Oct 17, 2023
Version 3
DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus V.3
- Chantal Vogels1,
- Verity Hill1,
- Mallery I Breban1,
- Chrispin Chaguza1,
- Afeez Sodeinde1,
- Emma Taylor-Salmon1,
- Abigail J. Porzucek1,
- Nathan D Grubaugh1
- 1Department of Epidemiology of Microbial Diseases, Yale School of Public Health
Protocol Citation: Chantal Vogels, Verity Hill, Mallery I Breban, Chrispin Chaguza, Afeez Sodeinde, Emma Taylor-Salmon, Abigail J. Porzucek, Nathan D Grubaugh 2023. DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39xxeg25/v3Version created by Nathan D Grubaugh
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: August 02, 2023
Last Modified: October 17, 2023
Protocol Integer ID: 85884
Keywords: dengue virus, genomic surveillance, next-generation sequencing, amplicon sequencing, whole-genome sequencing
Funders Acknowledgement:
NIAID
Grant ID: DP2AI176740
Abstract
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.
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.
Materials
DengueSeq preprint
Primers
Primer schemes for the four dengue virus serotypes (1-4) can be used individually as serotype-specific or combined as a single pan-serotype approach. Primers may be ordered from any oligonucleotide company. For primer preparation instructions for the serotype-specific and pan-serotype approaches, see Step 1 of the protocol. See files below for primer sequences.
Primer Schemes:
DENV1_Primer-Scheme.xlsx 
DENV2_Primer-Scheme.xlsx 
DENV3_Primer-Scheme.xlsx 
DENV4_Primer-Scheme.xlsx Equipment
- 96-well format Thermocycler (two needed for 49+ sample runs)
- Qubit
- Bioanalyzer
- Magnetic rack fit for a 96-well PCR plate
- Magnetic rack fit for 1.5 / 2-mL microcentrifuge tubes
- Cold block fit for a 96-well PCR plate
- Pipettes (assorted sizes)
Consumables
- 8-strip PCR tubes; may be substituted with PCR plates with heat-sealing film
- 1.5 / 2mL / 5 mL microcentrifuge tubes
- Filtered pipette tips (assorted sizes)
- Reservoirs
- Waste containers
Reagents
Bioinformatics pipeline
Reference genomes
DENV1.fasta10KB DENV2.fasta10KB DENV2_sylvatic.fasta10KB DENV3.fasta10KB DENV4.fasta10KB DENV4_sylvatic.fasta10KB BED files
DENV1.bed2KB DENV2.bed2KB DENV2_sylvatic.bed2KB DENV3.bed2KB DENV4.bed2KB DENV4_sylvatic.bed2KB Safety warnings
Before starting work with dengue virus samples, please contact your local EHS (environment, health and safety) or biosafety office for proper guidance on how to work with these samples in your laboratory.
Before start
If substituting 8-strip PCR tubes for PCR plates, plates must be heat-sealed before each thermocycling step.
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:
Serotype-specific approach61 steps
Identify the serotype of all samples being sequenced.
We recommend using the CDC DENV-1-4 Real-Time RT-PCR Multiplex Assay for serotype identification if an in-house assay is not available.
Organize samples by serotype. Step 4 requires a serotype-specific master mix to be added to the samples; order the samples to best accommodate this.
Each serotype-specific master mix will require its own negative template controls, one cDNA control (added in Step 3) and one PCR control (added in step 4). Be sure to account for these extra controls in the plate design.
Prepare Working Primer Solutions
Prepare Working Primer Solutions
Briefly centrifuge all primers
Within each serotype, organize odd and even-numbered primers into two separate groups:
Odd-numbered primers = Pool 1
Even-numbered primers = Pool 2
Note
Recommended to organize numerically, i.e. 1_left, 1_right, 3-left, 3 right...
If primers were ordered lyophilized, resuspend to 100 micromolar (µM) in nuclease-free water
Add 90 µL nuclease-free water to two sets of 8-strip PCR tubes for each serotype:
| Serotype | Tubes for Pool 1 | Tubes for Pool 2 | |
| DENV1 | 36 | 34 | |
| DENV2 | 38 | 36 | |
| DENV3 | 36 | 34 | |
| DENV4 | 36 | 34 |
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...
