TITLE: DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus
AUTHORS: Chantal Vogels, Chrispin Chaguza, Mallery I Breban, Afeez Sodeinde, Emma Taylor-Salmon, Abigail J. Porzucek, Nathan D Grubaugh

[DESCRIPTION]
Version 2 updates:
Updated the DENV3 primer file (DENV3_Primer-Scheme.xlsx) with correct version


Background
Amplicon-based sequencing (PrimalSeq) was developed in response to the Zika virus epidemic due to difficulties generating complete genomes using metagenomic approaches [1,2]. Later this approach was adapted as the primary sequencing method for SARS-CoV-2 (i.e. the “ARTIC” protocol) [3]. During the COVID-19 pandemic, investments in genomic infrastructure have resulted in a significant increase in sequencing capacity leading to a record number of over 14 million publicly available SARS-CoV-2 genomes. This expanded sequencing capacity can be utilized to improve genomic surveillance of other viruses, by swapping out components such as primer schemes. The COVID-19 pandemic further exemplifies how genomic surveillance can help to track the emergence and spread of variants, facilitate vaccine development, and inform diagnostics and therapeutics. Increased genomic surveillance of other viruses of public health concern, such as the mosquito-borne dengue virus, is needed to reduce the future burden of disease. Particularly, genomic surveillance can help to monitor the roll out of novel control strategies such as vaccines and release of mosquitoes carrying the virus-inhibiting Wolbachia bacteria. However, the majority of currently available sequences are partial, making them unsuitable to monitor and refine novel control tools. Here, we developed and validated primer schemes for all four dengue virus serotypes to improve whole-genome sequencing using an amplicon-based sequencing approach. The serotype-specific primers can be used individually or combined as a universal pan-serotype dengue virus amplicon-based sequencing approach. 

Overview of design 
We used PrimalScheme (https://primalscheme.com/) to generate four serotype-specific dengue virus primer schemes (serotypes 1-4). For each primer scheme, we selected 10 publicly available genomes that represent the genetic diversity within each serotype. As reference genomes, we used the four serotype-specific reference genomes available on GenBank (DENV1: NC_001477.1, DENV2: NC_001474.2, DENV3: NC_001475.2, DENV4: NC_002640.1). Each primer scheme consists of 35-37 primer pairs with an amplicon length of ~400 bp. 

Initial validation
We validated the four primer schemes with dengue virus stocks that we obtained from the Yale Arbovirus Research Unit (YARU) and World Reference Center for Emerging Viruses and Arboviruses (WRCEVA, UTMB) collections. These virus stocks spanned the defined genotypes within each of the four serotypes. Serotypes were confirmed and PCR cycle threshold (Ct) values were determined with the CDC real-time RT-PCR assay [4]. We sequenced dengue viruses using the Illumina COVIDSeq test (RUO version) and generated consensus genomes at a depth of coverage of 20X using iVar (version 1.3.1). Initially, we sequenced undiluted virus stocks with the serotype-specific assay and found high coverage across all genotypes within each serotype, except for sylvatic dengue virus serotype 2 (genotype VI; Figure 1). Next, we selected 1-2 viruses for each genotype and diluted these viruses until no RNA was detected. By sequencing these diluted viruses we found that we were able to generate near-complete genomes for samples containing at least ~100 RNA copies per μL (PCR Ct value of approximately 30) for each serotype. Some selected viruses had mismatches with the real-time RT-PCR assay, resulting in reduced sensitivity of the assay. As a result, there may be varying levels of genome coverage for samples that were not detected by the real-time RT-PCR assay. 


 
To facilitate high-throughput sequencing, we developed a single pan-serotype dengue virus approach that can be used for all four serotypes. By mixing the four primer schemes and increasing the working concentration of the primers to 20 μM, we were able to achieve similar genome coverage as compared to the serotype-specific approach (Figure 2). This pan-serotype protocol for dengue virus is particularly useful when the serotype is unknown, when there are co-infections with multiple serotypes, or when multiple serotypes are included in a single sequencing run. 


