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

[DESCRIPTION]
Version 3 updates:
Updated abstract
Included link to preprint and bioinformatics pipeline
Added links to additional library prep kits
Included DENV2 and DENV4 sylvatic reference genomes and BED files
Changed primer names in BED files 

DengueSeq preprint: https://www.medrxiv.org/content/10.1101/2023.10.13.23296997v1
Bioinformatics pipeline: https://github.com/grubaughlab/DENV_pipeline/tree/v1.0

Background
The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. 

Results
We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/μL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without  known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. 

Conclusions
DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Link to DengueSeq preprint.


[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 options available for use: Serotype-specific and Pan-serotype approach. 

Various library prep kits can be used with the DengueSeq primers, following the manufacturer's protocol. Below we describe detailed steps for the Illumina COVIDSeq Test Kit (RUO version), but other library prep kits for amplicon sequencing can be used as well.

For example:
Illumina COVIDSeq Test Kit 
Illumina Nextera XT DNA Library Preparation kit
New England Biolabs NEBNext ARTIC SARS-CoV-2 Library Prep Kit (Illumina)
Oxford Nanopore Technologies Rapid barcoding kit
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). 

Bioinformatics pipeline: https://github.com/grubaughlab/DENV_pipeline/tree/v1.0

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). 

Bioinformatics pipeline: https://github.com/grubaughlab/DENV_pipeline/tree/v1.0

Reference genomes:
  
 
   
  
 
   

BED files: