Viral Whole Genome Sequencing Using Illumina Microbial Amplicon Prep (iMAP) with ARTIC-Style Primer Schemes for RSV, SARS-CoV-2, and Dengue Virus (Serotypes 1–4)

Stepfan de Villiers; Kerwin Liedeman; Lucious Chabuka; Velda Wentzel; Tebogo Ramakutoane; Nondumiso Zakwe; Ottovan Dakurah; Eduan Wilkinson; Marije Hofstra; Lavanya Singh; Cheryl Baxter; Tulio De Oliveira

Apr 28, 2026

Viral Whole Genome Sequencing Using Illumina Microbial Amplicon Prep (iMAP) with ARTIC-Style Primer Schemes for RSV, SARS-CoV-2, and Dengue Virus (Serotypes 1–4)

DOI

https://dx.doi.org/10.17504/protocols.io.36wgqxz4xlk5/v1

Stepfan de Villiers¹,
Kerwin Liedeman¹,
Lucious Chabuka¹,
Velda Wentzel¹,
Tebogo Ramakutoane¹,
Nondumiso Zakwe¹,
Ottovan Dakurah¹,
Eduan Wilkinson¹,
Marije Hofstra¹,
Lavanya Singh¹,
Cheryl Baxter^1,2,
Tulio De Oliveira^1,3

¹Centre for Epidemic Response and Innovation, School for Data Science and Computational Thinking, Stellenbosch University;
²Division of Medical Virology, Faculty of Medicine and Health Science, Stellenbosch University;
³KwaZulu-Natal Research Innovation and Sequencing Platform (KRISP), University of KwaZulu-Natal

Kerwin Liedeman

Stellenbosch University/Centre for Epidemic Response and Inn...

DOI: https://dx.doi.org/10.17504/protocols.io.36wgqxz4xlk5/v1

Protocol Citation: Stepfan de Villiers, Kerwin Liedeman, Lucious Chabuka, Velda Wentzel, Tebogo Ramakutoane, Nondumiso Zakwe, Ottovan Dakurah, Eduan Wilkinson, Marije Hofstra, Lavanya Singh, Cheryl Baxter, Tulio De Oliveira 2026. Viral Whole Genome Sequencing Using Illumina Microbial Amplicon Prep (iMAP) with ARTIC-Style Primer Schemes for RSV, SARS-CoV-2, and Dengue Virus (Serotypes 1–4). protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqxz4xlk5/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: April 16, 2026

Last Modified: April 28, 2026

Protocol Integer ID: 315166

Keywords: amplicons across the viral genome, viral whole genome sequencing, viral genome, multiple rna virus, respiratory syncytial virus, including respiratory syncytial virus, using illumina microbial amplicon prep, wgs of multiple rna virus, illumina microbial amplicon prep, enabling comprehensive genome coverage, dengue virus, whole genome sequencing, comprehensive genome coverage suitable for genomic epidemiology, genome sequencing, understanding viral evolution, sequencing performance, viral evolution, genomic epidemiology, style primer schemes for rsv, supporting outbreak detection, overlapping amplicon, virus, viral, rsv

Funders Acknowledgements:

Global Health EDCTP3 Joint Undertaking

Grant ID: 101103171

Bill & Melinda Gates Foundation

Grant ID: 101103171

South African Medical Research Council

Grant ID: 96707/23139

Abstract

Viral whole genome sequencing (WGS) plays a pivotal role in understanding viral evolution, identifying emerging variants, and supporting outbreak detection and surveillance. This protocol describes the workflow for performing WGS of multiple RNA viruses, including Respiratory Syncytial Virus (RSV), SARS-CoV-2, and Dengue virus (DENV serotypes 1–4), using the Illumina Microbial Amplicon Prep (iMAP) protocol in combination with virus-specific ARTIC-style primer schemes. These primer sets generate overlapping amplicons across the viral genome, enabling comprehensive genome coverage suitable for genomic epidemiology and variant analysis.
The method has been optimised to enhance sensitivity, reduce reagent costs, and maintain robust sequencing performance. Key modifications include using half-volume (1/2) reactions throughout the workflow and implementing individual library clean-up steps prior to equimolar pooling for sequencing, thereby improving library quality and consistency.

Materials

Eppendorf, Twin.Tec, LoBind, 96-Well PCR PlatesEppendorfCatalog #EP0030129504  
Eppendorf 1.5ml, lo-bind, tubesEppendorfCatalog #EP0030108418  
Illumina Microbial Amplicon PrepIlluminaCatalog #20097857  
Library Quantification Kit (500 rxns)Takara BioCatalog #638324   
Agilent D1000 Reagents AgilentCatalog #(P/N 5067-5583)  
Agilent D1000 ScreenTape AgilentCatalog #(P/N 5067-5582)  
Qubit™ 1X dsDNA High Sensitivity (HS) Assay KitInvitrogen - Thermo FisherCatalog #Q33231  
1 Liter IDTE pH 8.0 (1X TE Solution)Integrated DNA Technologies, Inc. (IDT)Catalog #11-05-01-09  
 Ethyl alcohol, Pure 200 proof, for molecular biology Merck MilliporeSigma (Sigma-Aldrich)Catalog #E7023  
Adhesive sealing sheetsThermo ScientificCatalog #AB0558  
Adhesive Optical PCR Plate SealsThermo Fisher ScientificCatalog #4311971  
TrueMark™ SARS-CoV-2, Flu A, Flu B, RSV Select PanelApplied Biosystems (ThermoFisher Scientific)Catalog #A59526  
VWR 10ul, LR, Extended Filter TipsVWRCatalog #VWRA612-6260  
VWR 200ul, LR, Filter TipsVWRCatalog #VWRA612-6179  
VWR 1000ul Filter Tips, LR, Filter TipsVWRCatalog #VWRA612-6272  
EasyStrip&trade; Plus Tube Strip with Attached Flat CapsThermo FisherCatalog #AB2000  
Chemagic Viral DNA/RNA 300 Kit H96Perkin ElmerCatalog #CMG-1033-S  
QIAamp Viral RNA Mini KitQiagenCatalog #52904  
genesig Real-Time PCR kit for subtyping of Dengue virus in duplex, AdvancedPrimerdesignCatalog #348934  

Before start

This protocol starts with extracted RNA or TNA.

We use pre-sequencing sample qualification by RT-qPCR:
RNA extracts of SARS-CoV-2 and RSV are screened using the TrueMark SARS-CoV-2 Flu A Flu B RSV Select Panel.
RNA extracts of Dengue are typed prior to sequencing using the Genesig Dengue Virus subtypes 1, 2, 3 and 4 (Multiplex kit).
Samples with Ct values >30 are excluded from sequencing.

As quality controls, we include a positive control, consisting of a commercially prepared strain or a previously sequenced sample, and a negative control (nuclease-free water) in every run.

Preparation of Primer Pools

Prepare the primer pool for the viral pathogens as described below at step 2. 
The primer scheme is described at step 1.1 for SARS-CoV-2, step 1.2 for RSV and step 1.3 for Dengue virus. 

SARS-CoV-2 Primers
The SARS-CoV-2 primer scheme can be found at:
ARTIC Primer Scheme for nCoV-2019 retrieved from:
https://github.com/artic-network/primer-schemes/blob/3258a7fd11f39ad8b52f4743fe97aea4ec129544/nCoV-2019/V5.3.2/SARS-CoV-2.primer.bed

A predesigned panel can be purchased from IDT:
Artic V5.3.2 NCOV-2019 Panel – Catalog # 10016495

An overview of the primer sequences for SARS-CoV-2 is included at the bottom of the protocol. 

RSV Primers
The primers used in this assay were designed by the CDC, which include 19 (RSVA) and 20
(RSVB) amplicon pairs, targeting an amplicon size of ~925 bp and originally described by: Wang L, Ng TFF, Castro CJ, et al. Next-generation sequencing of human respiratory syncytial virus subgroups A and B genomes. J Virol Methods. 2022; 299:114335. doi:10.1016/j. jviromet.2021.114335. 

An overview of the primer sequences for RSV is included at the bottom of the protocol. 

Dengue Primers
The Dengue virus primer scheme can be found on the CERI CLIMADE consortium GitHub:
https://github.com/CERI-KRISP/CLIMADE/blob/master/Protocols/Arboviruses/AllPrimerSchemes.xlsx

An overview of the primer sequences for dengue virus is included at the bottom of the protocol. 

All primers are ordered from IDT and resuspended in 0.1× TE buffer to a final concentration of 100 micromolar (µM)   each. 

Prepare multiple working primer pools to minimise freeze-thaw cycles.

Note
Primers are typically resuspended in 1x TE buffer according to manufacturer recommendations. But we routinely resuspend primers in 0.1x TE buffer to minimise potential inhibitory effects of EDTA on downstream PCR reactions while still providing adequate primer stability.

Primers are then assembled into two pools to generate two overlapping sets of amplicons. 
For example, 5 µL   of each 100 micromolar (µM)   primer from a specific pool is transferred into a clean 1.5 mL microcentrifuge tube and mixed thoroughly.

Working 10 micromolar (µM)   primer pools are subsequently prepared by combining 20 µL   of the 100 micromolar (µM)   primer pool with 180 µL   of nuclease-free water (NFW). 

Anneal RNA

 
ReagentStorageInstructions
EPH3 (Elution Prime Fragment Fragment 3HC Mix)-25°C to -15°CBring to room temperature. Vortex thoroughly before use.
Overview of Reagents
 
Make a dilution of the EPH3 as follows. The table indicated the required volume for 1 sample. Calculate the volumes required for the amount of samples included in the run. 

ReagentVolume (μL) per sample Required volume for x samples
EPH31.5...
Nuclease free water3.0...
Total 4.5...
EPH3 Dilution Mix 


Note
The "Anneal RNA" step was modified to add a dilution of EPH3 instead of the default input. Previous testing has shown that reducing EPH3 input improves performance with amplicons larger than 400 bp.

Illumina. 2023. Illumina Microbial Amplicon Prep for Viral Surveillance. Application Note M-GL-02215 v10. Illumina Inc.

Add 4 µL   EPH3 dilution to each well of a new 96 wells PCR plate labelled cDNA. 
Add 4 µL   RNA to each well.

Seal, vortex (1600 rpm, 00:01:00 ), centrifuge (500 x g, 00:01:00 ).

Place on a thermal cycler and run the Anneal RNA program:

Temperature (°C)Time
653 minutes
4Hold
Anneal RNA Program

Synthesis First Strand cDNA

ReagentStorageInstructions
FSM (First Strand Mix)-25°C to -15°CThaw at room temperature. Invert to mix and keep on ice.
RVT (Reverse Transcriptase)-25°C to -15°CThaw at room temperature. Invert to mix and keep on ice.
Overview of Reagents

In a microcentrifuge tube, combine the following reagents to prepare the First Strand cDNA Master Mix:

ReagentVolume (μL) per sampleRequired volume for x samples
FSM4.5...
RVT0.5...
Total5.0...
First Strand Master Mix 

Add 4 µL   master mix to each well of the cDNA plate.

Seal, vortex (1600 rpm, 00:01:00 ), centrifuge (500 x g, 00:01:00 ).

Place on a thermal cycler and run the FSS Program.

Temperature (°C)Time
255 minutes
4230 minutes
805 minutes
4Hold
First Strand Synthesis Program

Note
Temperature and incubation times were modified as cDNA synthesis may be improved at lower temperatures (~42  °C).

Note
Safe Stopping Point - seal the plate and store at -25°C to -15°C

Amplify cDNA

ReagentStorageInstructions
Primer Pool 1-25°C to -15°CThaw on ice. Vortex to mix and keep on ice.
Primer Pool 2-25°C to -15°CThaw on ice. Invert to mix and keep on ice.
IPM (Illumina PCR Mix)-25°C to -25°CThaw on ice. Invert to mix and keep on ice.
Overview of Reagents

Label two new PCR plates, PCR1 and PCR2.