 

Conclusion
In this protocol we present the initial validation of a whole genome amplicon-based sequencing approach for all four dengue virus serotypes. We show that the four primer schemes can be used as serotype-specific or combined pan-serotype dengue virus whole genome sequencing protocol. The newly developed primer schemes can be used with currently established amplicon-based sequencing workflows, like COVIDseq, with minimal change to the protocol. Further testing involves sequencing of clinical samples. Our ultimate goal is to help increase capacity for whole genome dengue virus sequencing. 

Acknowledgements
We would like to acknowledge Philip Armstrong, Doug Brackney, and team (Connecticut Agricultural Experiment Station) for generously providing dengue virus samples from the Yale Arbovirus Research Unit collection, Kenneth Plante and team (The University of Texas Medical Branch) for generously providing dengue virus samples from the World Reference Center for Emerging Viruses and Arboviruses collection, and Mary Petrone and Isabel Ott for preparation of virus stocks. 

References
1. Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc. 2017;12: 1261–1276.
2. Grubaugh ND, Gangavarapu K, Quick J, Matteson NL, De Jesus JG, Main BJ, et al. An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar. Genome Biol. 2019;20: 8.
3. Artic Network. [cited 25 Jul 2022]. Available: https://artic.network/ncov-2019
4. Santiago GA, Vergne E, Quiles Y, Cosme J, Vazquez J, Medina JF, et al. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus. PLoS Negl Trop Dis. 2013;7: e2311.


[BEFORE_START]
If substituting 8-strip PCR tubes for PCR plates, plates must be heat-sealed before each thermocycling step.

[GUIDELINES]
It is recommended that steps performed up to amplicon generation be performed at a different workstation and with different equipment than steps post-amplicon generation.

[STEPS]
SECTION: Dengue Serotype Identification / Plate Setup
1. There are two primer scheme options available for use: Single-Serotype and Pan-Serotype.
SECTION: Prepare Working Primer Solutions
2. 
Briefly centrifuge all primers
SECTION: Prepare Working Primer Solutions
2.1. Within each serotype, organize odd and even-numbered primers into two separate groups:
Odd-numbered primers = Pool 1
Even-numbered primers = Pool 2
 

SECTION: Prepare Working Primer Solutions
2.2. If primers were ordered lyophilized, resuspend to 100 micromolar (µM) 
SECTION: Prepare Working Primer Solutions
2.3. Add 90 µL to two sets of 8-strip PCR tubes for each serotype:

 

SECTION: Prepare Working Primer Solutions
2.4. Label the tops of each tube with the corresponding primer that will be added
For readability, recommended this format: D1 1L, D1 1R, D1 3L, D2 3R...
SECTION: Prepare Working Primer Solutions
2.5. Add 10 µL to each corresponding tube to reach 10 micromolar (µM) 
Recommend to either mix tubes with a new pipette tip or gently vortex

SECTION: Prepare Working Primer Solutions
2.6. Working pool-by-pool, transfer 10 µL into 1.5mL microcentrifuge tubes
8 tubes total, with 2 tubes (Pool 1 and Pool 2) per serotype
Recommended to label each tube with serotype, concentration, pool #, and date of dilution
This will be the working primer solution that will be added during the amplicon generation step

SECTION: Prepare Working Primer Solutions
2.7. Store all primers at -20ºC until use
SECTION: cDNA Synthesis
3. Prepare the following reagents:
 



SECTION: cDNA Synthesis
3.1. Add 8.5 µL to new PCR tubes according to the number of samples
Include one additional reaction for a cDNA NTC for each serotype-specific master mix (NTC), which will be included through the entirety of the protocol


SECTION: cDNA Synthesis
3.2. Add 8.5 µL to each tube
Add 8.5 µL to the cDNA NTC
Mix by pipetting up and down 10 times
Briefly centrifuge tubes


SECTION: cDNA Synthesis
3.3. Load tubes into thermocycler and run the following program to generate first-strand cDNA:
 

SECTION: cDNA Synthesis
3.4. In a new 1.5mL tube, prepare the following master mix according to the number of samples:
 

 