Add 10 µL 100 µM primer to each corresponding tube to reach 10 micromolar (µM) /primer
- Recommend to either mix tubes with a new pipette tip or gently vortex
Working pool-by-pool, transfer 10 µL 10µM primer 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
Store all primers at -20ºC until use
cDNA Synthesis
cDNA Synthesis
Prepare the following reagents:
| Reagent | Storage | Instructions | |
| EPH3 HT | -20°C | Thaw at room temperature, invert to mix | |
| FSM HT | -20°C | Thaw at room temperature, invert to mix | |
| RVT HT | -20°C | Invert to mix, keep on ice | |
| Nuclease-free water | Room Temp. | Keep at room temperature |
Add 8.5 µL EPH3 HT 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
Add 8.5 µL RNA to each tube
Add 8.5 µL nuclease-free water to the cDNA NTC
- Mix by pipetting up and down 10 times
- Briefly centrifuge tubes
Load tubes into thermocycler and run the following program to generate first-strand cDNA:
| Temperature | Time | |
| 65°C | 3 minutes | |
| 4°C | Hold |
17µL reaction, lid temp = 105°C, preheat lid = on
In a new 1.5mL tube, prepare the following master mix according to the number of samples:
| Reagent | µL per Sample | |
| FSM HT | 7.2 | |
| RVT HT | 0.8 | |
| Total | 8 |
Note
All master mix volumes in this protocol are for one reaction and do not account for lost volume due to pipetting. Multiply volumes by reaction number accordingly.
Add 8 µL Master Mix to each tube
Mix by pipetting up and down 10 times
Briefly centrifuge tubes
Load tubes into thermocycler and run the following program to generate second-strand cDNA:
| Temperature | Time | |
| 25°C | 5 minutes | |
| 50°C | 10 minutes | |
| 80°C | 5 minutes | |
| 4°C | ∞ |
25µL reaction, lid temp = 105°C, preheat lid = on
Remove tubes from thermocycler and briefly centrifuge
Note
This is a safe stopping point. cDNA can be stored long-term at -20°C.
Amplicon Generation
Amplicon Generation
Prepare the following reagents:
| Reagent | Storage | Instructions | |
| Primer Pool 1 | -20°C | Thaw at room temperature, vortex to mix | |
| Primer Pool 2 | -20°C | Thaw at room temperature, vortex to mix | |
| IPM HT | -20°C | Thaw at room temperature, invert to mix | |
| Nuclease-free water | Room temp. | Keep at room temperature |
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
| Reagent | µL per Sample | |
| IPM HT | 12.5 | |
| Primer Pool 1 | 3.6 | |
| Nuclease-free Water | 3.9 | |
| Total | 20 |
Pool 2
| Reagent | µL per Sample | |
| IPM HT | 12.5 | |
| Primer Pool 2 | 3.6 | |
| Nuclease-free Water | 3.9 | |
| Total | 20 |
Distribute the master mixes:
Add 20 µL Pool 1 Master Mix to a new set of PCR tubes, according to the number of samples
Add 20 µL Pool 2 Master Mix 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
Add 5 µL cDNA to each tube in both Pool 1 and Pool 2.
Add 5 µL nuclease-free water to the PCR NTC in both Pool 1 and Pool 2.
- Mix by pipetting up and down 10 times
- Briefly centrifuge tubes
Load tubes into thermocycler and run the following program:
| Step(s) | Temperature | Time | Cycles | |
| Initial denaturation | 98°C | 3 minutes | 1 | |
| Denaturation | 98°C | 15 seconds | 35 | |
| Anneal and extension | 63°C | 5 minutes | ||
| Hold | 4°C | ∞ | ∞ |
25µL reaction, lid temp = 105°C, preheat lid = on
When program is complete, samples can remain in the thermocycler at 4°C or be stored at -20°C
- Briefly centrifuge tubes before use
Note
This is a safe stopping point. Amplicons can be stored long-term at -20°C.
Amplicon Tagmentation and Cleanup
Amplicon Tagmentation and Cleanup
Prepare the following reagents:
| Reagent | Storage | Instructions | |
| EBLTS HT | 4°C | Vortex to mix | |
| TB1 HT | -20°C | Thaw at room temperature, vortex to mix | |
| ST2 HT | Room temp. | Vortex before use | |
| TWB HT | 4°C | Vortex before use | |
| Nuclease-free water | Room temp. | Keep at room temperature |
Quantify samples on a Qubit to confirm proper amplification in both amplicon pools
Prepare the following master mix:
| Reagent | µL per sample | |
| TB1 HT | 10 | |
| EBLTS HT | 3.3 | |
| Nuclease-free water | 16.7 | |
| Total | 30 |
Add 30 µL Master Mix to a new set of PCR tubes, according to the number of samples
For each sample, combine amplicon pools:
Add 10 µL Pool 1 to the Master Mix tubes
Add 10 µL Pool 2 to the Master Mix tubes
- Mix by pipetting up and down 10 times
- Briefly centrifuge tubes
Note
Remaining amplicon pools can be stored long-term at -20°C
Load tubes into thermocycler and run the following program:
| Temperature | Time | |
| 55°C | 3 minutes | |
| 10°C | ∞ |
50µL reaction, lid temp = 105°C, preheat lid = on
When the program is complete, immediately remove the tubes from the thermocycler
- Briefly centrifuge tubes
Add 10 µL ST2 to each tube
- Mix by pipetting up and down
- Briefly centrifuge tubes
Incubate at Room temperature for 5 minutes
Place tubes on a magnetic stand and wait until the liquid is clear before continuing
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
Remove tubes from the magnetic stand and add 100 µL TWB HT to each tube (first wash)
- Mix by pipetting up and down 10 times
- Briefly centrifuge tubes
- Be careful not to introduce bubbles
Repeat steps 5.9 - 5.11 (second wash)
- Do not remove supernatant after the second wash at this point
Place the tubes on the magnetic stand
Amplify Tagmented Amplicons
Amplify Tagmented Amplicons
Prepare the following reagents:
| Reagent | Storage | Instructions | |
| EPM HT | -20°C | Thaw at room temperature, invert to mix | |
| Index adapters | -20°C | Thaw at room temperature, vortex to mix | |
| Nuclease-free water | Room temp. | Keep at room temperature |
Prepare the following Master Mix:
| Reagent | µL per sample | |
| EPM | 20 | |
| Nuclease-free water | 20 | |
| Total | 40 |
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.
