# Targeted full-genome amplification and sequencing of dengue virus types 1–4 from South America

Cristopher D. Cruz, Armando Torre, Gilda Troncos, Louis Lambrechts, Mariana Leguia

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Cristopher D. Cruz, Armando Torre, Gilda Troncos, Louis Lambrechts, Mariana Leguia. Targeted full-genome amplification and sequencing of dengue virus types 1–4 from South America. Journal of Virological Methods, 2016, 235, pp.158 - 167. 10.1016/j.jviromet.2016.06.001. hal-01445723

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# Targeted full-genome amplification and sequencing of dengue virus types 1–4 from South America

<figure><img src="image_2.png" /></figure>

CrossMark

Cristopher D. Cruzª, Armando Torreª, Gilda Troncosª, Louis Lambrechtsᵇ,ᶜ, Mariana Leguiaª,⁺

**Article history:**

Received 31 March 2016

Received in revised form 1 June 2016

Accepted 3 June 2016

Available online 19 June 2016

**Keywords:**

Dengue virus

Full-genome sequencing

Next-generation sequencing

Amplicon sequencing

## ABSTRACT

We report optimized workflows for full-genome sequencing of dengue viruses (DENVs) 1–4. Based on alignments of publicly available complete genomes we modified and expanded existing primers sets to amplify DENV genotypes that were previously difficult or impossible to sequence. We also report improvements to streamline laboratory handling, including a dual amplification strategy for easy and difficult to sequence “high-copy” and “low-copy” templates, respectively, and normalization of PCR cycling conditions across serotypes. High-copy templates can be sequenced following amplification of as few as 5 overlapping segments covering the complete viral genome, whereas low-copy templates can be sequenced following amplification of no more than 10 overlapping segments of smaller size. These changes have been validated using a balanced set of wild-type DENV genomes (11 of DENV1, 14 of DENV2, 13 of DENV3 and 7 of DENV4) derived from human serum samples collected throughout South America over the past 15 years. The changes described enable generation of complete DENV genomes from wild-type samples without the need for viral enrichment via passaging through laboratory cell lines. This should facilitate quick and cost-effective generation of DENV full-genome sequences of the type needed for accurate epidemiological surveillance and thorough evolutionary studies of wild-type DENVs.

Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

Dengue fever remains a serious public health threat throughout the world. According to the World Health Organization, dengue viruses (DENVs) (family *Flaviviridae*; genus *Flavivirus*) infect ~390 million people each year, result in 0.5 million hospitalizations, and lead to death in 2.5% of those affected (WHO, 2015). Using modeling frameworks to map the global distribution of dengue risk others have estimated the dengue burden to be three times higher than the estimate of the WHO (Bhatt et al., 2013). Despite the recent historic licensing of the first dengue vaccine approved for use in Mexico (Sanofi-Pasteur, 2015), robust countermeasures

against DENVs continue to rely on a combination of vector control and surveillance efforts. All four DENV serotypes circulate in South America, where almost all countries are hyper-endemic for DENV transmission (Cafferata et al., 2016). Surveillance efforts in these countries depend heavily on laboratory diagnostics that are sensitive (such as PCR and ELISA based assays) and accurate (such as direct sequencing) in order to track viral evolution and inform public health interventions.

Several phylogenetic studies have documented the evolution of DENVs in South America, highlighting the importance of continuous monitoring of circulating DENV strains that could be linked to severe forms of the disease and/or to clade displacement (Aquino et al., 2008; Cruz et al., 2013; Dettogni and Louro, 2011; Forshey et al., 2009; Kochel et al., 2008; Mendez et al., 2012; Oliveira et al., 2010; Roca et al., 2009; Romano et al., 2010; Uzcategui et al., 2001; Uzcategui et al., 2003). Many of these studies have focused on envelope (E) gene sequences generated using traditional Sanger sequencing (Sanger and Coulson, 1975; Sanger et al., 1977). Despite being a major antigenic determinant, the E gene (~1484 bp in length) only provides limited phylogenetic information compared to the complete viral genome (~11 kb in length). The development

Abbreviations: DENVs, dengue viruses; NGS, next-generation sequencing; PGM, personal genome machine.

* Corresponding author.

E-mail addresses: cristopher.d.cruz.ln@mail.mil (C.D. Cruz), armando.torre@med.navy.mil (A. Torre), gilda.troncos.fn@mail.mil (G. Troncos), louis.lambrechts@pasteur.fr (L. Lambrechts), mariana.leguia.fn@mail.mil (M. Leguia).