SECTION: cDNA Synthesis
3.5. Add 8 µL to each tube
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
SECTION: cDNA Synthesis
3.6. Load tubes into thermocycler and run the following program to generate second-strand cDNA:
 

SECTION: cDNA Synthesis
3.7. Remove tubes from thermocycler and briefly centrifuge
 

SECTION: Amplicon Generation
4. Prepare the following reagents:
 



SECTION: Amplicon Generation
4.1. In two separate tubes, prepare the following master mixes according to the number of samples:
If processing more than one serotype in a run, prepare separate master mixes for each serotype, using the proper primer pools.
Include one additional reaction for a PCR NTC for each serotype-specific master mix, which will be included through the entirety of the protocol. 

Pool 1
 
Pool 2
 

SECTION: Amplicon Generation
4.2. Distribute the master mixes:
Add 20 µL to a new set of PCR tubes, according to the number of samples
Add 20 µL to a new set of PCR tubes, according to the number of samples
Include one additional tube at the end of each set for the PCR NTC
Be sure to match the serotype of each master mix to the previously-identified serotype of each sample
SECTION: Amplicon Generation
4.3. Add 5 µL to each tube in both Pool 1 and Pool 2. 
Add 5 µL to the PCR NTC in both Pool 1 and Pool 2. 
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
SECTION: Amplicon Generation
4.4. Load tubes into thermocycler and run the following program:
 

SECTION: Amplicon Generation
4.5. When program is complete, samples can remain in the thermocycler at 4°C or be stored at -20°C
Briefly centrifuge tubes before use
 


SECTION: Amplicon Tagmentation and Cleanup
5. Prepare the following reagents:
 

SECTION: Amplicon Tagmentation and Cleanup
5.1. Quantify samples on a Qubit to confirm proper amplification in both amplicon pools
SECTION: Amplicon Tagmentation and Cleanup
5.2. Prepare the following master mix:
 

SECTION: Amplicon Tagmentation and Cleanup
5.3. Add 30 µL to a new set of PCR tubes, according to the number of samples

SECTION: Amplicon Tagmentation and Cleanup
5.4. For each sample, combine amplicon pools:
Add 10 µL to the Master Mix tubes
Add 10 µL to the Master Mix tubes
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
 

SECTION: Amplicon Tagmentation and Cleanup
5.5. Load tubes into thermocycler and run the following program:
 

SECTION: Amplicon Tagmentation and Cleanup
5.6. When the program is complete, immediately remove the tubes from the thermocycler
Briefly centrifuge tubes
SECTION: Amplicon Tagmentation and Cleanup
5.7. Add 10 µL to each tube
Mix by pipetting up and down
Briefly centrifuge tubes

SECTION: Amplicon Tagmentation and Cleanup
5.8. Incubate at Room temperature 
SECTION: Amplicon Tagmentation and Cleanup
5.9. Place tubes on a magnetic stand and wait until the liquid is clear before continuing
SECTION: Amplicon Tagmentation and Cleanup
5.10. Remove and discard all supernatant
Do not allow pipette tip to come into contact with the inner walls of the tube, as it can scrape away the magnetic beads
SECTION: Amplicon Tagmentation and Cleanup
5.11. Remove tubes from the magnetic stand and add 100 µL to each tube (first wash)
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
Be careful not to introduce bubbles
SECTION: Amplicon Tagmentation and Cleanup
5.12. Repeat steps 5.9 - 5.11 (second wash)
Do not remove supernatant after the second wash at this point
SECTION: Amplicon Tagmentation and Cleanup
5.13. Place the tubes on the magnetic stand
SECTION: Amplify Tagmented Amplicons
6. Prepare the following reagents:
 

SECTION: Amplify Tagmented Amplicons
6.1. Prepare the following Master Mix:
 

SECTION: Amplify Tagmented Amplicons
6.2. When the tubes with tagmented amplicons (on the magnetic stand) are clear, remove and discard all supernatant
Remove any remaining supernatant with a smaller-sized pipette.
SECTION: Amplify Tagmented Amplicons
6.3. Remove tubes from the magnetic stand and add 40 µL to each tube
SECTION: Amplify Tagmented Amplicons
6.4. Briefly centrifuge the plate containing the dual-barcoded index adapters
Clean the surface of the sealing foil with RNase Away
Remove residual RNase away by wiping with 70% EtOH
SECTION: Amplify Tagmented Amplicons
6.5. Add 10 µL to each well
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
SECTION: Amplify Tagmented Amplicons
6.6. Load tubes into thermocycler and run the following program:
 
When program is complete, samples can remain in the thermocycler at 10°C or be stored at -20°C. 
 