In two microcentrifuge tubes, combine the following reagents to prepare a PCR 1 Master Mix and a PCR 2 Master Mix. The indicated volumes are per sample. 

ReagentPCR 1 Master Mix (μL)PCR 2 Master Mix (μL)
IPM7.57.5
Nuclease free water2.32.3
PP1 (10 µM)2.2-
PP2 (10 µM)-2.2
Total1212
PCR Master Mixes

Add 10 µL   PCR 1 Master Mix to each well of the PCR1 plate.
Add 10 µL   PCR 2 Master Mix to each well of the PCR2 plate.
Add 2.5 µL   from the cDNA plate to the corresponding wells of the PCR1 and PCR2 plates.

Seal, vortex (1600 rpm, 00:01:00 ), centrifuge (500 x g, 00:01:00 ).

Place on a thermocycler and run the relevant PCR program depending on the pathogen of interest. 
Preheat Lid: 105 °C   and Reaction Volume: 15 µL  

SARS-CoV-2 PCR program 
Temperature (°C)TimeCycle
983 minutes
9815 seconds35 cycles
645 minutes
4Hold

RSV PCR
Temperature (°C)TimeCycle
983 minutes
9815 seconds35 cycles
5630 seconds
723 minutes
4Hold
 
Note
The annealing temperature for the RSV PCR program was reduced to 56 °C to improve primer annealing.

Dengue PCR
Temperature (°C)TimeCycle
983 minutes
9815 seconds35 cycles
645 minutes
4Hold

At least 10% of the samples should be checked post-PCR using a Qubit Fluorometer and Qubit DNA HS Assay to ensure successful amplification. 

Note
Safe Stopping Point – seal the plate and store at 2°C to 8°C or leave on the thermal cycler overnight.

Tagment PCR Amplicons

ReagentStorageInstructions
EBLT (Enriched Bead-Linked Transposomes)2°C to 8°CBring to room temperature. Vortex thoroughly before use.
TB1 (Tagmentation Buffer 1)-25°C to -15°CBring to room temperature. Vortex thoroughly before use.
Overview of Reagents
 

Label a new PCR plate TAG.

Combine PCR1 and PCR2 as follows:
Transfer 5 µL   from each well of the PCR1 plate to the corresponding wells of the TAG plate.
Transfer 5 µL   from each well of the PCR2 plate to the corresponding wells of the TAG plate.

In a microcentrifuge tube, combine the following reagents to prepare the Tagmentation
Master Mix. The indicated volume is per sample. 
 
ReagentVolume (μL) n=1
EBLT2
TB16
Nuclease free water10
Total18
Tagmentation Master Mix 
 

Add 15 µL   of the master mix to each well of the TAG plate.

Seal, vortex (1600 rpm, 00:01:00 ), centrifuge (500 x g, 00:01:00 ).

Place on a thermal cycler and run the TAG program:

Preheat Lid: 105 °C   and Reaction Volume: 25 µL  
 
Temperature (°C)Time
555 minutes
10Hold
TAG program 
 

Post tagmentation clean up step. 

ReagentStorageInstructions
ST2 (Stop Tagment Buffer 2)Room TempVortex before use.
TWB (Tagmentation Wash Buffer)2°C to 8°CInvert to mix.
Overview of Reagents

After the TAG program, immediately add 5 µL   ST2 to each well of the TAG plate.

Seal and shake.

Incubate at Room temperature   for 00:05:00  .

Centrifuge briefly.

Place it on a magnetic stand and wait until the liquid is clear.

Remove and discard the supernatant.

Wash beads as follows: 
a. Remove the plate from the magnetic stand.
b. Add 50 µL   TWB to each well.

Seal, vortex (1600 rpm, 00:01:00 ), centrifuge (500 x g, 00:01:00 ).

Place it on the magnetic stand and wait until the liquid is clear.

Remove and discard the supernatant.

Wash beads a second time. (repeat steps 7.7-7.9) 

Place it on the magnetic stand and wait until the liquid is clear. Leave supernatant in the plate after the second wash to prevent beads from overdrying.
    

Amplify Tagmented Amplicons

ReagentStorageInstructions
EPM (Enriched PCR Mix)-25°C to -15°CInvert to mix. Keep on ice until use.
Index Adapters-25°C to -15°CThaw at room temperature. Vortex to mix and then centrifuge.
Overview of Reagents

In a microcentrifuge tube, combine the following reagents to prepare the TAG
PCR Master Mix: 
 
ReagentVolume (μL) n=1
EPM12
Nuclease free water12
Total24
TAG PCR Master Mix 
 

Vortex the TAG PCR Master Mix. 
With the plate on the magnetic stand, remove and discard the supernatant.
Then use a P20 pipette to remove any remaining TWB.

Remove the TAG plate from the magnetic stand.

Add 20 µL   TAG PCR Master Mix to each well of the TAG plate.
Add 5 µL   Index Adapters to each well of the TAG plate.

Seal, vortex (1600 rpm, 00:01:00 ), centrifuge (500 x g, 00:01:00 ).

Place on a thermocycler and run the TAG PCR Program. 

Preheat Lid: 100 °C   and Reaction Volume: 25 µL  

Temperature (°C)TimeCycle
723 minutes
983 minutes
9820 seconds7 cycles*
6030 seconds
721 minutes
723 minutes
10Hold
TAG PCR Program


Total cDNA Input (ng)Number of PCR cycles
1-2412
25-999
100-5007
To compensate for lower cDNA input amounts, the number of PCR cycles can be increased as above.

Clean-Up Libraries

20m 30s

ReagentStorageInstructions
IPB (Illumina Purification Beads)Room tempVortex thoroughly to mix.
RSB (Resuspension Buffer)2°C to 8°CBring to room temperature. Vortex and invert to mix.
EthanolRoom TempPrepare a fresh solution of 80% EtOH
Overview of Reagents

20m 30s

Centrifuge the TAG plate at 500 x g, 00:01:00 .

Place it on a magnetic stand and wait until the liquid is clear (00:03:00  ).

Label a new PCR plate IPB.
Vortex the IPB tube to resuspend and add 22.5 µL   of IPB to each well of the IPB plate.

Transfer 25 µL   supernatant from each well of the TAG plate to the corresponding wells of the IPB plate.

Vortex to mix and centrifuge briefly.

Incubate at Room temperature   for 00:05:00  .

Place it on a magnetic stand and wait for the liquid to clear (00:05:00  ).

Remove and discard the supernatant.

Wash beads as follows:
a. Keep the plate on the magnetic stand and add 100 µL   80% EtOH to each well.
b. Wait 00:00:30  , then remove and discard the supernatant.

Wash the beads a second time.

Centrifuge the tube briefly to collect any remaining EtOH at the bottom of the plate.

Place it on the magnetic stand and wait for the liquid to clear.
Use a P20 pipette to remove ALL residual EtOH.

Allow to air-dry on the magnetic stand for 00:01:00  .

Add 21 µL   to resuspend the beads.

Vortex to mix and then centrifuge briefly.

Incubate at Room temperature   for 00:02:00  .

Place the tube on the magnetic rack and wait for the liquid to clear (00:03:00  ).

Transfer 20 µL   of supernatant to a new PCR plate labelled LIB.


Note
Safe Stopping Point – seal the plate and store at -25°C to -15°C

Quantify and Normalise Libraries

Quantify and normalise libraries 

Analyse 1 µL   of each prepared library using the Qubit dsDNA HS Assay on a Qubit fluorometer.

Calculate the molarity of each prepared library (use 350 bp as the average library size).

Dilute each library to a normalised concentration of 4 nanomolar (nM)   using RSB.

Pool 5 µL   from each library into a clean 1.5 mL microcentrifuge tube.

Quantify the pool by qPCR using the Takara Library Quantification Kit.

[Optional] Run 1 µL   of the library pool on a TapeStation using the D1000 ScreenTape assay to confirm the average library size.

Using the qPCR quantification, dilute the library to the starting concentration for your chosen sequencing platform/kit.

Follow the denature and dilute instructions for your chosen sequencing platform by referring to the manufacturer's user guides.

Sequencing Platform/KitStarting Concentration (nM)Final Loading Concentration (pM)
NextSeq (Standard-SBS)2750
NextSeq (XLEAP-SBS)2650
MiSeq i100280
MiSeq V2410-12
MiSeq V3412-14
Loading Concentration Recommendations by Platform/ Kit 