<footnote>ª U.S. Naval Medical Research Unit No. 6 (NAMRU-6), Av. Venezuela c36s/n, Bellavista, Callao 2, Peru</footnote>

<footnote>ᵇ Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, Paris, France</footnote>

<footnote>ᶜ Centre National de la Recherche Scientifique, Unité de Recherche Associée 3012, Paris, France</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Dual amplification strategy for DENV1-4.</figcaption></figure>

Gels show representative full-genome amplifications of all DENV serotypes, using both the 5- and 10-overlapping fragment approaches. Amplicons from the 5-fragment approach from three serum samples of different Ct value (representing high-, medium- and low-copy templates) are shown in red, labeled in 1–5. Amplicons from the 10-fragment approach from low-copy templates only are shown in blue, labeled in A–J. M indicates the 1 kb plus DNA ladder.
of robust next-generation sequencing (NGS) platforms has signif-
icantly lowered sequencing costs, enabling the use of complete
genome sequences to provide greater resolution of viral evolution-
ary dynamics. As a result, NGS technologies are now increasingly
used for epidemiological surveillance and studies of viral genetic
diversity (Lequime et al., 2016; Rodriguez-Roche et al., 2016; Sim
et al., 2015). Unfortunately, the protocols developed for NGS of
DENVs have mainly focused on viral strains that have circulated
in Asia (Parameswaran et al., 2012; Sim et al., 2015; Zhao et al.,
2014). Given that strains of DENVs circulating in South America can
show significant genetic variation (Rodriguez-Roche et al., 2016;
Romano et al., 2013), and especially when compared to those from
Asia, existing protocols for full-genome amplification of DENVs can
fail to generate complete genomes from American samples.

Here we describe changes made to existing sets of primers (Christenbury et al., 2010; Rodriguez-Roche et al., 2016) in order to optimize workflows and reliably generate full genomes of South American DENV strains. Specifically, we have implemented three sets of changes: the first ifs a complete re-design and expansion of primers used for amplicon sequencing of DENV1-4; the second is the development of a dual approach strategy for both high- and low-copy viral templates; the last is the inclusion of an initial viral quantification step that informs whether the high- or low-copy amplification approach should be used. Together, these changes streamline laboratory handling, reduce costs, and enable generation of complete genome data from DENV samples that were previously difficult, or even impossible to sequence.

**2. Materials and methods**

2.1. Amplicon generation

Nucleic acids were extracted using QIAamp Viral RNA Mini Kit (Qiagen 52906) according to the manufacturer's instructions and converted to cDNA using serotype-specific primers targeting the 3'-ends of viral genomes (Table 1). Briefly, 20-µL reactions containing 10 µL nucleic acids, 1 µL 10 µM gene-specific primer, 1 µL SuperScript™ III (Invitrogen 18080-044), 4 µL 5X RT buffer, 1 µL 0.1 M DTT, 1 µL 10 mM of each dNTPs, 1 µL RNaseOUT™ (Invitrogen 10777-019), and 1 µL nuclease-free water were incubated at 50 °C for 60 min and then terminated at 75 °C for 15 min. Amplicons were generated in 50-µL reactions containing 2 µL DENV cDNA, 1 µL each 10 µM forward and reverse primers, 0.5 µL Platinum™ Taq DNA Polymerase High Fidelity (Invitrogen 11304-029), 5 µL 10X HiFi PCR Buffer, 1.5 µL 50 mM MgSO4, 1 µL 10 mM dNTPs, 10 µL 5X Q solution (supplied with Taq DNA Polymerase, Qiagen 201203), and 28 µL nuclease-free water. The cycling conditions consisted of 40 cycles of denaturation (94 °C for 30 s), annealing (47 °C (DENV1), 53 °C (DENV2), 42 °C (DENV3) and 52 °C (DENV4) for 30 s), and extension (72 °C for 3 min), with a final extension at 72 °C for 5 min. Amplicons were visualized by agarose gel electrophoresis and purified either directly or by gel extraction using QIAquick Gel Extraction Kits (Qiagen 28706).

2.2. Sequencing

Prior to library preparation, overlapping amplicons were quant-
tified by NanoDrop 1000 (Thermo-Fisher) and pooled in equimolar
amounts in order to cover the length of each genome. Pools
were used to prepare sequencing libraries optimized for both
PGM and MiSeq sequencing platforms, according to the man-
ufacturer's instructions. Briefly, PGM libraries were constructed
using Bioruptor (Diagenode) fragmented samples, Ion Xpress Bar-
code Adapters (Thermo-Fisher 4474517) and Ion Plus Fragment
Library Kits (Thermo-Fisher 4471252). MiSeq libraries were con-