SECTION: Pooling and Cleanup
7. Prepare the following reagents:
 

SECTION: Pooling and Cleanup
7.1. Place tubes on a magnetic stand and wait until the liquid is clear
SECTION: Pooling and Cleanup
7.2. Pool libraries together in a new 1.5mL microcentrifuge tube by equal volume:
 

 

SECTION: Pooling and Cleanup
7.3. Calculate the volume of ITB needed to reach 0.9x ITB : total pooled volume

 

SECTION: Pooling and Cleanup
7.4. Add the calculated volume of ITB to the pooled sample tube
Vortex for 10 seconds to mix
Briefly centrifuge
SECTION: Pooling and Cleanup
7.5. Incubate at Room temperature 
SECTION: Pooling and Cleanup
7.6. Place tube on a single-tube magnetic stand and wait until the liquid is clear
SECTION: Pooling and Cleanup
7.7. Remove and discard all supernatant
SECTION: Pooling and Cleanup
7.8. Add 1000 µL to the tube and incubate at Room temperature 
SECTION: Pooling and Cleanup
7.9. Repeat steps 7.7 - 7.8 and remove and discard supernatant after the second wash
Remove any residual EtOH with a smaller-sized pipette
SECTION: Pooling and Cleanup
7.10. Add RSB HT to the tube according to the number of samples pooled:
For 1 - 24 samples add 30 µL 
For 25 - 48 samples add 40 µL 
For 49 - 96 samples add 50 µL 
SECTION: Pooling and Cleanup
7.11. Vortex tube to mix 
Briefly centrifuge tube
SECTION: Pooling and Cleanup
7.12. Incubate at Room temperature 
SECTION: Pooling and Cleanup
7.13. Place on the magnetic stand and wait until the liquid is clear
SECTION: Pooling and Cleanup
7.14. Transfer [total volume RSB] - 5µL to a new 1.5mL microcentrifuge tube
SECTION: Pooling and Cleanup
7.15. Quantify library on a Qubit and obtain fragment distribution using a Bioanalyzer/Tape Station
SECTION: Sequencing
8. Protocol validated on the Illumina NovaSeq (2x150).

 

SECTION: Bioinformatics/Analysis
9. Sequencing results may be analyzed utilizing a standard amplicon sequencing bioinformatics pipeline, including those employed for SARS-CoV-2 sequencing (e.g. iVar). 

Reference Sequences:
  
  
  
  
BED files:
  
  
  
  

SECTION: Prepare Working Primer Solutions
2. 
Briefly centrifuge all primers
SECTION: Prepare Working Primer Solutions
2.1. Organize odd and even-numbered primers into two separate groups:
Odd-numbered primers = Pool 1
Even-numbered primers = Pool 2
 

SECTION: Prepare Working Primer Solutions
2.2. If primers were ordered lyophilized, resuspend to 100 micromolar (µM) 
SECTION: Prepare Working Primer Solutions
2.3. Add 80 µL to two sets of 8-strip PCR tubes for each serotype:

 

SECTION: Prepare Working Primer Solutions
2.4. Label the tops of each tube with the corresponding primer that will be added
For readability, recommended this format: 1L, 1R, 3L, 3R...
SECTION: Prepare Working Primer Solutions
2.5. Add 20 µL to each corresponding tube to reach 20 micromolar (µM) 
Recommend to either mix tubes with a new pipette tip or gently vortex

SECTION: Prepare Working Primer Solutions
2.6. Combine 10 µL of each Pool 1 primer from all serotypes in a new 1.5mL microcentrifuge tube
A total of 146 primers will be combined
This tube will contain all Pool 1 primers for all serotypes
Recommended to label each tube with concentration, pool #, and date of dilution
This will be the working primer solution that will be added during the amplicon generation step