Appendix: SARS-CoV-2, RSV and Dengue primer pools

SARS-CoV-2 primers
Primer NameSequence (5'-3')Primer Pool
SARS-CoV-2_1_LEFTCTCTTGTAGATCTGTTCTCTAAACGAACTTT1
SARS-CoV-2_1_RIGHTAAAACGCCTTTTTCAACTTCTACTAAGC1
SARS-CoV-2_3_LEFTAGAACGGTAATAAAGGAGCTGGT1
SARS-CoV-2_3_RIGHTTTTGCCAATTTAATTTCAAAAGGTGTCTG1
SARS-CoV-2_5_LEFTTTTGTGGCACTGAGAATTTGACTAAAGA1
SARS-CoV-2_5_RIGHTATAATGGCGATCTCTTCATTAAGTTTAAAGTC1
SARS-CoV-2_7_LEFTGCTGCTCGTGTTGTACGATCAAT1
SARS-CoV-2_7_RIGHTCTCCTTAATTTCCTTTGCACAGGTG1
SARS-CoV-2_9_LEFTGTCTTGAAAACTGGTGATTTACAACCATTA1
SARS-CoV-2_9_RIGHTGTAATTCAGATACTGGTTGCAAAGTTTTTATGA1
SARS-CoV-2_11_LEFTGAGCAAGAAGAAGATTGGTTAGATGATGA1
SARS-CoV-2_11_RIGHTCGCTTAAAACACAACTACCACCCA1
SARS-CoV-2_13_LEFTCTGTCTTTGATAAAAATCTCTATGACAAACTTG1
SARS-CoV-2_13_RIGHTCTTTTTAGTAGGTATAACCACAGCAGTTAAAAC1
SARS-CoV-2_15_LEFTCACATGCAGAAGAAACACGCAA1
SARS-CoV-2_15_RIGHTTCTTTATAGGAACCAGCAAGTGAGATG1
SARS-CoV-2_17_LEFTGACAACAGTTTGGTCCAACTTATTTGG1
SARS-CoV-2_17_RIGHTCTGTCTTATTACAGTAGGCTAAGATAAGTGC1
SARS-CoV-2_19_LEFTCCTTTTGTTATGATGTCAGCACCAC1
SARS-CoV-2_19_RIGHTCACATACAAACTTAAAATTATCGAAGCTTGC1
SARS-CoV-2_21_LEFTGAAAGGAGCTAAATTGTTACATAAACCTATTGT1
SARS-CoV-2_21_RIGHTAGGTTTCTTAATAGTAAGACTAGAATTGTCTAC1
SARS-CoV-2_23_LEFTAATTCTAGAATTAAAGCATCTATGCCGACTA1
SARS-CoV-2_23_RIGHTCAAAAGCAGTTAAATCCCATTTAAAAGATG1
SARS-CoV-2_25_LEFTGTGCATGTTGTAGACGGTTGTAATTC1
SARS-CoV-2_25_RIGHTCTCTCAGGTTGTCTAAGTTAACAAAATGAGA1
SARS-CoV-2_27_LEFTAAAACTCAAAACACTAGTTGCAACTGC1
SARS-CoV-2_27_RIGHTCACATGTCAACTTAAAAGGTAAGTTATTCT1
SARS-CoV-2_29_LEFTCAGATACTTGTTTTGCTAACAAACATGCT1
SARS-CoV-2_29_RIGHTACATAACGTGTGTCAGGGCGTA1
SARS-CoV-2_31_LEFTGATCTTTACCAGGAGTTTTCTGTGG1
SARS-CoV-2_31_RIGHTACAAATGATATAAGCAATTGTTATCCAGAAAGG1
SARS-CoV-2_33_LEFTATAATAAGTACAAGTATTTTAGTGGAGCAATGG1
SARS-CoV-2_33_RIGHTAATGTCCAATAACCCTGAGTTGAACATTA1
SARS-CoV-2_35_LEFTTTTTTGTTACATGCACCATATGGAATTACC1
SARS-CoV-2_35_RIGHTAAATTCATCTTCTAATAAAGCACTACCCAA1
SARS-CoV-2_37_LEFTAAACATAAGCATGCATTTCTCTGTTTGTT1
SARS-CoV-2_37_RIGHTACCTCTGGCCAAAAACATGACA1
SARS-CoV-2_39_LEFTAACATTAAATTGTTGGGTGTTGGTGG1
SARS-CoV-2_39_RIGHTCAGAATCACCATTAGCAACAGCCT1
SARS-CoV-2_41_LEFTCAAGAGATGGTTGTGTTCCCTTGAA1
SARS-CoV-2_41_RIGHTAACAGTGCAAGTACAAACCTACCTC1
SARS-CoV-2_43_LEFTGCTTTTGCTGTAGATGCTGCTAAA1
SARS-CoV-2_43_RIGHTCACCGCAAACCCGTTTAAAAAC1
SARS-CoV-2_45_LEFTACTTCTTTAAGTTTAGAATAGACGGTGAC1
SARS-CoV-2_45_RIGHTTCTACAACAGGAACTCCACTACCT1
SARS-CoV-2_47_LEFTCTTTAATGTTTTATTCTCTACAGTGTTCCCAC1
SARS-CoV-2_47_RIGHTATCCTGAGCAAAGAAGAAGTGTTTTAATTC1
SARS-CoV-2_49_LEFTCACTTTTCGCATATACAAAACGTAATGTC1
SARS-CoV-2_49_RIGHTCACTCATTAGCTAATCTATAGAAACGGTGT1
SARS-CoV-2_51_LEFTCTTTGTGAATGAGTTTTACGCATATTTGC1
SARS-CoV-2_51_RIGHTAGGATGTTTAGTAAGTGGGTAAGCATCTATA1
SARS-CoV-2_53_LEFTGTGCTTGCATACGTAGACCATTCTTA1
SARS-CoV-2_53_RIGHTGACAGTTTAAATGTCTCCTCAGTAGCTTT1
SARS-CoV-2_55_LEFTCAAGAGCACTATGTTAGAATTACTGGCTTATA1
SARS-CoV-2_55_RIGHTTTTCATCAAAGACAACTATATCTGCTGTC1
SARS-CoV-2_57_LEFTGGCACACTAGAACCAGAATATTTCAATTC1
SARS-CoV-2_57_RIGHTAGTGGTTTGAGTGAATATGACATAGTC1
SARS-CoV-2_59_LEFTGTGTTGACACTAAATTCAAAACTGAAGGTTTA1
SARS-CoV-2_59_RIGHTCGCACTACATTCCAAGGAAGTCC1
SARS-CoV-2_61_LEFTGGTAACCTACAAAGCAACCATGATCT1
SARS-CoV-2_61_RIGHTTCCAAAATAGGCATACACCATCTGT1
SARS-CoV-2_63_LEFTAGTGTCAGATATAGATTATGTACCACTAAAGTCT1
SARS-CoV-2_63_RIGHTGCCCAAAGCTCAAATGCTACATTAAC1
SARS-CoV-2_65_LEFTGCCCGTAATGGTGTTCTTATTACAGA1
SARS-CoV-2_65_RIGHTGTACTGTCCATAGGAATAAAATCTTCTAATTCA1
SARS-CoV-2_67_LEFTAATTACAATCTAGTCAAGCGTGGCAA1
SARS-CoV-2_67_RIGHTCATATCACTAATAATGAGATCCCATTTATTAGC1
SARS-CoV-2_69_LEFTATGTCATGCATGCAAATTACATATTTTGGA1
SARS-CoV-2_69_RIGHTAGTCCTGAGTTGAATGTAAAACTGAG1
SARS-CoV-2_71_LEFTGAGGCTGGATTTTTGGTACTACTTTAGA1
SARS-CoV-2_71_RIGHTATGTTAATACCTATTGGCAAATCTACCA1
SARS-CoV-2_73_LEFTCGTTGAAATCCTTCACTGTAGAAAAAGG1
SARS-CoV-2_73_RIGHTGCTATAACGCAGCCTGTAAAATCATCT1
SARS-CoV-2_75_LEFTAACCATACAGAGTAGTAGTACTTTCTTTTGA1
SARS-CoV-2_75_RIGHTCCTGTAGAATAAACACGCCAAGTAGG1
SARS-CoV-2_77_LEFTGCAGGTATATGCGCTAGTTATCAGAC1
SARS-CoV-2_77_RIGHTAATTGGTGGTGTTTTGTAAATTTGTTTGAC1
SARS-CoV-2_79_LEFTGATGAAATGATTGCTCAATACACTTCTGC1
SARS-CoV-2_79_RIGHTTGCCTGTGATCAACCTATCAATTTG1
SARS-CoV-2_81_LEFTCATGTGACTTATGTCCCTGCACA1
SARS-CoV-2_81_RIGHTGAGATTCATTTAAATTCTTGGCAACCTCATT1
SARS-CoV-2_83_LEFTCATTACACATAAACGAACTTATGGATTTGT1
SARS-CoV-2_83_RIGHTGCCAAAGCCTCATTATTATTCTTACAAAGTTTA1
SARS-CoV-2_85_LEFTTCACTTCAGACTATTACCAGCTGTACTC1
SARS-CoV-2_85_RIGHTAAAGAAGGTTTTACAAGACTCACGTTAAC1
SARS-CoV-2_87_LEFTAGGTTTCCTATTCCTTACATGGATTTGT1
SARS-CoV-2_87_RIGHTCCTTGATGTCACAGCGTCCT1
SARS-CoV-2_89_LEFTGATGTTTCATCTCGTTGACTTTCAGG1
SARS-CoV-2_89_RIGHTGCCGTCAGGACAAGCAAAAG1
SARS-CoV-2_91_LEFTTATCTTTTGGTTCTCACTTGAACTGCAA1
SARS-CoV-2_91_RIGHTCGAACGTCATGATACTCTAAAAAGTCTT1
SARS-CoV-2_93_LEFTTCGAGGACAAGGCGTTCCAA1
SARS-CoV-2_93_RIGHTGCCTGGAGTTGAATTTCTTGAACT1
SARS-CoV-2_95_LEFTCTGATTACAAACATTGGCCGCAAA1
SARS-CoV-2_95_RIGHTATCTGCCTTGTGTGGTCTGCA1
SARS-CoV-2_2_LEFTTCGTACGTGGCTTTGGAGACTC2
SARS-CoV-2_2_RIGHTTCTTCATAAGGATCAGTGCCAAGCT2
SARS-CoV-2_4_LEFTCATGAAATTGCTTGGTACACGGAAC2
SARS-CoV-2_4_RIGHTAAATTTTAACAACAGCATTTTGGGGTAAGT2
SARS-CoV-2_6_LEFTGTGTTGTTGGAGAAGGTTCCGA2
SARS-CoV-2_6_RIGHTTATTGTTATAGCGGCCTTCTGTAAAAC2
SARS-CoV-2_8_LEFTTGCTTGTGAAATTGTCGGTGGAC2
SARS-CoV-2_8_RIGHTAAGCCCGTTAATACAAACTGGTGTAC2
SARS-CoV-2_10_LEFTCTCGGTACAGAAGTAAATGAGTTCGC2
SARS-CoV-2_10_RIGHTTGTCCTCACTGCCGTCTTGTT2
SARS-CoV-2_12_LEFTCATGCAAGTTGAATCTGATGATTACATAGC2
SARS-CoV-2_12_RIGHTAGTTATAAATGGCTTAACTTCCTCTTTAGG2
SARS-CoV-2_14_LEFTGTGACATTGACATCACTTTCTTAAAGAAA2
SARS-CoV-2_14_RIGHTCCCTTATATTTACGCTGTATAGTTGAAACTA2
SARS-CoV-2_16_LEFTAATGGTTATCTTACTTCTTCTTCTAAAACACCT2
SARS-CoV-2_16_RIGHTAAATGTTTTACCTTCATGTGAATTATGAGG2
SARS-CoV-2_18_LEFTGCTCTACAAGATGCTTATTACAGAGC2
SARS-CoV-2_18_RIGHTACTCACTAGCACAAGTAAATGTACCATG2
SARS-CoV-2_20_LEFTCCATAAAACCAGTTACTTATAAATTGGATGGT2
SARS-CoV-2_20_RIGHTCAGGTATTTGGTTTATACGTGGCTTTATTAG2
SARS-CoV-2_22_LEFTTAAAAATTACAGAAGAGGTTGGCCACA2
SARS-CoV-2_22_RIGHTTAATTAAATGAAGCCTCTAGACAAAATTTACCG2
SARS-CoV-2_24_LEFTGTTTAGATTCTTTAGACACCTATCCTTCTTTAGA2
SARS-CoV-2_24_RIGHTACAATAGTTGTACATTCGACTCTTGTT2
SARS-CoV-2_26_LEFTCATCTTTACTTTGATAAAGCTGGTCAAAA2
SARS-CoV-2_26_RIGHTGAAATAAAAGTAGATAAGACATTGTCTAAGGAC2
SARS-CoV-2_28_LEFTCGTTAAAGATTTCATGTCATTGTCTGAACA2
SARS-CoV-2_28_RIGHTATTAGTATAACTACCACCACGCTGG2
SARS-CoV-2_30_LEFTCAATTTTTAAAGATGCTTCTGGTAAGCCA2
SARS-CoV-2_30_RIGHTACAATACCACCAGCTACTATAGATGC2
SARS-CoV-2_32_LEFTGGTGTTTATTCTGTTATTTACTTGTACTTGACA2
SARS-CoV-2_32_RIGHTTTGCGAGATGACAACAAGCAGC2
SARS-CoV-2_34_LEFTTGAAGATTTACTCATTCGTAAGTCTAATCA2
SARS-CoV-2_34_RIGHTGTCAACAAAAGGTCCATAAAAGTTACCT2
SARS-CoV-2_36_LEFTCCGTTTTAGATATGTGTGCTTCATTAAAAGAAT2
SARS-CoV-2_36_RIGHTGGCATATAGACCATATTAAAATAAGCTACAG2
SARS-CoV-2_38_LEFTGTGGGCTCTTATAATCTCTGTTACTTCTAAC2
SARS-CoV-2_38_RIGHTCTTTACATCTGACATTTTAGACTGTACAGTG2
SARS-CoV-2_40_LEFTTCCCTTCCATCATATGCAGCTTTTG2
SARS-CoV-2_40_RIGHTTTTTTATATGTGTTATAGTCTGGTATGACAACC2
SARS-CoV-2_42_LEFTGCACTGATGACAATGCGTTAGC2
SARS-CoV-2_42_RIGHTCAATTAGTGATTGGTTGTCCCCCAC2
SARS-CoV-2_44_LEFTATCAACTCCGCGAACCCATG2
SARS-CoV-2_44_RIGHTTGCCATTGTGTATTTAGTAAGACGTTGA2
SARS-CoV-2_46_LEFTCTCAATGGTAACTGGTATGATTTCGGT2
SARS-CoV-2_46_RIGHTTGGAACACCATCAACAAATATTTTTCTCAC2
SARS-CoV-2_48_LEFTGACTTTGCTGTGTCTAAGGGTTTCT2
SARS-CoV-2_48_RIGHTTTCTTTGCACTAATGGCATACTTAAGATTC2
SARS-CoV-2_50_LEFTTCACTTGTTCTTGCTCGCAAACAT2
SARS-CoV-2_50_RIGHTTATTGAAACACACAACAGCATCGTCA2
SARS-CoV-2_52_LEFTTGGTACACTTATGATTGAACGGTTCG2
SARS-CoV-2_52_RIGHTGTGACATCACAACCTGGAGCATT2
SARS-CoV-2_54_LEFTTCAAGCTTTTTGCAGCAGAAACG2
SARS-CoV-2_54_RIGHTACATTGCTAGAAAACTCATCTGAGATATT2
SARS-CoV-2_56_LEFTCACTATGTGAGAAGGCATTAAAATATTTGC2
SARS-CoV-2_56_RIGHTACAATTTCAGCAGGACAACGCC2
SARS-CoV-2_58_LEFTGGGACTACCAACTCAAACTGTTGATT2
SARS-CoV-2_58_RIGHTATCATAGAGATGAGTCTTCTATAGGTCATGT2
SARS-CoV-2_60_LEFTCGCCTGGAGATCAATTTAAACACC2
SARS-CoV-2_60_RIGHTGCATCACAACTAGCTACATGTGCAT2
SARS-CoV-2_62_LEFTTCTATGATGCACAGCCTTGTAGTGA2
SARS-CoV-2_62_RIGHTGTCTACAGACAGCACCACCTAAATTG2
SARS-CoV-2_64_LEFTAGTTGATGGTGTTGATGTAGAATTGTTTG2
SARS-CoV-2_64_RIGHTTAATGTGACTCCATTAAGACTAGCTTGTTT2
SARS-CoV-2_66_LEFTGGTTTACATCTACTGATTGGACTAGCTAAA2
SARS-CoV-2_66_RIGHTCACTATCACCATAATTTTGAAGGTCACAC2
SARS-CoV-2_68_LEFTATTGGTGATTGTGCAACTGTACATACA2
SARS-CoV-2_68_RIGHTACTCATGTCAAATAAAGAATAGGAAGACAA2
SARS-CoV-2_70_LEFTTTTATTGCCACTAGTCTCTAGTCAGTGT2
SARS-CoV-2_70_RIGHTAATTCACAGACTTTAATAACAACATTAGTAGCG2
SARS-CoV-2_72_LEFTGGTTATTTTAAAATATATTCTAAGCACACGCCT2
SARS-CoV-2_72_RIGHTATCTAACAATAGATTCTGTTGGTTGGACTC2
SARS-CoV-2_74_LEFTATGTCTATGCAGATTCATTTGTAATTAGAGGT2
SARS-CoV-2_74_RIGHTTCCACAAACAGTTGCTGGTGC2
SARS-CoV-2_76_LEFTCAAAAAGTTTCTGCCTTTCCAACAATTTG2
SARS-CoV-2_76_RIGHTTGACTAGCTACACTACGTGCCC2
SARS-CoV-2_78_LEFTGCAATATGGCAGTTTTTGTACACAATTAAA2
SARS-CoV-2_78_RIGHTGCACCAAAGGTCCAACCAGAAG2
SARS-CoV-2_80_LEFTACGCTTGTTAAACAACTTAGCTCCAA2
SARS-CoV-2_80_RIGHTCTTCACGAGGAAAGTGTGCTTTTC2
SARS-CoV-2_82_LEFTTGATTTAGGTGACATCTCTGGCATTAATG2
SARS-CoV-2_82_RIGHTGATTTCACCTTGCTTCAAAGTTACAGTTC2
SARS-CoV-2_84_LEFTGTAACAGTTTACTCACACCTTTTGCTC2
SARS-CoV-2_84_RIGHTTGTTCAACACCARTGTCTGTACTC2
SARS-CoV-2_86_LEFTCATCCTTACTGCGCTTCGATTGT2
SARS-CoV-2_86_RIGHTACCGGTGATCCAATTTATTCTGTAAA2
SARS-CoV-2_88_LEFTGTGGACATCTTCGTATTGCTGGA2
SARS-CoV-2_88_RIGHTCCATTGGTTGCTCTTCATCTAATTGAG2
SARS-CoV-2_90_LEFTTCATCCTCTAGCTGATAACAAATTTGCA2
SARS-CoV-2_90_RIGHTTTCTTGGTGAAATGCAGCTACAG2
SARS-CoV-2_92_LEFTTTCCTGTTTACCTTTTACAATTAATTGCCAG2
SARS-CoV-2_92_RIGHTCTTCGGTAGTAGCCAATTTGGTCATC2
SARS-CoV-2_94_LEFTGCAGTCAAGCCTCTTCTCGTT2
SARS-CoV-2_94_RIGHTCATTCCGAAGAACGCTGAAGC2
SARS-CoV-2_96_LEFTCTGCAGATTTGGATGATTTCTCCA2
SARS-CoV-2_96_RIGHTTTTGTCATTCTCCTAAGAAGCTATTAAAATCAC2
 

RSV primers
 
Primer NameSequence (5'-3')Primer Pool
RSVA-CDC20_1_LEFTACGSGAAAAAATGCGTACAAC1
RSVA-CDC20_1_RIGHTAGTGCCATATTTTGTGTTGTATTCAG1
RSVA-CDC20_3_LEFTGTAACAACACATCGTCAAGACATTA1
RSVA-CDC20_3_RIGHTCTGTTGTTTGCRTCTTCTCCA1
RSVA-CDC20_5_LEFTTGTCTCTCAATCCAACATCAGAG1
RSVA-CDC20_5_RIGHTGCTCCAAGATCTACTATGAATTGAC1
RSVA-CDC20_8_LEFTGAAGTGTTCAAYTTTGTACCCTGC1
RSVA-CDC20_8_RIGHTACTGGCGATTGCAGATCCA1
RSVA-CDC20_10_LEFTGTRTTTTGTGACACAATGAACAGT1
RSVA-CDC20_10_RIGHTCCATTCAAGCAATGACCTCG1
RSVA-CDC20_12_LEFTGATACTACCTGACAAATATCCTTGTAG1
RSVA-CDC20_12_RIGHTTTACCTCAYTWGATCGATAYTGTGT1
RSVA-CDC20_14_LEFTCTTGCAGGTGACAATAACCT1
RSVA-CDC20_14_RIGHTTGCCTATATGTGCATATTATTGTGAC1
RSVA-CDC20_16_LEFTAGAATCTATAGGTAGTTTGACACAAG1
RSVA-CDC20_16_RIGHTGCTTGTTGTATATTTKATGTCCATTG1
RSVA-CDC20_18_LEFTCACAGGTGATGTTGATATTCACAAG1
RSVA-CDC20_18_RIGHTAGCTTCTTATTAGATAACRATGGTAAC1
RSVA-CDC20_20_LEFTATAGAGTGGAGCAAGCATGT1
RSVA-CDC20_20_RIGHTACGAGAAAAAAAGTGTCAAAAACTAA1
RSVB-CDC20_2_LEFTTGATGATCACAGACATGAGACC1
RSVB-CDC20_2_RIGHTTTATCATCCCACAGTCTGGAG1
RSVB-CDC20_4_LEFTGAGCAACTCAAAGAAAATGGAG1
RSVB-CDC20_4_RIGHTGTCTGGCARTTGATTCAATGGA1
RSVB-CDC20_6_LEFTGATCTWACCATGAAGACATTCAACC1
RSVB-CDC20_6_RIGHTACTACTCTATGCTACCATRATRGT1
RSVB-CDC20_8_LEFTGATGATTACCATTTTGAAGTGTTCAA1
RSVB-CDC20_8_RIGHTCTTCGTTTCCTCTTCTTGCTTAT1
RSVB-CDC20_10_LEFTGCAATGTTCAGATAGTAAGGCAAC1
RSVB-CDC20_10_RIGHTTGACTGTAGTGRCATCTTCTACC1
RSVB-CDC20_12_LEFTCACCTAAGATAAGAGTRTACAATACTGT1
RSVB-CDC20_12_RIGHTAGTACTGAGTTTTCATCACCTGA1
RSVB-CDC20_14_LEFTAAGAAGTAGARGGATTTATTATGTCTT1
RSVB-CDC20_14_RIGHTATCTTGTCAAACTCTCAGGGAA1
RSVB-CDC20_16_LEFTAGCACAATGGAGTGTACTATCC1
RSVB-CDC20_16_RIGHTCTTTCTCTTACATACTTGCTTAATTCAG1
RSVB-CDC20_18_LEFTGTTGTGGAACAATTCACAAACAT1
RSVB-CDC20_18_RIGHTTCCACTTATACAAAATTTAGGTTTGTT1
RSVB-CDC20_20_LEFTTGYGATGCTGAATTACCTGT1
RSVB-CDC20_20_RIGHTACGAGAAAAAAAGTGTCAAAAACTAATG1
RSVA-CDC20_2_LEFTCWCTAACCAGAGAYATCATAACACA2
RSVA-CDC20_2_RIGHTATCAGGAGAGTCATGCCTG2
RSVA-CDC20_4_LEFTGAAAATGGTGTGATTAACTACAGTGT2
RSVA-CDC20_4_RIGHTGCAGATRGATGTTTGGTTGGAT2
RSVA-CDC20_21_LEFTGTCAATTCATAGTAGATCTTGGAGC2
RSVA-CDC20_21_RIGHTGCAGGGTACAAARTTGAACACTTC2
RSVA-CDC20_9_LEFTCTGTAACAGAATTGCAGTTGCT2
RSVA-CDC20_9_RIGHTTTTGCCATAGCATGACACAAT2
RSVA-CDC20_11_LEFTCAGAAGCACACCAGTCACA2
RSVA-CDC20_11_RIGHTATCATCAGTAATACCTAGATGTTGTAG2
RSVA-CDC20_13_LEFTCAGTYATTACWACCATAATCAAAGATG2
RSVA-CDC20_13_RIGHTCTTAAAGTAGGCCATCTGTTGTA2
RSVA-CDC20_15_LEFTACAACAATTACATYAGTAAGTGCTC2
RSVA-CDC20_15_RIGHTGTGAACTATAGCCTCTGTGAGG2
RSVA-CDC20_17_LEFTCCAATGTCCAGCTAAATTAGTACT2
RSVA-CDC20_17_RIGHTGAGAAATATTGAGTATGGAAAACCTAAG2
RSVA-CDC20_19_LEFTCATATAAGGATTGCTAATTCYGAATTAG2
RSVA-CDC20_19_RIGHTGCAGGACCTATTGTAAGGAC2
RSVB-CDC20_1_LEFTACGCGAAAAAATGCGTACTACA2
RSVB-CDC20_1_RIGHTGCATRGGGAAWGTGCCATATT2
RSVB-CDC20_3_LEFTTGGAAAGGAAATGAAATTCGAAGT2
RSVB-CDC20_3_RIGHTGCTTTGTTATTTGCATCTTCTCCA2
RSVB-CDC20_5_LEFTGACTTAGGAATGAGGAAAGCGA2
RSVB-CDC20_5_RIGHTGGAATAATTTTAGCATTGGTGATAGCA2
RSVB-CDC20_7_LEFTGCAATACTAAATAAGCTAAGTGAACAT2
RSVB-CDC20_7_RIGHTGGGTCTCTTTTGTTTGTGGTTTTG2
RSVB-CDC20_9_LEFTAGTGCTTTAAGAACAGGTTGG2
RSVB-CDC20_9_RIGHTCACAAAATACTCGATTGGACTGTAC2
RSVB-CDC20_11_LEFTGTAAATACTGGMAAATCTACTACAAAT2
RSVB-CDC20_11_RIGHTGTTGACTCTTTTGGGTTGCTTATG2
RSVB-CDC20_13_LEFTGGTGAACTGAAATTAGAAGAACCAAC2
RSVB-CDC20_13_RIGHTATGATATTATCAGACACTGTCTTGTC2
RSVB-CDC20_15_LEFTAATCAAAGCTATCTCAACAACTC2
RSVB-CDC20_15_RIGHTCCAAATRTTCCTAAATATTAAACTGC2
RSVB-CDC20_17_LEFTCTTATATCAGGAACAAAATCCATAACTA2
RSVB-CDC20_17_RIGHTTGGGTTAAACTTATTTTATCTGGTAGG2
RSVB-CDC20_19_LEFTCAATGATGAATTTTACACATCAAATCTC2
RSVB-CDC20_19_RIGHTACTTGCTACCTAGGCACAC2
 

Dengue virus primers (Serotypes 1-4)

 

 DENV1
Primer NameSequence (5'-3')Pool
DENV1_1_LEFTAATATGCTGAAACGCGCGAGAA1
DENV1_1_RIGHTCCGTCTTCAAGAGTTCAATGTCCA1
DENV1_3_LEFTGGAAATACAGCTGACCGACTACG1
DENV1_3_RIGHTACTGCAATGCACGTCATCGAAA1
DENV1_5_LEFTTGGAACATTTGGGAAGTTGAGGAC1
DENV1_5_RIGHTACTTCTCTGGATGTTAGTCTGCG1
DENV1_7_LEFTAGTTGGCCCCTCAATGAAGGAA1
DENV1_7_RIGHTGCACTGACGTAGGTTCCACTTG1
DENV1_9_LEFTATAGCGGCCAGAGGGTACATCT1
DENV1_9_RIGHTTGTTCTCCTCCAACACCTGGTT1
DENV1_11_LEFTTGTGGTGATAGGTTTGTTATTCATGATACT1
DENV1_11_RIGHTCTTTGGCTTCGGATCTGTCCAC1
DENV1_13_LEFTTGGAGCAAATGCAAAGAAAACATGG1
DENV1_13_RIGHTTGCACGACTTCCTTTTGCCTTT1
DENV1_15_LEFTAGACGTGACCAGAGAGGAAGTG1
DENV1_15_RIGHTTCACTTGGTTTATGGCCACTTGT1
DENV1_2_LEFTACCCAGGATTCACGGTGATAGC2
DENV1_2_RIGHTACCAGCAAATCTTGTCTGTTCCA2
DENV1_4_LEFTCAAGAAAGGAAGCAGCATAGGGA2
DENV1_4_RIGHTTTGATGGCAGCTGACATTAGCC2
DENV1_6_LEFTTGGATGAACATTGTGGAAATCGAGG2
DENV1_6_RIGHTGCATGCCTCCAGCTATTAGTGG2
DENV1_8_LEFTTCTCATATGGAGGAGGTTGGAGG2
DENV1_8_RIGHTAGCCTGAGTTCCATGATCTCTCA2
DENV1_10_LEFTAAAGAGTGCAGCAATAGACGGG2
DENV1_10_RIGHTATAGAGGGTCCAGGCTGAAGCT2
DENV1_12_LEFTGGGAAACACTGGGAGAGAAATGG2
DENV1_12_RIGHTTGATCCTGATGGCTTGACCTCA2
DENV1_14_LEFTACTCAGCAAAAGAAGCAGTGGA2
DENV1_14_RIGHTGCATGGCACCACTATTTCCCTC2
 

 DENV2
Primer NameSequence (5'-3')Pool
DENV2_1_LEFTAGCAGATCTCTGATGAATAACCAACG1
DENV2_1_RIGHTTTTTTGCCATCGTCGTCACACA1
DENV2_3_LEFTACATTGGTCACTTTCAAAAATCCCC1
DENV2_3_RIGHTTGAAGGGGATTCTGGTTGGAACT1
DENV2_5_LEFTTCATGCAGGCAGGAAAACGATC1
DENV2_5_RIGHTTCTCAAGAGTAGTCCAGCTGCA1
DENV2_7_LEFTCCAATCCTGTCAATAACAATATCAGAAGAT1
DENV2_7_RIGHTTGATGGCTGGGGTTTGGTATCT1
DENV2_9_LEFTATGCCAGTGACCCACTCTAGTG1
DENV2_9_RIGHTCCACCACTGTGAGGATGGCTAT1
DENV2_11_LEFTGGAGCTGGACTTCTCTTTTCCAT1
DENV2_11_RIGHTGACGTCCCAAGGTTTTGTCAGC1
DENV2_13_LEFTTGGGACACAAGAATCACACTAGAAG1
DENV2_13_RIGHTCCGCACCATTGGTCTTCTCTTT1
DENV2_2_LEFTTCGCTCCTTCAATGACAATGCG2
DENV2_2_RIGHTCCATTCTCAGCCTGCACTTGAG2
DENV2_4_LEFTATAGTGGTTGCGTTGTGAGCTG2
DENV2_4_RIGHTCGGCAGCACCATTCTGTTATGA2
DENV2_6_LEFTTGGAAATCAGACCATTGAAAGAGAAAGA2
DENV2_6_RIGHTTGGTCAGTGTTTGTTCTTCCTCTT2
DENV2_8_LEFTAGATCGAAGATGACATTTTCCGAAAGA2
DENV2_8_RIGHTCCCATGTATATGTACTGGTCATTTTCATT2
DENV2_10_LEFTACCAGAAAAACAGAGAACACCCC2
DENV2_10_RIGHTCCACTTCCTGGATTCCACTTTTCT2
DENV2_12_LEFTAGAGCATGAAACATCATGGCACT2
DENV2_12_RIGHTGTGCCTCTTGGTGTTGGTCTTT2
 

DENV3
Primer NameSequence (5'-3')Primer Pool
DENV3_1_LEFTTCAATATGCTGAAACGCGTGAGA1
DENV3_1_RIGHTGCCTCGGTCTTCTGAAGCTCTA1
DENV3_3_LEFTACCAATAGAGGGAAAAGTGGTGC1
DENV3_3_RIGHTTGGCCTCGAACATCTTCCCAAT1
DENV3_5_LEFTGGCAAAAATAGTGACAGCTGAAACA1
DENV3_5_RIGHTTCTCCATGTTATTTGCCCTGAGAGA1
DENV3_7_LEFTGCCAGTCTTCGAGCATGAGGAA1
DENV3_7_RIGHTTTCCCAGGCTCTACGGCAATAA1
DENV3_9_LEFTGCAACAAAATCTGAACACACAGGA1
DENV3_9_RIGHTCCCTCCTCATGAGTTCCACGAA1
DENV3_11_LEFTGGAAAGACTTCAATAGGACTCATTTGTG1
DENV3_11_RIGHTCAGGTGATCCTTCCCAGAGTGT1
DENV3_13_LEFTAGTGGAAGAAAGCAGAACTATAAGAGT1
DENV3_13_RIGHTTGGAGTTCACGTTCTCTGTCCA1
DENV3_15_LEFTTGGACATCATATCTAGGAAAGACCAAAG1
DENV3_15_RIGHTAGGTTGCTCTGGAAGTGAGACC1
DENV3_2_LEFTGGGAGTAGGAAACAGAGATTTTGTGG2
DENV3_2_RIGHTGAGTATTGTCCCATGCTGCGTT2
DENV3_4_LEFTGCTGAACCTCCTTTTGGGGAAA2
DENV3_4_RIGHTTTCCACCTCCCACACATTCCAT2
DENV3_6_LEFTAGGTGGACAACTTCACAATGGG2
DENV3_6_RIGHTCTCAGCCTCTTCCTCCCATGTT2
DENV3_8_LEFTAAAGACTGGAACCAAACTGGGC2
DENV3_8_RIGHTTGCCTGAATTCCATGAGCGTTC2
DENV3_10_LEFTAGACCATGCTCACTGGACAGAA2
DENV3_10_RIGHTTATGCGAGTTGGTTGTCTTGGG2
DENV3_12_LEFTGTGGATGGGATAATGACAATAGACCT2
DENV3_12_RIGHTGGTGCTCAATCACAGTTGGCAT2
DENV3_14_LEFTGGAGAACCCTGGGAAGGAACAA2
DENV3_14_RIGHTATGGCTGCCATTGAGGTATGTC2
 

DENV4
ABC
DENV4_5_LEFTGTGCACACTTGGACAGAACAGT1
DENV4_5_RIGHTGCAGTGGTGGTCCTCAAAGATG1
DENV4_7_LEFTGGAAGAATGCTTGAGGAGAAGAGT1
DENV4_7_RIGHTGTCCCGTATGGAGAAAGAGCCA1
DENV4_9_LEFTTATCATACGGTGGGGGATGGAG1
DENV4_9_RIGHTATGCTGGGAACAAACCACACAG1
DENV4_11_LEFTACATTGTTTGGTCCGGAAAGGG1
DENV4_11_RIGHTGTGGGGTCCTTTGTTTTTCTGGT1
DENV4_13_LEFTAGATCTAGAACCAATATCCTATGACCCA1
DENV4_13_RIGHTAGGTTTTGAAGAGAGCCATGGC1
DENV4_15_LEFTTATGAAGCTCCTTCGACAGGCT1
DENV4_15_RIGHTGGGTCTGAGGACTTTCACCACT1
DENV4_4_LEFTAGATGGCAGAAACACAGCATGG2
DENV4_4_RIGHTCATTTTCCAGCCTCGTGGTTGA2
DENV4_6_LEFTGTGAAAACATGTCTGTGGCCCA2
DENV4_6_RIGHTGTTGTCATGGCCATTCCTATTACCA2
DENV4_8_LEFTAATGGTGGCAGGAGGCTTACTT2
DENV4_8_RIGHTTCCTCATCCACTTCATAATCTGGCT2
DENV4_10_LEFTACAGCCCAATAGAAGACATCGAGA2
DENV4_10_RIGHTACCTCCATGTTTTCTTCTAAAATTTGGT2
DENV4_12_LEFTAGGAATAGGGAAATTGTCAATGGGT2
DENV4_12_RIGHTGATTGGTCCTGTGGCCAAAGTC2
DENV4_14_LEFTTTGGTCAAACTCCATTCAGGGG2
DENV4_14_RIGHTACTCTTCCCTTGTGCACAGTCT2
DENV4_16_LEFTATGGAGTGGAGTGGAAGGGGAA2
DENV4_16_RIGHTTGCTGAGCATATGGCCATGGAG2
DENV4_16_LEFTATGGAGTGGAGTGGAAGGGGAA2
DENV4_16_RIGHTTGCTGAGCATATGGCCATGGAG2
 

Protocol references

Wang L, Ng TFF, Castro CJ, et al. Next-generation sequencing of human respiratory syncytial virus subgroups A and B genomes. J Virol Methods. 2022; 299:114335. doi:10.1016/j. jviromet.2021.114335
https://github.com/artic-network/primer-schemes/blob/3258a7fd11f39ad8b52f4743fe97aea4ec129544/nCoV-2019/V5.3.2/SARS-CoV-2.primer.bed
https://github.com/CERI-KRISP/CLIMADE/blob/master/Protocols/Arboviruses/AllPrimerSchemes.xlsx
Illumina. 2023. Illumina Microbial Amplicon Prep. Document #200039808 v00. San Diego, CA: Illumina, Inc.
Illumina. 2023. Illumina Microbial Amplicon Prep for viral surveillance. Application Note M-GL-02215 v1.0. Illumina, Inc

Acknowledgements

The GenPath Africa project is funded by the Global Health EDCTP3 Joint Undertaking and its members, as well as the Bill & Melinda Gates Foundation (101103171) and the South African Medical Research Council.

	Reagent	Storage	Instructions
	EPH3 (Elution Prime Fragment Fragment 3HC Mix)	-25°C to -15°C	Bring to room temperature. Vortex thoroughly before use.

Reagent	Volume (μL) per sample	Required volume for x samples
EPH3	1.5	...
Nuclease free water	3.0	...
Total	4.5	...