structed using enzymatically-fragmented samples, Nextera DNA
Indexes (Illumina FC-121-1011) and Nextera DNA Library Prepa-
ration Kits (Illumina FC-121-1030). QC was done by Bioanalyzer®
(Agilent) using High Sensitivity DNA chips (Agilent 5067-4627)
and by Qubit® (Thermo-Fisher) using Qubit® dsDNA HS Assay Kits
(Thermo-Fisher Q32854). Prior to sequencing, barcoded libraries
were pooled as recommended for each sequencing platform.
Briefly, PGM libraries were diluted to 12 pM, amplified by emul-
sion PCR on the Ion OneTouch™ 2 Instrument (Thermo-Fisher)
using Ion PGM™ Template OT2 200 Kits v2 (Thermo-Fisher
4480974), and enriched on the Ion OneTouch™ ES enrichment
System (Thermo-Fisher), according to the manufacturer's instruc-
tions. PGM libraries were sequenced on the Ion PGM platform
(Thermo-Fisher) using Ion PGM™ sequencing 200 kits v2 (Thermo-
Fisher 4482006) and Ion 314™ Chips (Thermo-Fisher 4482261).
MiSeq libraries were diluted to 12 pM and sequenced on the MiSeq
platform (Illumina) using MiSeq v3 600 cycle kits (Illumina MS-
102-3003). Bioinformatics processing was carried out using the
EDGE platform (Lo, personal communication;) and custom scripts.

**3. Results**

3.1. Primer design

Sequences of all primers used for targeted amplification of
DENV1-4 are listed in Table 1. The primers for each serotype were
designed to share annealing temperatures so that they could be
used interchangeably within each set. The primer sets for DENV1,
2 and 3 consist of 20 primers each with an annealing tempera-
ture of 47 °C, 53 °C and 42 °C, respectively; the DENV4 set consists
of 18 primers with an annealing temperature of 52 °C. To ensure
that each set would amplify South American DENV strains we first
downloaded representative full genome sequences for DENVs that
have circulated in the region since 2000 (Table 2). Specifically,
81 representative complete genomes (23 of DENV1, 18 of DENV2,
29 of DENV3 and 11 of DENV4) from Argentina, Brazil, Colombia,
Paraguay, Peru and Venezuela available from public databases were
used as references. These were carefully selected to maximize
diversity in terms of serotype, year of isolation, and geographical
origin. Previously published sets of DENV primers (Christenbury
et al., 2010; Rodriguez-Roche et al., 2016) were also used to guide
our design. In some cases, primers included degenerate or semi-
degenerate bases. Primers were positioned to generate overlapping
amplicons spanning the complete length of each DENV genome in
as few steps as possible (Fig. 1). Given that amplification success is
linked to target amplicon size and to template concentration, we
anticipated that long amplicons from low-copy templates would be
harder to generate than short amplicons from high-copy templates.
Thus, our design considers both high- and low-copy amplification
strategies in order to ensure full-genome amplification in a range
of starting template concentrations. High-copy templates are fully
covered by as few as 5 overlapping amplicons, whereas low-copy
templates are fully covered by no more than 10 amplicons. Overall,
target amplicon size ranges between ~1 and ~3 kb.

3.2. Targeted full-genome amplification and sequencing

To test the modifications introduced in our primers, we selected a set of 43 DENV positive samples (9 of DENV1, 14 of DENV2, 13 of DENV3 and 7 of DENV4) consisting of acute-phase sera (Table 3) collected during routine febrile surveillance programs conducted between 2000 and 2015 by investigators from the U.S. Naval Medical Research Unit No.6 in Peru and neighboring countries (Bolivia, Ecuador, Paraguay, Venezuela), with institutional review board approval from all implicated partners (proto-
<caption>**Table 1**</caption>

Primer sequences used for full-genome amplification of DENV1-4.

All primers, with the exception of d3a10 (""), are either new or modified for this work. Primers for each serotype share annealing temperatures, which enables interchangeable use within each set. Genome locations are reported with respect to GenBank isolates JX669464 for DENV1, KC294222 for DENV2, JX669501 for DENV3, and JN983813 for DENV4. The last primer of the 3' end in each set (shown in red) was used for reverse transcription (note that all reverse transcription reactions were carried out at 50 °C regardless of primer annealing temperature). In the 5-fragment amplification strategy individual overlapping amplicons are named 1–5, whereas in the 10-fragment amplification strategy individual overlapping amplicons are named A-J (except for DENV4, where the last overlapping amplicon (named 5 and/or I) is shared by both the 5- and 10-fragment amplification strategies.