Combine 10 µL of each Pool 2 primer from all serotypes in a new 1.5mL microcentrifuge tube
A total of 138 primers will be combined
This tube will contain all Pool 2 primers for all serotypes
Recommended to label each tube with concentration, pool #, and date of dilution
This will be the working primer solution that will be added during the amplicon generation step

SECTION: Prepare Working Primer Solutions
2.7. Store all primers at -20ºC until use
SECTION: cDNA Synthesis
3. Prepare the following reagents:
 



SECTION: cDNA Synthesis
3.1. Add 8.5 µL to new PCR tubes according to the number of samples
Include one additional tube at the end of the set for a cDNA negative template control (NTC), which will be included through the entirety of the protocol


SECTION: cDNA Synthesis
3.2. Add 8.5 µL to each tube
Add 8.5 µL to the cDNA NTC
Mix by pipetting up and down 10 times
Briefly centrifuge tubes


SECTION: cDNA Synthesis
3.3. Load tubes into thermocycler and run the following program to generate first-strand cDNA:
 

SECTION: cDNA Synthesis
3.4. In a new 1.5mL tube, prepare the following master mix according to the number of samples:
 

 


SECTION: cDNA Synthesis
3.5. Add 8 µL to each tube
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
SECTION: cDNA Synthesis
3.6. Load tubes into thermocycler and run the following program to generate second-strand cDNA:
 

SECTION: cDNA Synthesis
3.7. Remove tubes from thermocycler and briefly centrifuge
 

SECTION: Amplicon Generation
4. Prepare the following reagents:
 



SECTION: Amplicon Generation
4.1. In two separate tubes, prepare the following master mixes according to the number of samples:
Include one additional reaction for a PCR NTC which will be included through the entirety of the protocol. 

Pool 1
 
Pool 2
 

SECTION: Amplicon Generation
4.2. Distribute the master mixes:
Add 20 µL to a new set of PCR tubes, according to the number of samples
Add 20 µL to a new set of PCR tubes, according to the number of samples
Include one additional tube at the end of each set for the PCR NTC
SECTION: Amplicon Generation
4.3. Add 5 µL to each tube in both Pool 1 and Pool 2. 
Add 5 µL to the PCR NTC in both Pool 1 and Pool 2. 
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
SECTION: Amplicon Generation
4.4. Load tubes into thermocycler and run the following program:
 

SECTION: Amplicon Generation
4.5. When program is complete, samples can remain in the thermocycler at 4°C or be stored at -20°C
Briefly centrifuge tubes before use
 


SECTION: Amplicon Tagmentation and Cleanup
5. Prepare the following reagents:
 

SECTION: Amplicon Tagmentation and Cleanup
5.1. Quantify samples on a Qubit to confirm proper amplification in both amplicon pools
SECTION: Amplicon Tagmentation and Cleanup
5.2. Prepare the following master mix:
 

SECTION: Amplicon Tagmentation and Cleanup
5.3. Add 30 µL to a new set of PCR tubes, according to the number of samples

SECTION: Amplicon Tagmentation and Cleanup
5.4. For each sample, combine amplicon pools:
Add 10 µL to the Master Mix tubes
Add 10 µL to the Master Mix tubes
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
 

SECTION: Amplicon Tagmentation and Cleanup
5.5. Load tubes into thermocycler and run the following program:
 

SECTION: Amplicon Tagmentation and Cleanup
5.6. When the program is complete, immediately remove the tubes from the thermocycler
Briefly centrifuge tubes
SECTION: Amplicon Tagmentation and Cleanup
5.7. Add 10 µL to each tube
Mix by pipetting up and down
Briefly centrifuge tubes