Remove tubes from the magnetic stand and add 40 µL Master Mix to each tube
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
Add 10 µL dual-coded index adapters to each well
- Mix by pipetting up and down 10 times
- Briefly centrifuge tubes
Load tubes into thermocycler and run the following program:
| Temperature | Time | Cycles | |
| 72°C | 3 minutes | 1 | |
| 98°C | 3 minutes | 1 | |
| 98°C | 20 seconds | 7 | |
| 60°C | 30 seconds | ||
| 72°C | 1 minute | ||
| 72°C | 3 minutes | 1 | |
| 10°C | ∞ | ∞ |
When program is complete, samples can remain in the thermocycler at 10°C or be stored at -20°C.
Note
This is a safe stopping point. Individual libraries can be stored long-term at -20°C.
Pooling and Cleanup
Pooling and Cleanup
Prepare the following reagents:
| Reagent | Storage | Instructions | |
| ITB HT | Room temp. | Vortex to mix | |
| RSB HT | 4°C | Keep at room temperature, vortex to mix | |
| 80% EtOH | Room temp. | Prepare 2.5 - 3mL using nuclease free water and molecular-grade 100% EtOH immediately before use |
Place tubes on a magnetic stand and wait until the liquid is clear
Pool libraries together in a new 1.5mL microcentrifuge tube by equal volume:
| Number of samples | µL per sample | |
| 1 - 24 | 15 | |
| 25 - 48 | 10 | |
| 49 - 96 | 5 |
Note
For easier pooling of a large number of samples, it is recommended to multichannel the libraries into a new 8-strip of 0.2mL PCR tubes, then transfer the total volume to the microcentrifuge tube.
Calculate the volume of ITB needed to reach 0.9x ITB : total pooled volume
Note
Example:
34 samples; 10µL of each sample pooled
34 * 10 = 340µL total pooled volume
340µL * 0.9 = 306µL ITB
Add the calculated volume of ITB to the pooled sample tube
- Vortex for 10 seconds to mix
- Briefly centrifuge
Incubate at Room temperature for 5 minutes
Place tube on a single-tube magnetic stand and wait until the liquid is clear
Remove and discard all supernatant
Add 1000 µL 80% EtOH to the tube and incubate at Room temperature for 30 seconds
Repeat steps 7.7 - 7.8 and remove and discard supernatant after the second wash
- Remove any residual EtOH with a smaller-sized pipette
Add RSB HT to the tube according to the number of samples pooled:
- For 1 - 24 samples add 30 µL RSB HT
- For 25 - 48 samples add 40 µL RSB HT
- For 49 - 96 samples add 50 µL RSB HT
Vortex tube to mix
Briefly centrifuge tube
Incubate at Room temperature 2 minutes
Place on the magnetic stand and wait until the liquid is clear
Transfer [total volume RSB] - 5µL to a new 1.5mL microcentrifuge tube
Quantify library on a Qubit and obtain fragment distribution using a Bioanalyzer/Tape Station
Sequencing
Sequencing
Protocol validated on the Illumina NovaSeq (2x150).
Note
For sequencing we recommend generating at least 1 million reads per sample for optimal sequencing coverage. Sequencing may be performed on Illumina and Oxford Nanopore Technologies sequencing platforms following standard protocols.
Bioinformatics/Analysis
Bioinformatics/Analysis
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:
DENV1.fasta10KB DENV2.fasta10KB DENV2_sylvatic.fasta10KB DENV3.fasta10KB DENV4.fasta10KB DENV4_sylvatic.fasta10KB BED files:
DENV1.bed2KB DENV2.bed2KB DENV2_sylvatic.bed2KB DENV3.bed2KB DENV4.bed2KB DENV4_sylvatic.bed2KB Protocol references
DengueSeq preprint: https://www.medrxiv.org/content/10.1101/2023.10.13.23296997v1
Bioinformatics pipeline: https://github.com/grubaughlab/DENV_pipeline/tree/v1.0