	Temperature (°C)	Time
	25	5 minutes
	42	30 minutes
	80	5 minutes
	4	Hold

Reagent	PCR 1 Master Mix (μL)	PCR 2 Master Mix (μL)
IPM	7.5	7.5
Nuclease free water	2.3	2.3
PP1 (10 µM)	2.2	-
PP2 (10 µM)	-	2.2
Total	12	12

Temperature (°C)	Time	Cycle
98	3 minutes
98	15 seconds	35 cycles
64	5 minutes	35 cycles
4	Hold

	Reagent	Volume (μL) n=1
	EBLT	2
	TB1	6
	Nuclease free water	10
	Total	18

	Total cDNA Input (ng)	Number of PCR cycles
	1-24	12
	25-99	9
	100-500	7

Sequencing Platform/Kit	Starting Concentration (nM)	Final Loading Concentration (pM)
NextSeq (Standard-SBS)	2	750
NextSeq (XLEAP-SBS)	2	650
MiSeq i100	2	80
MiSeq V2	4	10-12
MiSeq V3	4	12-14

Primer Name	Sequence (5'-3')	Primer Pool
SARS-CoV-2_1_LEFT	CTCTTGTAGATCTGTTCTCTAAACGAACTTT	1
SARS-CoV-2_1_RIGHT	AAAACGCCTTTTTCAACTTCTACTAAGC	1
SARS-CoV-2_3_LEFT	AGAACGGTAATAAAGGAGCTGGT	1
SARS-CoV-2_3_RIGHT	TTTGCCAATTTAATTTCAAAAGGTGTCTG	1
SARS-CoV-2_5_LEFT	TTTGTGGCACTGAGAATTTGACTAAAGA	1
SARS-CoV-2_5_RIGHT	ATAATGGCGATCTCTTCATTAAGTTTAAAGTC	1
SARS-CoV-2_7_LEFT	GCTGCTCGTGTTGTACGATCAAT	1
SARS-CoV-2_7_RIGHT	CTCCTTAATTTCCTTTGCACAGGTG	1
SARS-CoV-2_9_LEFT	GTCTTGAAAACTGGTGATTTACAACCATTA	1
SARS-CoV-2_9_RIGHT	GTAATTCAGATACTGGTTGCAAAGTTTTTATGA	1
SARS-CoV-2_11_LEFT	GAGCAAGAAGAAGATTGGTTAGATGATGA	1
SARS-CoV-2_11_RIGHT	CGCTTAAAACACAACTACCACCCA	1
SARS-CoV-2_13_LEFT	CTGTCTTTGATAAAAATCTCTATGACAAACTTG	1
SARS-CoV-2_13_RIGHT	CTTTTTAGTAGGTATAACCACAGCAGTTAAAAC	1
SARS-CoV-2_15_LEFT	CACATGCAGAAGAAACACGCAA	1
SARS-CoV-2_15_RIGHT	TCTTTATAGGAACCAGCAAGTGAGATG	1
SARS-CoV-2_17_LEFT	GACAACAGTTTGGTCCAACTTATTTGG	1
SARS-CoV-2_17_RIGHT	CTGTCTTATTACAGTAGGCTAAGATAAGTGC	1
SARS-CoV-2_19_LEFT	CCTTTTGTTATGATGTCAGCACCAC	1
SARS-CoV-2_19_RIGHT	CACATACAAACTTAAAATTATCGAAGCTTGC	1
SARS-CoV-2_21_LEFT	GAAAGGAGCTAAATTGTTACATAAACCTATTGT	1
SARS-CoV-2_21_RIGHT	AGGTTTCTTAATAGTAAGACTAGAATTGTCTAC	1
SARS-CoV-2_23_LEFT	AATTCTAGAATTAAAGCATCTATGCCGACTA	1
SARS-CoV-2_23_RIGHT	CAAAAGCAGTTAAATCCCATTTAAAAGATG	1
SARS-CoV-2_25_LEFT	GTGCATGTTGTAGACGGTTGTAATTC	1
SARS-CoV-2_25_RIGHT	CTCTCAGGTTGTCTAAGTTAACAAAATGAGA	1
SARS-CoV-2_27_LEFT	AAAACTCAAAACACTAGTTGCAACTGC	1
SARS-CoV-2_27_RIGHT	CACATGTCAACTTAAAAGGTAAGTTATTCT	1
SARS-CoV-2_29_LEFT	CAGATACTTGTTTTGCTAACAAACATGCT	1
SARS-CoV-2_29_RIGHT	ACATAACGTGTGTCAGGGCGTA	1
SARS-CoV-2_31_LEFT	GATCTTTACCAGGAGTTTTCTGTGG	1
SARS-CoV-2_31_RIGHT	ACAAATGATATAAGCAATTGTTATCCAGAAAGG	1
SARS-CoV-2_33_LEFT	ATAATAAGTACAAGTATTTTAGTGGAGCAATGG	1
SARS-CoV-2_33_RIGHT	AATGTCCAATAACCCTGAGTTGAACATTA	1
SARS-CoV-2_35_LEFT	TTTTTGTTACATGCACCATATGGAATTACC	1
SARS-CoV-2_35_RIGHT	AAATTCATCTTCTAATAAAGCACTACCCAA	1
SARS-CoV-2_37_LEFT	AAACATAAGCATGCATTTCTCTGTTTGTT	1
SARS-CoV-2_37_RIGHT	ACCTCTGGCCAAAAACATGACA	1
SARS-CoV-2_39_LEFT	AACATTAAATTGTTGGGTGTTGGTGG	1
SARS-CoV-2_39_RIGHT	CAGAATCACCATTAGCAACAGCCT	1
SARS-CoV-2_41_LEFT	CAAGAGATGGTTGTGTTCCCTTGAA	1
SARS-CoV-2_41_RIGHT	AACAGTGCAAGTACAAACCTACCTC	1
SARS-CoV-2_43_LEFT	GCTTTTGCTGTAGATGCTGCTAAA	1
SARS-CoV-2_43_RIGHT	CACCGCAAACCCGTTTAAAAAC	1
SARS-CoV-2_45_LEFT	ACTTCTTTAAGTTTAGAATAGACGGTGAC	1
SARS-CoV-2_45_RIGHT	TCTACAACAGGAACTCCACTACCT	1
SARS-CoV-2_47_LEFT	CTTTAATGTTTTATTCTCTACAGTGTTCCCAC	1
SARS-CoV-2_47_RIGHT	ATCCTGAGCAAAGAAGAAGTGTTTTAATTC	1
SARS-CoV-2_49_LEFT	CACTTTTCGCATATACAAAACGTAATGTC	1
SARS-CoV-2_49_RIGHT	CACTCATTAGCTAATCTATAGAAACGGTGT	1
SARS-CoV-2_51_LEFT	CTTTGTGAATGAGTTTTACGCATATTTGC	1
SARS-CoV-2_51_RIGHT	AGGATGTTTAGTAAGTGGGTAAGCATCTATA	1
SARS-CoV-2_53_LEFT	GTGCTTGCATACGTAGACCATTCTTA	1
SARS-CoV-2_53_RIGHT	GACAGTTTAAATGTCTCCTCAGTAGCTTT	1
SARS-CoV-2_55_LEFT	CAAGAGCACTATGTTAGAATTACTGGCTTATA	1
SARS-CoV-2_55_RIGHT	TTTCATCAAAGACAACTATATCTGCTGTC	1
SARS-CoV-2_57_LEFT	GGCACACTAGAACCAGAATATTTCAATTC	1
SARS-CoV-2_57_RIGHT	AGTGGTTTGAGTGAATATGACATAGTC	1
SARS-CoV-2_59_LEFT	GTGTTGACACTAAATTCAAAACTGAAGGTTTA	1
SARS-CoV-2_59_RIGHT	CGCACTACATTCCAAGGAAGTCC	1
SARS-CoV-2_61_LEFT	GGTAACCTACAAAGCAACCATGATCT	1
SARS-CoV-2_61_RIGHT	TCCAAAATAGGCATACACCATCTGT	1
SARS-CoV-2_63_LEFT	AGTGTCAGATATAGATTATGTACCACTAAAGTCT	1
SARS-CoV-2_63_RIGHT	GCCCAAAGCTCAAATGCTACATTAAC	1
SARS-CoV-2_65_LEFT	GCCCGTAATGGTGTTCTTATTACAGA	1
SARS-CoV-2_65_RIGHT	GTACTGTCCATAGGAATAAAATCTTCTAATTCA	1
SARS-CoV-2_67_LEFT	AATTACAATCTAGTCAAGCGTGGCAA	1
SARS-CoV-2_67_RIGHT	CATATCACTAATAATGAGATCCCATTTATTAGC	1
SARS-CoV-2_69_LEFT	ATGTCATGCATGCAAATTACATATTTTGGA	1
SARS-CoV-2_69_RIGHT	AGTCCTGAGTTGAATGTAAAACTGAG	1
SARS-CoV-2_71_LEFT	GAGGCTGGATTTTTGGTACTACTTTAGA	1
SARS-CoV-2_71_RIGHT	ATGTTAATACCTATTGGCAAATCTACCA	1
SARS-CoV-2_73_LEFT	CGTTGAAATCCTTCACTGTAGAAAAAGG	1
SARS-CoV-2_73_RIGHT	GCTATAACGCAGCCTGTAAAATCATCT	1
SARS-CoV-2_75_LEFT	AACCATACAGAGTAGTAGTACTTTCTTTTGA	1
SARS-CoV-2_75_RIGHT	CCTGTAGAATAAACACGCCAAGTAGG	1
SARS-CoV-2_77_LEFT	GCAGGTATATGCGCTAGTTATCAGAC	1
SARS-CoV-2_77_RIGHT	AATTGGTGGTGTTTTGTAAATTTGTTTGAC	1
SARS-CoV-2_79_LEFT	GATGAAATGATTGCTCAATACACTTCTGC	1
SARS-CoV-2_79_RIGHT	TGCCTGTGATCAACCTATCAATTTG	1
SARS-CoV-2_81_LEFT	CATGTGACTTATGTCCCTGCACA	1
SARS-CoV-2_81_RIGHT	GAGATTCATTTAAATTCTTGGCAACCTCATT	1
SARS-CoV-2_83_LEFT	CATTACACATAAACGAACTTATGGATTTGT	1
SARS-CoV-2_83_RIGHT	GCCAAAGCCTCATTATTATTCTTACAAAGTTTA	1
SARS-CoV-2_85_LEFT	TCACTTCAGACTATTACCAGCTGTACTC	1
SARS-CoV-2_85_RIGHT	AAAGAAGGTTTTACAAGACTCACGTTAAC	1
SARS-CoV-2_87_LEFT	AGGTTTCCTATTCCTTACATGGATTTGT	1
SARS-CoV-2_87_RIGHT	CCTTGATGTCACAGCGTCCT	1
SARS-CoV-2_89_LEFT	GATGTTTCATCTCGTTGACTTTCAGG	1
SARS-CoV-2_89_RIGHT	GCCGTCAGGACAAGCAAAAG	1
SARS-CoV-2_91_LEFT	TATCTTTTGGTTCTCACTTGAACTGCAA	1
SARS-CoV-2_91_RIGHT	CGAACGTCATGATACTCTAAAAAGTCTT	1
SARS-CoV-2_93_LEFT	TCGAGGACAAGGCGTTCCAA	1
SARS-CoV-2_93_RIGHT	GCCTGGAGTTGAATTTCTTGAACT	1
SARS-CoV-2_95_LEFT	CTGATTACAAACATTGGCCGCAAA	1
SARS-CoV-2_95_RIGHT	ATCTGCCTTGTGTGGTCTGCA	1
SARS-CoV-2_2_LEFT	TCGTACGTGGCTTTGGAGACTC	2
SARS-CoV-2_2_RIGHT	TCTTCATAAGGATCAGTGCCAAGCT	2
SARS-CoV-2_4_LEFT	CATGAAATTGCTTGGTACACGGAAC	2
SARS-CoV-2_4_RIGHT	AAATTTTAACAACAGCATTTTGGGGTAAGT	2
SARS-CoV-2_6_LEFT	GTGTTGTTGGAGAAGGTTCCGA	2
SARS-CoV-2_6_RIGHT	TATTGTTATAGCGGCCTTCTGTAAAAC	2
SARS-CoV-2_8_LEFT	TGCTTGTGAAATTGTCGGTGGAC	2
SARS-CoV-2_8_RIGHT	AAGCCCGTTAATACAAACTGGTGTAC	2
SARS-CoV-2_10_LEFT	CTCGGTACAGAAGTAAATGAGTTCGC	2
SARS-CoV-2_10_RIGHT	TGTCCTCACTGCCGTCTTGTT	2
SARS-CoV-2_12_LEFT	CATGCAAGTTGAATCTGATGATTACATAGC	2
SARS-CoV-2_12_RIGHT	AGTTATAAATGGCTTAACTTCCTCTTTAGG	2
SARS-CoV-2_14_LEFT	GTGACATTGACATCACTTTCTTAAAGAAA	2
SARS-CoV-2_14_RIGHT	CCCTTATATTTACGCTGTATAGTTGAAACTA	2
SARS-CoV-2_16_LEFT	AATGGTTATCTTACTTCTTCTTCTAAAACACCT	2
SARS-CoV-2_16_RIGHT	AAATGTTTTACCTTCATGTGAATTATGAGG	2
SARS-CoV-2_18_LEFT	GCTCTACAAGATGCTTATTACAGAGC	2
SARS-CoV-2_18_RIGHT	ACTCACTAGCACAAGTAAATGTACCATG	2
SARS-CoV-2_20_LEFT	CCATAAAACCAGTTACTTATAAATTGGATGGT	2
SARS-CoV-2_20_RIGHT	CAGGTATTTGGTTTATACGTGGCTTTATTAG	2
SARS-CoV-2_22_LEFT	TAAAAATTACAGAAGAGGTTGGCCACA	2
SARS-CoV-2_22_RIGHT	TAATTAAATGAAGCCTCTAGACAAAATTTACCG	2
SARS-CoV-2_24_LEFT	GTTTAGATTCTTTAGACACCTATCCTTCTTTAGA	2
SARS-CoV-2_24_RIGHT	ACAATAGTTGTACATTCGACTCTTGTT	2
SARS-CoV-2_26_LEFT	CATCTTTACTTTGATAAAGCTGGTCAAAA	2
SARS-CoV-2_26_RIGHT	GAAATAAAAGTAGATAAGACATTGTCTAAGGAC	2
SARS-CoV-2_28_LEFT	CGTTAAAGATTTCATGTCATTGTCTGAACA	2
SARS-CoV-2_28_RIGHT	ATTAGTATAACTACCACCACGCTGG	2
SARS-CoV-2_30_LEFT	CAATTTTTAAAGATGCTTCTGGTAAGCCA	2
SARS-CoV-2_30_RIGHT	ACAATACCACCAGCTACTATAGATGC	2
SARS-CoV-2_32_LEFT	GGTGTTTATTCTGTTATTTACTTGTACTTGACA	2
SARS-CoV-2_32_RIGHT	TTGCGAGATGACAACAAGCAGC	2
SARS-CoV-2_34_LEFT	TGAAGATTTACTCATTCGTAAGTCTAATCA	2
SARS-CoV-2_34_RIGHT	GTCAACAAAAGGTCCATAAAAGTTACCT	2
SARS-CoV-2_36_LEFT	CCGTTTTAGATATGTGTGCTTCATTAAAAGAAT	2
SARS-CoV-2_36_RIGHT	GGCATATAGACCATATTAAAATAAGCTACAG	2
SARS-CoV-2_38_LEFT	GTGGGCTCTTATAATCTCTGTTACTTCTAAC	2
SARS-CoV-2_38_RIGHT	CTTTACATCTGACATTTTAGACTGTACAGTG	2
SARS-CoV-2_40_LEFT	TCCCTTCCATCATATGCAGCTTTTG	2
SARS-CoV-2_40_RIGHT	TTTTTATATGTGTTATAGTCTGGTATGACAACC	2
SARS-CoV-2_42_LEFT	GCACTGATGACAATGCGTTAGC	2
SARS-CoV-2_42_RIGHT	CAATTAGTGATTGGTTGTCCCCCAC	2
SARS-CoV-2_44_LEFT	ATCAACTCCGCGAACCCATG	2
SARS-CoV-2_44_RIGHT	TGCCATTGTGTATTTAGTAAGACGTTGA	2
SARS-CoV-2_46_LEFT	CTCAATGGTAACTGGTATGATTTCGGT	2
SARS-CoV-2_46_RIGHT	TGGAACACCATCAACAAATATTTTTCTCAC	2
SARS-CoV-2_48_LEFT	GACTTTGCTGTGTCTAAGGGTTTCT	2
SARS-CoV-2_48_RIGHT	TTCTTTGCACTAATGGCATACTTAAGATTC	2
SARS-CoV-2_50_LEFT	TCACTTGTTCTTGCTCGCAAACAT	2
SARS-CoV-2_50_RIGHT	TATTGAAACACACAACAGCATCGTCA	2
SARS-CoV-2_52_LEFT	TGGTACACTTATGATTGAACGGTTCG	2
SARS-CoV-2_52_RIGHT	GTGACATCACAACCTGGAGCATT	2
SARS-CoV-2_54_LEFT	TCAAGCTTTTTGCAGCAGAAACG	2
SARS-CoV-2_54_RIGHT	ACATTGCTAGAAAACTCATCTGAGATATT	2
SARS-CoV-2_56_LEFT	CACTATGTGAGAAGGCATTAAAATATTTGC	2
SARS-CoV-2_56_RIGHT	ACAATTTCAGCAGGACAACGCC	2
SARS-CoV-2_58_LEFT	GGGACTACCAACTCAAACTGTTGATT	2
SARS-CoV-2_58_RIGHT	ATCATAGAGATGAGTCTTCTATAGGTCATGT	2
SARS-CoV-2_60_LEFT	CGCCTGGAGATCAATTTAAACACC	2
SARS-CoV-2_60_RIGHT	GCATCACAACTAGCTACATGTGCAT	2
SARS-CoV-2_62_LEFT	TCTATGATGCACAGCCTTGTAGTGA	2
SARS-CoV-2_62_RIGHT	GTCTACAGACAGCACCACCTAAATTG	2
SARS-CoV-2_64_LEFT	AGTTGATGGTGTTGATGTAGAATTGTTTG	2
SARS-CoV-2_64_RIGHT	TAATGTGACTCCATTAAGACTAGCTTGTTT	2
SARS-CoV-2_66_LEFT	GGTTTACATCTACTGATTGGACTAGCTAAA	2
SARS-CoV-2_66_RIGHT	CACTATCACCATAATTTTGAAGGTCACAC	2
SARS-CoV-2_68_LEFT	ATTGGTGATTGTGCAACTGTACATACA	2
SARS-CoV-2_68_RIGHT	ACTCATGTCAAATAAAGAATAGGAAGACAA	2
SARS-CoV-2_70_LEFT	TTTATTGCCACTAGTCTCTAGTCAGTGT	2
SARS-CoV-2_70_RIGHT	AATTCACAGACTTTAATAACAACATTAGTAGCG	2
SARS-CoV-2_72_LEFT	GGTTATTTTAAAATATATTCTAAGCACACGCCT	2
SARS-CoV-2_72_RIGHT	ATCTAACAATAGATTCTGTTGGTTGGACTC	2
SARS-CoV-2_74_LEFT	ATGTCTATGCAGATTCATTTGTAATTAGAGGT	2
SARS-CoV-2_74_RIGHT	TCCACAAACAGTTGCTGGTGC	2
SARS-CoV-2_76_LEFT	CAAAAAGTTTCTGCCTTTCCAACAATTTG	2
SARS-CoV-2_76_RIGHT	TGACTAGCTACACTACGTGCCC	2
SARS-CoV-2_78_LEFT	GCAATATGGCAGTTTTTGTACACAATTAAA	2
SARS-CoV-2_78_RIGHT	GCACCAAAGGTCCAACCAGAAG	2
SARS-CoV-2_80_LEFT	ACGCTTGTTAAACAACTTAGCTCCAA	2
SARS-CoV-2_80_RIGHT	CTTCACGAGGAAAGTGTGCTTTTC	2
SARS-CoV-2_82_LEFT	TGATTTAGGTGACATCTCTGGCATTAATG	2
SARS-CoV-2_82_RIGHT	GATTTCACCTTGCTTCAAAGTTACAGTTC	2
SARS-CoV-2_84_LEFT	GTAACAGTTTACTCACACCTTTTGCTC	2
SARS-CoV-2_84_RIGHT	TGTTCAACACCARTGTCTGTACTC	2
SARS-CoV-2_86_LEFT	CATCCTTACTGCGCTTCGATTGT	2
SARS-CoV-2_86_RIGHT	ACCGGTGATCCAATTTATTCTGTAAA	2
SARS-CoV-2_88_LEFT	GTGGACATCTTCGTATTGCTGGA	2
SARS-CoV-2_88_RIGHT	CCATTGGTTGCTCTTCATCTAATTGAG	2
SARS-CoV-2_90_LEFT	TCATCCTCTAGCTGATAACAAATTTGCA	2
SARS-CoV-2_90_RIGHT	TTCTTGGTGAAATGCAGCTACAG	2
SARS-CoV-2_92_LEFT	TTCCTGTTTACCTTTTACAATTAATTGCCAG	2
SARS-CoV-2_92_RIGHT	CTTCGGTAGTAGCCAATTTGGTCATC	2
SARS-CoV-2_94_LEFT	GCAGTCAAGCCTCTTCTCGTT	2
SARS-CoV-2_94_RIGHT	CATTCCGAAGAACGCTGAAGC	2
SARS-CoV-2_96_LEFT	CTGCAGATTTGGATGATTTCTCCA	2
SARS-CoV-2_96_RIGHT	TTTGTCATTCTCCTAAGAAGCTATTAAAATCAC	2