SECTION: Amplicon Tagmentation and Cleanup
5.8. Incubate at Room temperature 
SECTION: Amplicon Tagmentation and Cleanup
5.9. Place tubes on a magnetic stand and wait until the liquid is clear before continuing
SECTION: Amplicon Tagmentation and Cleanup
5.10. Remove and discard all supernatant
Do not allow pipette tip to come into contact with the inner walls of the tube, as it can scrape away the magnetic beads
SECTION: Amplicon Tagmentation and Cleanup
5.11. Remove tubes from the magnetic stand and add 100 µL to each tube (first wash)
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
Be careful not to introduce bubbles
SECTION: Amplicon Tagmentation and Cleanup
5.12. Repeat steps 5.9 - 5.11 (second wash)
Do not remove supernatant after the second wash at this point
SECTION: Amplicon Tagmentation and Cleanup
5.13. Place the tubes on the magnetic stand
SECTION: Amplify Targeted Amplicons
6. Prepare the following reagents:
 

SECTION: Amplify Targeted Amplicons
6.1. Prepare the following Master Mix:
 

SECTION: Amplify Targeted Amplicons
6.2. When the tubes with tagmented amplicons (on the magnetic stand) are clear, remove and discard all supernatant
Remove any remaining supernatant with a smaller-sized pipette.
SECTION: Amplify Targeted Amplicons
6.3. Remove tubes from the magnetic stand and add 40 µL to each tube
SECTION: Amplify Targeted Amplicons
6.4. Briefly centrifuge the plate containing the dual-barcoded index adapters
Clean the surface of the sealing foil with RNase Away
Remove residual RNase away by wiping with 70% EtOH
SECTION: Amplify Targeted Amplicons
6.5. Add 10 µL to each well
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
SECTION: Amplify Targeted Amplicons
6.6. Load tubes into thermocycler and run the following program:
 
When program is complete, samples can remain in the thermocycler at 10°C or be stored at -20°C. 
 

SECTION: Pooling and Cleanup
7. Prepare the following reagents:
 

SECTION: Pooling and Cleanup
7.1. Place tubes on a magnetic stand and wait until the liquid is clear
SECTION: Pooling and Cleanup
7.2. Pool libraries together in a new 1.5mL microcentrifuge tube by equal volume:
 

 

SECTION: Pooling and Cleanup
7.3. Calculate the volume of ITB needed to reach 0.9x ITB : total pooled volume

 

SECTION: Pooling and Cleanup
7.4. Add the calculated volume of ITB to the pooled sample tube
Vortex for 10 seconds to mix
Briefly centrifuge
SECTION: Pooling and Cleanup
7.5. Incubate at Room temperature 
SECTION: Pooling and Cleanup
7.6. Place tube on a single-tube magnetic stand and wait until the liquid is clear
SECTION: Pooling and Cleanup
7.7. Remove and discard all supernatant
SECTION: Pooling and Cleanup
7.8. Add 1000 µL to the tube and incubate at Room temperature 
SECTION: Pooling and Cleanup
7.9. Repeat steps 7.7 - 7.8 and remove and discard supernatant after the second wash
Remove any residual EtOH with a smaller-sized pipette
SECTION: Pooling and Cleanup
7.10. Add RSB HT to the tube according to the number of samples pooled:
For 1 - 24 samples add 30 µL 
For 25 - 48 samples add 40 µL 
For 49 - 96 samples add 50 µL 
SECTION: Pooling and Cleanup
7.11. Vortex tube to mix 
Briefly centrifuge tube
SECTION: Pooling and Cleanup
7.12. Incubate at Room temperature 
SECTION: Pooling and Cleanup
7.13. Place on the magnetic stand and wait until the liquid is clear
SECTION: Pooling and Cleanup
7.14. Transfer [total volume RSB] - 5µL to a new 1.5mL microcentrifuge tube
SECTION: Pooling and Cleanup
7.15. Quantify library on a Qubit and obtain fragment distribution using a Bioanalyzer/Tape Station
SECTION: Sequencing
8. Protocol validated on the Illumina NovaSeq (2x150).

 

SECTION: Bioinformatics/Analysis
9. Sequencing results may be analyzed utilizing a standard amplicon sequencing bioinformatics pipeline, including those employed for SARS-CoV-2 sequencing (e.g. iVar). 

Reference Sequences:
  
  
  
  
BED files: