.g. Quick-RNA Viral Kit (Zymo Research).


Note
QIAamp‱ Viral RNA Mini Handbook

Download HB-0354-008_HB_QA_Viral_RNA_Mini_0623_WW.pdfHB-0354-008_HB_QA_Viral_RNA_Mini_0623_WW.pdf


qRT-PCR
qRT-PCR with RealStar‱ WNV RT-PCR Kit 2.0 based on manufacturers recommendation.

Component Volume
Master A 5 µL
Master B 15 µL
Sample 10 µL

Final volume 30 µL

Reaction conditions

Step Temperature Time Cycles

Reverse transcription 55 °C 00:20:00 1
Denaturation 95 °C 00:02:00 1
95 °C 00:00:15 45
Amplification 55 °C 00:00:45 45
72 °C 00:00:15 45


Note
Manual RealStar‱ WNV RT-PCR Kit 2.0

Download RealStar-WNV-RT-PCR-Kit-2.0_WEB_CE_EN-S01.pdfRealStar-WNV-RT-PCR-Kit-2.0_WEB_CE_EN-S01.pdf



23m 15s
Reverse transcription
Reverse transcription is conducted with SuperScript IV Reverse Transcriptase.

Mix the following components in a 0.2 mL tube.

Component Volume

50µM random hexamers 1 µL
10mM dNTPs mix (10mM each) 1 µL
Template RNA 11 µL

Total volume 13 µL


Gently mix by pipetting, and spin down afterwards if needed. Incubate the reaction as follows:

Temperature Time
65 °C 00:05:00
Place on ice for 00:01:00

Note
The master mix should be prepared in a clean cabinet while the sample should be added in another one.

6m
Prepare the following master mix:

Component Volume
SSIV Buffer 4 µL
100mM DTT 1 µL
RNaseOUT RNase Inhibitor 1 µL
SSIV Reverse Transcriptase 1 µL

Total volume 7 µL

Add 7 µL of master mix to the 13 µL annealed template RNA. The final volume is 20 µL . Gently mix by pipetting, and spin down afterwards if needed.

Incubate the reaction as follows:

Temperature Time
25 °C 00:05:00
50 °C 00:50:00
70 °C 00:20:00

Note
The master mix should be prepared in a clean cabinet while the sample should be added in another one.

1h 15m
Primer pool preparation
Primer pool preparation from preordered primers. The primers were designed with the usage of Primalscheme using the sequences from the list below:
Download wnv_lineage2_sequences.csvwnv_lineage2_sequences.csv

The list of designed primers and pool ID (1 or 2) are available in the attached table.
Download WNV_lineage2_primers.xlsxWNV_lineage2_primers.xlsx

This table contains the necessary amount and ratios of individual primers what should be used for primer pool generation.
Primers should be sorted based on there pool ID and resuspend lyophilised oligos in nuclease free water to 100 µM concentration. We tried to optimise the primer concentrations and the current ratios are written in the table above. Use the mentioned amount (5 µL to 12.5 µL ) from unique primers to generate primer stocks (Pool 1-2).

Dilute the primer stocks (100 µM cc) 1:10 in nuclease free water to get the reaction ready primer solution (10 µM).


Note
Primer pools should be prepared in a clean mastermix cabinet!

For one reaction 3.2 µL reaction ready primer solution (10 µM) is needed. It can be prepared and aliquoted in 100 µL batches to avoid contamination and degradation.


Multiplex-PCR
Set up the multiplex PCR reaction in 0.2 mL PCR tube.


Reactions with Pool 1 and 2 primers:

Component Pool 1 Pool 2
5X Q5 Reaction Buffer 5 µL 5 µL
10 mM dNTPs 0.5 µL 0.5 µL
Q5 Hot Start DNA Polymerase 0.25 µL 0.25 µL
Primer Pool 1 or 2 (10µM) 3.2 µL 3.2 µL
Nuclease-free water 13.55 µL 13.55 µL

Total volume: 22.5 µL 22.5 µL


Note
The master mix should be prepared in a clean cabinet while the sample should be added in another one.

Add 2.5 µL cDNA directly to each tube and mix well by pipetting. The final volume is 25 µL .

Note
Inside the extraction and sample addition cabinet

Set-up the following program on the thermal cycler:

Step Temperature Time Cycles

Initial Denaturation 98 °C 00:00:30 1
Denaturation 98 °C 00:00:05 20-35
Annealing + Extension 60 °C 00:01:30 20-35
Hold 4 °C Till further processes


Note
The cycle number are determined based on the qPCR result. In our case the Ct number + ~3 cycles formula gave the best result (e.g. The Ct value is 26,54 use 30 cycles). In general, use higher number of cycles than the obtained Ct value.

This is a safe stopping point. The amplicons can be stored at 4°C till further processes.

2m 5s
Quantification and normalisation
Quantify 1 µL directly from the two different PCR reaction (Pool 1, Pool 2) with the 1x dsDNA HS Assaykit.


Aliquot 199 µL 1x Working solution to Qubit assay tube (equal to the number of your samples). Prepare two extra tubes for the standards with 190 µL 1x Working solution.

Add 1 µL sample for each test tube and 10 µL from the Standards (Standard 1, Standard 2). The final volume is 200 µL .

Mix each sample vigorously by vortexing and pulse centrifuge to collect the liquid.
Incubate at room temperature for 00:02:00 before measuring.

2m
Calibrate Qubit fluorometer with the standards based on manufacturers recommendation.
Read your sample.
Normalization is not required if the concentrations of the two reactions (Pool 1, Pool 2) from the same sample differ by 20 % or less. If the difference exceeds this threshold, use the entire volume of the low concentration pool and add an equivalent concentration from the other pool.


Note
Alternative dsDNA quantification systems can be used as well (e.g.QuantiFluor‱ ONE dsDNA System).

Amplicon clean-up
Conduct clean-up with Ampure XP Beads.

Sample/Bead ratio = 1:1 (50 µL )
Washing: two times with 200 µL 80% EtOH.
Elution: 28 µL nuclease-free water.

Merge the two pools prior to adding the magnetic beads.
Add 50 µL AMPure XP beads to the sample and mix gently. The sample/magnetic bead ratio is 1:1.

Incubate for 00:05:00 at room temperature.

5m
Place the tube on a magnetic rack for 00:02:00 (until supernatant is completely clear).

2m
Remove supernatant without touching the bead pellet.
Add 200 µL freshly prepared 80% ethanol to the tube. Float the beads through ethanol either by replacing to another magnetic rack or mix via pipetting.

Remove supernatant and discard.
Repeat washing step with 80% ethanol (step 9.6-9.7).
Carefully remove the supernatant and dry the beads for around00:02:00 by leaving the lid open. (Dull pellet indicates dryness)

2m
Remove tube from the magnetic rack and resuspend the pellets in 28 µL nuclease-free water and incubate at room temperature for 00:02:00 .

2m
Place the tube back to a magnetic rack for 00:02:00 until supernatant is completely clear and transfer eluate into clean tube. Make sure that no beads are transferred as well.

2m
Quantification and normalisation
Quantify 1 µL directly from the two different PCR reaction (Pool 1, Pool 2) with the 1x dsDNA HS Assaykit.


Aliquot 199 µL 1x Working solution to Qubit assay tube (equal to the number of your samples). Prepare two extra tubes for the standards with 190 µL 1x Working solution.

Add 1 µL sample for each test tube and 10 µL from the Standards (Standard 1, Standard 2). The final volume is 200 µL .

Mix each sample vigorously by vortexing and pulse centrifuge to collect the liquid.
Incubate at room temperature for 00:02:00 before measuring.

Calibrate Qubit fluorometer with the standards based on manufacturers recommendation.
Read your sample.
End Repair and dA Tailing
Amplicon end preparation with NEBNext Ultra II End Repair/dA-Tailing Module.


Add the following components to a sterile nuclease-free tube:

Component Volume
NEBNext Ultra II End Prep Reaction Buffer 1.75 µL
NEBNext Ultra II End Prep Enzyme Mix 0.75 µL
50 ng Amplicons x µL x
Nuclease-free water 12.5-x µL

Total volume 15 µL


Note
If the concentration does not reach the 4 ng / µl use the maximum amount (12.5 µL ) from the cleaned up amplicons.


Place sample in a thermal cycler, with the heated lid set to 75 °C , and run the following program:

Temperature Time
20 °C 00:30:00
70 °C 00:15:00

45m
Adapter Ligation
Adapter ligation with QIAseq FX DNA Library Kit.
Component Volume
DNA Ligase Buffer, 5x 10 µL
DNA Ligase 5 µL
UDI Adapter 2.5 µL
Nuclease-free water 17.5 µL
Previous reaction 15 µL

Final volume 50 µL

Place sample in a thermal cycler without heated lid, set and incubate:

Temperature Time
20 °C 00:15:00


Note
We used QIAseq FX DNA Library Kit as ligation module. Applied concentrations and reaction conditions are optimised for this protocol. Alternative ligase modules could be use after optimisation (e.g. from NEB).

15m
Library clean up
Conduct clean-up with Ampure XP Beads.

Sample/Bead ratio = 1 : 0.6 (30 µL )
Washing: two times with 200 µL 80% EtOH.
Elution: 25 µL nuclease-free water.

Add 30 µL AMPure XP beads to the sample and mix gently. The sample/magnetic bead ratio is 1:0.6.

Incubate for 00:05:00 at room temperature.

Place the tube on a magnetic rack for 00:02:00 (until the supernatant is completely clear).

Remove supernatant without touching the bead pellet.
Add 200 µL freshly prepared 80% ethanol to the tube. Float the beads through ethanol either by replacing to another magnetic rack or mix via pipetting.

Remove supernatant and discard.
Repeat washing step with 80% ethanol (step 12.5-12.6).
Carefully remove the supernatant and dry the beads for around00:02:00 by leaving the lid open. (Dull pellet indicates dryness)


Remove tube from the magnetic rack and resuspend the pellets in 25 µL nuclease-free water and incubate at room temperature for 00:02:00 .
Place the tube back to a magnetic rack for 00:02:00 until the supernatant is completely clear and transfer eluate into clean tube. Make sure that no beads are transferred as well.

Measure the concentration of the product with Qubit. As previously described.
Library amplification
Conduct library amplification with the usage of HiFi PCR Master Mix.

Mix the following components in an 0.2 mL tube.

Component Volume
HiFi PCR Master Mix, 2x 12.5 µL
Primer Mix Illumina Library Amp 0.75 µL
Adapter ligated amplicons 11.75 µL

Final volume 25 µL

Step Temperature Time Cycles

Initial Denaturation 98 °C 00:02:00 1
Denaturation 98 °C 00:00:20 4-8
Annealing 60 °C 00:01:30 4-8
Extension 72 °C 00:00:30 4-8
Final extension 72 °C 00:01:00 1
Hold 4 °C Till further processes



Note
Under 0,5 ng the applied cycle number should be 8 while above 4 ng 4 cycles is suitable. Reduced number of PCR cycles can result balanced amplicon distribution. Concentration measurment is recommended at this point.


5m 20s
Final Library clean up
Conduct clean-up with Ampure XP Beads.

Sample/Bead ratio = 1 : 0.7 (17.5 µL )
Washing: two times with 200 µL 80% EtOH.
Elution: 25 µL Elution Buffer.

Add 17.5 µL AMPure XP beads to the sample and mix it gently. The sample/magnetic bead ratio is 1:0.7.

Incubate for 00:05:00 at room temperature.

Place the tube on magnetic rack for 00:02:00 (until the supernatant is completely clear).

Remove supernatant without touching the bead pellet.
Add 200 µL freshly prepared 80% ethanol to the tube. Float the beads through ethanol either by replacing to another magnetic rack or mix via pipetting.

Remove supernatant and discard.
Repeat washing step with 80% ethanol (step 14.5-14.6).
Carefully remove the supernatant and dry the beads for around00:02:00 by leaving the lid open. (Dull pellet indicates dryness)

Remove tube from the magnetic rack and resuspend the pellets in 30 µL elution buffer and incubate at room temperature for 00:02:00 .
Place the tube back to the magnetic rack for 00:02:00 until the supernatant is completely clear and transfer the eluate into clean tube. Make sure that no beads are transferred as well.

Library Size check
Run 10 µL from the cleaned up final library on 1,5% agarose gel. Low concentration of WNV genome in the sample could affect the multiplex PCR effectivity. Missing template genome results high amount of primer dimers. The expected final library size is 400bp to 420 bp. The dimers are appearing around 200 bp.


Note

Examples of clean and primer dimer containing libraries.

Fragment size should be considered in the case of pooling to be sure about the equal sequencing depth of individual samples.Fragment size should be considered in the case of pooling to be sure about the equal sequencing depth of individual samples. Fragment size should be considered in the case of pooling to be sure about the equal sequencing depth of individual samples.

Final library quantification
Quantify 1 µL directly from the two different PCR reaction (Pool 1, Pool 2) with the 1x dsDNA HS Assaykit.


Aliquot 199 µL 1x Working solution to Qubit assay tube (equal to the number of your samples). Prepare two extra tubes for the standards with 190 µL 1x Working solution.

Add 1 µL sample for each test tube and 10 µL from the Standards (Standard 1, Standard 2). The final volume is 200 µL .

Mix each sample vigorously by vortexing and pulse centrifuge to collect the liquid.
Incubate at room temperature for 00:02:00 before measuring.

Calibrate Qubit fluorometer with the standards based on manufacturers recommendation.
Read your sample.
Normalization is not required if the concentrations of the two reactions (Pool 1, Pool 2) from the same sample differ by 20 % or less. If the difference exceeds this threshold, use the entire volume of the low concentration pool and add an equivalent concentration from the other pool.


Note
Alternative dsDNA quantification systems can be used as well (e.g.QuantiFluor‱ ONE dsDNA System).

Pooling and loading final libraries
Fragment size should be considered in the case of pooling to be sure about the equal sequencing depth of individual samples.
Pool all samples together in a 1.5 mL Eppendorf Low Binding tube in equimolar concentration.

Dilute the pooled libraries in RSB to 100 pM final concentration. Add PhiX control.
Load 20 µL onto an iSeq 100 2x150 bp flow cell.

Start the sequencing.
Protocol references
Amplicon based targeted sequencing:

Quick, J., Grubaugh, N. D., Pullan, S. T., Claro, I. M., Smith, A. D., Gangavarapu, K., Oliveira, G., Robles-Sikisaka, R., Rogers, T. F., Beutler, N. A., Burton, D. R., Lewis-Ximenez, L. L., de Jesus, J. G., Giovanetti, M., Hill, S. C., Black, A., Bedford, T., Carroll, M. W., Nunes, M., … Loman, N. J. (2017). Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nature Protocols, 12(6). https://doi.org/10.1038/nprot.2017.066

ARTIC SARS-CoV-2 sequencing protocol v4 (LSK114) V.4
DOI: dx.doi.org/10.17504/protocols.io.bp2l6n26rgqe/v4
https://www.protocols.io/view/artic-sars-cov-2-sequencing-protocol-v4-lsk114-bp2l6n26rgqe/v4

Koskela von Sydow, A., Lindqvist, C. M., Asghar, N., Johansson, M., Sundqvist, M., Mölling, P., & Stenmark, B. (2023). Comparison of SARS-CoV-2 whole genome sequencing using tiled amplicon enrichment and bait hybridization. Scientific Reports, 13(1). https://doi.org/10.1038/s41598-023-33168-1

Vogels, C. B. F., Hill, V., Breban, M. I., Chaguza, C., Paul, L. M., Sodeinde, A., Taylor-Salmon, E., Ott, I. M., Petrone, M. E., Dijk, D., Jonges, M., Welkers, M. R. A., Locksmith, T., Dong, Y., Tarigopula, N., Tekin, O., Schmedes, S., Bunch, S., Cano, N., … Grubaugh, N. D. (2024). DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus. BMC Genomics, 25(1). https://doi.org/10.1186/s12864-024-10350-x

AmpliSeq for Illumina SARS-CoV-2 Research Panel
https://emea.illumina.com/products/by-brand/ampliseq/community-panels/sars-cov-2.html

West Nile virus (WNV):
Habarugira, G., Suen, W. W., Hobson-Peters, J., Hall, R. A., & Bielefeldt-Ohmann, H. (2020). West nile virus: An update on pathobiology, epidemiology, diagnostics, control and “One health” implications. In Pathogens (Vol. 9, Issue 7). https://doi.org/10.3390/pathogens9070589

Bakonyi, T., & Haussig, J. M. (2020). West nile virus keeps on moving up in Europe. In Eurosurveillance (Vol. 25, Issue 46). https://doi.org/10.2807/1560-7917.ES.2020.25.46.2001938

Erazo, D., Grant, L., Ghisbain, G., Marini, G., Colón-González, F. J., Wint, W., Rizzoli, A., van Bortel, W., Vogels, C. B. F., Grubaugh, N. D., Mengel, M., Frieler, K., Thiery, W., & Dellicour, S. (2024). Contribution of climate change to the spatial expansion of West Nile virus in Europe. Nature Communications, 15(1). https://doi.org/10.1038/s41467-024-45290-3


Amplicon based targeted sequencing for WNV:

Chaintoutis, S. C., Papadopoulou, E., Melidou, A., Papa, A., & Dovas, C. I. (2019). A PCR-based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological specimens. Molecular and Cellular Probes, 46. https://doi.org/10.1016/j.mcp.2019.06.002

Pappa, S., Chaintoutis, S. C., Dovas, C. I., & Papa, A. (2021). PCR-based next-generation West Nile virus sequencing protocols. Molecular and Cellular Probes, 60. https://doi.org/10.1016/j.mcp.2021.101774

Tešović, B., Nišavić, J., Banović Đeri, B., Petrović, T., Radalj, A., Šekler, M., Matović, K., Debeljak, Z., Vasković, N., Dmitrić, M., & Vidanović, D. (2023). Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform. Diagnostic Microbiology and Infectious Disease, 105(2). https://doi.org/10.1016/j.diagmicrobio.2022.115852

Diagne, M. M., Ndione, M. H. D., Mencattelli, G., Diallo, A., Ndiaye, E. hadji, di Domenico, M., Diallo, D., Kane, M., Curini, V., Top, N. M., Marcacci, M., Mbanne, M., Ancora, M., Secondini, B., di Lollo, V., Teodori, L., Leone, A., Puglia, I., Gaye, A., … Faye, O. (2023). Novel Amplicon-Based Sequencing Approach to West Nile Virus. Viruses, 15(6). https://doi.org/10.3390/v15061261