Primer Name	Sequence (5'-3')	Pool
DENV1_1_LEFT	AATATGCTGAAACGCGCGAGAA	1
DENV1_1_RIGHT	CCGTCTTCAAGAGTTCAATGTCCA	1
DENV1_3_LEFT	GGAAATACAGCTGACCGACTACG	1
DENV1_3_RIGHT	ACTGCAATGCACGTCATCGAAA	1
DENV1_5_LEFT	TGGAACATTTGGGAAGTTGAGGAC	1
DENV1_5_RIGHT	ACTTCTCTGGATGTTAGTCTGCG	1
DENV1_7_LEFT	AGTTGGCCCCTCAATGAAGGAA	1
DENV1_7_RIGHT	GCACTGACGTAGGTTCCACTTG	1
DENV1_9_LEFT	ATAGCGGCCAGAGGGTACATCT	1
DENV1_9_RIGHT	TGTTCTCCTCCAACACCTGGTT	1
DENV1_11_LEFT	TGTGGTGATAGGTTTGTTATTCATGATACT	1
DENV1_11_RIGHT	CTTTGGCTTCGGATCTGTCCAC	1
DENV1_13_LEFT	TGGAGCAAATGCAAAGAAAACATGG	1
DENV1_13_RIGHT	TGCACGACTTCCTTTTGCCTTT	1
DENV1_15_LEFT	AGACGTGACCAGAGAGGAAGTG	1
DENV1_15_RIGHT	TCACTTGGTTTATGGCCACTTGT	1
DENV1_2_LEFT	ACCCAGGATTCACGGTGATAGC	2
DENV1_2_RIGHT	ACCAGCAAATCTTGTCTGTTCCA	2
DENV1_4_LEFT	CAAGAAAGGAAGCAGCATAGGGA	2
DENV1_4_RIGHT	TTGATGGCAGCTGACATTAGCC	2
DENV1_6_LEFT	TGGATGAACATTGTGGAAATCGAGG	2
DENV1_6_RIGHT	GCATGCCTCCAGCTATTAGTGG	2
DENV1_8_LEFT	TCTCATATGGAGGAGGTTGGAGG	2
DENV1_8_RIGHT	AGCCTGAGTTCCATGATCTCTCA	2
DENV1_10_LEFT	AAAGAGTGCAGCAATAGACGGG	2
DENV1_10_RIGHT	ATAGAGGGTCCAGGCTGAAGCT	2
DENV1_12_LEFT	GGGAAACACTGGGAGAGAAATGG	2
DENV1_12_RIGHT	TGATCCTGATGGCTTGACCTCA	2
DENV1_14_LEFT	ACTCAGCAAAAGAAGCAGTGGA	2
DENV1_14_RIGHT	GCATGGCACCACTATTTCCCTC	2

Primer Name	Sequence (5'-3')	Primer Pool
RSVA-CDC20_1_LEFT	ACGSGAAAAAATGCGTACAAC	1
RSVA-CDC20_1_RIGHT	AGTGCCATATTTTGTGTTGTATTCAG	1
RSVA-CDC20_3_LEFT	GTAACAACACATCGTCAAGACATTA	1
RSVA-CDC20_3_RIGHT	CTGTTGTTTGCRTCTTCTCCA	1
RSVA-CDC20_5_LEFT	TGTCTCTCAATCCAACATCAGAG	1
RSVA-CDC20_5_RIGHT	GCTCCAAGATCTACTATGAATTGAC	1
RSVA-CDC20_8_LEFT	GAAGTGTTCAAYTTTGTACCCTGC	1
RSVA-CDC20_8_RIGHT	ACTGGCGATTGCAGATCCA	1
RSVA-CDC20_10_LEFT	GTRTTTTGTGACACAATGAACAGT	1
RSVA-CDC20_10_RIGHT	CCATTCAAGCAATGACCTCG	1
RSVA-CDC20_12_LEFT	GATACTACCTGACAAATATCCTTGTAG	1
RSVA-CDC20_12_RIGHT	TTACCTCAYTWGATCGATAYTGTGT	1
RSVA-CDC20_14_LEFT	CTTGCAGGTGACAATAACCT	1
RSVA-CDC20_14_RIGHT	TGCCTATATGTGCATATTATTGTGAC	1
RSVA-CDC20_16_LEFT	AGAATCTATAGGTAGTTTGACACAAG	1
RSVA-CDC20_16_RIGHT	GCTTGTTGTATATTTKATGTCCATTG	1
RSVA-CDC20_18_LEFT	CACAGGTGATGTTGATATTCACAAG	1
RSVA-CDC20_18_RIGHT	AGCTTCTTATTAGATAACRATGGTAAC	1
RSVA-CDC20_20_LEFT	ATAGAGTGGAGCAAGCATGT	1
RSVA-CDC20_20_RIGHT	ACGAGAAAAAAAGTGTCAAAAACTAA	1
RSVB-CDC20_2_LEFT	TGATGATCACAGACATGAGACC	1
RSVB-CDC20_2_RIGHT	TTATCATCCCACAGTCTGGAG	1
RSVB-CDC20_4_LEFT	GAGCAACTCAAAGAAAATGGAG	1
RSVB-CDC20_4_RIGHT	GTCTGGCARTTGATTCAATGGA	1
RSVB-CDC20_6_LEFT	GATCTWACCATGAAGACATTCAACC	1
RSVB-CDC20_6_RIGHT	ACTACTCTATGCTACCATRATRGT	1
RSVB-CDC20_8_LEFT	GATGATTACCATTTTGAAGTGTTCAA	1
RSVB-CDC20_8_RIGHT	CTTCGTTTCCTCTTCTTGCTTAT	1
RSVB-CDC20_10_LEFT	GCAATGTTCAGATAGTAAGGCAAC	1
RSVB-CDC20_10_RIGHT	TGACTGTAGTGRCATCTTCTACC	1
RSVB-CDC20_12_LEFT	CACCTAAGATAAGAGTRTACAATACTGT	1
RSVB-CDC20_12_RIGHT	AGTACTGAGTTTTCATCACCTGA	1
RSVB-CDC20_14_LEFT	AAGAAGTAGARGGATTTATTATGTCTT	1
RSVB-CDC20_14_RIGHT	ATCTTGTCAAACTCTCAGGGAA	1
RSVB-CDC20_16_LEFT	AGCACAATGGAGTGTACTATCC	1
RSVB-CDC20_16_RIGHT	CTTTCTCTTACATACTTGCTTAATTCAG	1
RSVB-CDC20_18_LEFT	GTTGTGGAACAATTCACAAACAT	1
RSVB-CDC20_18_RIGHT	TCCACTTATACAAAATTTAGGTTTGTT	1
RSVB-CDC20_20_LEFT	TGYGATGCTGAATTACCTGT	1

Primer Name	Sequence (5'-3')	Pool
DENV2_1_LEFT	AGCAGATCTCTGATGAATAACCAACG	1
DENV2_1_RIGHT	TTTTTGCCATCGTCGTCACACA	1
DENV2_3_LEFT	ACATTGGTCACTTTCAAAAATCCCC	1
DENV2_3_RIGHT	TGAAGGGGATTCTGGTTGGAACT	1
DENV2_5_LEFT	TCATGCAGGCAGGAAAACGATC	1
DENV2_5_RIGHT	TCTCAAGAGTAGTCCAGCTGCA	1
DENV2_7_LEFT	CCAATCCTGTCAATAACAATATCAGAAGAT	1
DENV2_7_RIGHT	TGATGGCTGGGGTTTGGTATCT	1
DENV2_9_LEFT	ATGCCAGTGACCCACTCTAGTG	1
DENV2_9_RIGHT	CCACCACTGTGAGGATGGCTAT	1
DENV2_11_LEFT	GGAGCTGGACTTCTCTTTTCCAT	1
DENV2_11_RIGHT	GACGTCCCAAGGTTTTGTCAGC	1
DENV2_13_LEFT	TGGGACACAAGAATCACACTAGAAG	1
DENV2_13_RIGHT	CCGCACCATTGGTCTTCTCTTT	1
DENV2_2_LEFT	TCGCTCCTTCAATGACAATGCG	2
DENV2_2_RIGHT	CCATTCTCAGCCTGCACTTGAG	2
DENV2_4_LEFT	ATAGTGGTTGCGTTGTGAGCTG	2
DENV2_4_RIGHT	CGGCAGCACCATTCTGTTATGA	2
DENV2_6_LEFT	TGGAAATCAGACCATTGAAAGAGAAAGA	2
DENV2_6_RIGHT	TGGTCAGTGTTTGTTCTTCCTCTT	2
DENV2_8_LEFT	AGATCGAAGATGACATTTTCCGAAAGA	2
DENV2_8_RIGHT	CCCATGTATATGTACTGGTCATTTTCATT	2
DENV2_10_LEFT	ACCAGAAAAACAGAGAACACCCC	2
DENV2_10_RIGHT	CCACTTCCTGGATTCCACTTTTCT	2
DENV2_12_LEFT	AGAGCATGAAACATCATGGCACT	2
DENV2_12_RIGHT	GTGCCTCTTGGTGTTGGTCTTT	2

Primer Name	Sequence (5'-3')	Primer Pool
DENV3_1_LEFT	TCAATATGCTGAAACGCGTGAGA	1
DENV3_1_RIGHT	GCCTCGGTCTTCTGAAGCTCTA	1
DENV3_3_LEFT	ACCAATAGAGGGAAAAGTGGTGC	1
DENV3_3_RIGHT	TGGCCTCGAACATCTTCCCAAT	1
DENV3_5_LEFT	GGCAAAAATAGTGACAGCTGAAACA	1
DENV3_5_RIGHT	TCTCCATGTTATTTGCCCTGAGAGA	1
DENV3_7_LEFT	GCCAGTCTTCGAGCATGAGGAA	1
DENV3_7_RIGHT	TTCCCAGGCTCTACGGCAATAA	1
DENV3_9_LEFT	GCAACAAAATCTGAACACACAGGA	1
DENV3_9_RIGHT	CCCTCCTCATGAGTTCCACGAA	1
DENV3_11_LEFT	GGAAAGACTTCAATAGGACTCATTTGTG	1
DENV3_11_RIGHT	CAGGTGATCCTTCCCAGAGTGT	1
DENV3_13_LEFT	AGTGGAAGAAAGCAGAACTATAAGAGT	1
DENV3_13_RIGHT	TGGAGTTCACGTTCTCTGTCCA	1
DENV3_15_LEFT	TGGACATCATATCTAGGAAAGACCAAAG	1
DENV3_15_RIGHT	AGGTTGCTCTGGAAGTGAGACC	1
DENV3_2_LEFT	GGGAGTAGGAAACAGAGATTTTGTGG	2
DENV3_2_RIGHT	GAGTATTGTCCCATGCTGCGTT	2
DENV3_4_LEFT	GCTGAACCTCCTTTTGGGGAAA	2
DENV3_4_RIGHT	TTCCACCTCCCACACATTCCAT	2
DENV3_6_LEFT	AGGTGGACAACTTCACAATGGG	2
DENV3_6_RIGHT	CTCAGCCTCTTCCTCCCATGTT	2
DENV3_8_LEFT	AAAGACTGGAACCAAACTGGGC	2
DENV3_8_RIGHT	TGCCTGAATTCCATGAGCGTTC	2
DENV3_10_LEFT	AGACCATGCTCACTGGACAGAA	2
DENV3_10_RIGHT	TATGCGAGTTGGTTGTCTTGGG	2
DENV3_12_LEFT	GTGGATGGGATAATGACAATAGACCT	2
DENV3_12_RIGHT	GGTGCTCAATCACAGTTGGCAT	2
DENV3_14_LEFT	GGAGAACCCTGGGAAGGAACAA	2
DENV3_14_RIGHT	ATGGCTGCCATTGAGGTATGTC	2

A	B	C
DENV4_5_LEFT	GTGCACACTTGGACAGAACAGT	1
DENV4_5_RIGHT	GCAGTGGTGGTCCTCAAAGATG	1
DENV4_7_LEFT	GGAAGAATGCTTGAGGAGAAGAGT	1
DENV4_7_RIGHT	GTCCCGTATGGAGAAAGAGCCA	1
DENV4_9_LEFT	TATCATACGGTGGGGGATGGAG	1
DENV4_9_RIGHT	ATGCTGGGAACAAACCACACAG	1
DENV4_11_LEFT	ACATTGTTTGGTCCGGAAAGGG	1
DENV4_11_RIGHT	GTGGGGTCCTTTGTTTTTCTGGT	1
DENV4_13_LEFT	AGATCTAGAACCAATATCCTATGACCCA	1
DENV4_13_RIGHT	AGGTTTTGAAGAGAGCCATGGC	1
DENV4_15_LEFT	TATGAAGCTCCTTCGACAGGCT	1
DENV4_15_RIGHT	GGGTCTGAGGACTTTCACCACT	1
DENV4_4_LEFT	AGATGGCAGAAACACAGCATGG	2
DENV4_4_RIGHT	CATTTTCCAGCCTCGTGGTTGA	2
DENV4_6_LEFT	GTGAAAACATGTCTGTGGCCCA	2
DENV4_6_RIGHT	GTTGTCATGGCCATTCCTATTACCA	2
DENV4_8_LEFT	AATGGTGGCAGGAGGCTTACTT	2
DENV4_8_RIGHT	TCCTCATCCACTTCATAATCTGGCT	2
DENV4_10_LEFT	ACAGCCCAATAGAAGACATCGAGA	2
DENV4_10_RIGHT	ACCTCCATGTTTTCTTCTAAAATTTGGT	2
DENV4_12_LEFT	AGGAATAGGGAAATTGTCAATGGGT	2
DENV4_12_RIGHT	GATTGGTCCTGTGGCCAAAGTC	2
DENV4_14_LEFT	TTGGTCAAACTCCATTCAGGGG	2
DENV4_14_RIGHT	ACTCTTCCCTTGTGCACAGTCT	2
DENV4_16_LEFT	ATGGAGTGGAGTGGAAGGGGAA	2
DENV4_16_RIGHT	TGCTGAGCATATGGCCATGGAG	2
DENV4_16_LEFT	ATGGAGTGGAGTGGAAGGGGAA	2
DENV4_16_RIGHT	TGCTGAGCATATGGCCATGGAG	2