<table><thead><tr><th>Serotype</th><th>Primer Name</th><th>Sequence 5'–3'</th><th>Location</th><th>10 Products</th><th>5 Products</th><th>Annealing Temperature</th></tr></thead><tbody><tr><td rowspan="23">DENV-1</td><td>DEN1 CC AF</td><td>ACGTGGACCGACAAGAACAGTTTCG</td><td>13–37</td><td>A ~1.65Kb</td><td>1 ~2.55Kb</td><td rowspan="23">47 °C</td></tr><tr><td>DEN1 CC 1R</td><td>CTTCCTGTTTCTTTGCATGAGCTGTC</td><td>1657–1682</td><td></td><td></td></tr><tr><td>DEN1 CC 1F</td><td>GAGCATGGAAGCTGCGTCACCAC</td><td>1010–1032</td><td>B ~1.55Kb</td><td></td></tr><tr><td>DEN1 CC BR</td><td>CTTYCCAATGGCTGCTGACAGTCTT</td><td>2539–2563</td><td></td><td></td></tr><tr><td>DENV1-LAB-5F-CC</td><td>CGCATGGGACTTCGGYTCATAAGG</td><td>2191–2213</td><td>C ~1.40Kb</td><td>2 ~2.50Kb</td></tr><tr><td>DEN1 CC 2R</td><td>TGTTCCAGTCATCAGCATCTTTCTAC</td><td>3558–3583</td><td></td><td></td></tr><tr><td>DEN1 CC 2F</td><td>ATTGCGTGACTCCYACACCCAAATG</td><td>2930–2953</td><td>D ~1.75Kb</td><td></td></tr><tr><td>DEN1 CC DR</td><td>CCTGGTGACGTGCCACATTGTGTG</td><td>4658–4681</td><td></td><td></td></tr><tr><td>DENV1-LAB-8F-CC</td><td>CAAAGAGGACTGTTGGGCAGGTCC</td><td>4600–4620</td><td>E ~1.65Kb</td><td>3 ~2.35Kb</td></tr><tr><td>DEN1 CC 3R</td><td>ATCTCCACGTCCATGTTCTCCTCC</td><td>6241–6264</td><td></td><td></td></tr><tr><td>DEN1 CC 3F</td><td>TCATGGAACTCAGGCTACGACTGG</td><td>5549–5572</td><td>F ~1.40Kb</td><td></td></tr><tr><td>DENV1-LAB-10R-CC</td><td>CTGCATAGAGRGTCAGGCTGAAG</td><td>6933–6956</td><td></td><td></td></tr><tr><td>DENV1-LAB-12F-CC</td><td>CACAAAGAAAGACCTGGGGATTGGC</td><td>6850–6874</td><td>G ~1.50Kb</td><td>4 ~2.25Kb</td></tr><tr><td>DEN1 CC 4R</td><td>GTTCCACTGCCACATGTCTTGTTCC</td><td>8351–8375</td><td></td><td></td></tr><tr><td>DEN1 CC 4F</td><td>GAGAAACAACCAAACATGCAGTGTCG</td><td>7716–7741</td><td>H ~1.40Kb</td><td></td></tr><tr><td>DENV1-LAB-14R-CC</td><td>TTGTGCAGTCCTTCTCCTTCCACTC</td><td>9087–9111</td><td></td><td></td></tr><tr><td>DENV1-LAB-14F-CC</td><td>TACGTGTTCTAAARATGGTGGAACCAT</td><td>8058–8084</td><td>I ~1.35Kb</td><td>5 ~2.65Kb</td></tr><tr><td>DEN1 CC 5R</td><td>AGCCATAAGTTCGACCTGTCCAC</td><td>9372–9395</td><td></td><td></td></tr><tr><td>DEN1 CC 5F</td><td>ACTGACATCATGGAGCCTGAACATG</td><td>9230–9254</td><td>J ~1.45Kb</td><td></td></tr><tr><td>DEN1 CC JR</td><td>CGTTCTGTGCCTGGAATGATGCTG</td><td>10684–10707</td><td></td><td></td></tr><tr><td>d2s1C—CC</td><td>AGTTGTTAGTCTACGTGGACCGACA</td><td>1–25</td><td>A ~1.50Kb</td><td>1 ~2.45Kb</td><td rowspan="10">53 °C</td></tr><tr><td>DEN2 CC 1R</td><td>AGCACCATCTCATTGAAGTCGAGG</td><td>1506–1529</td><td></td><td></td></tr><tr><td>DEN2 CC 1F</td><td>CAAACACTCCATGGTAGACAGAGG</td><td>1212–1235</td><td>B ~1.25Kb</td><td></td></tr><tr><td>d2a18-CC</td><td>TCCCRCTGCCACATTTCARTTCTTT</td><td>2455–2474</td><td></td><td></td></tr><tr><td>d2s4-CC</td><td>ACGCSAAGAAACAGGATGTGYTTGT</td><td>1669–1690</td><td>C ~1.55Kb</td><td>2 ~2.75Kb</td></tr><tr><td>DEN2 CC 2R</td><td>TCCTGCTGTTTGTGTATGGTAGCC</td><td>3196–3219</td><td></td><td></td></tr><tr><td>DEN2 CC 2F</td><td>CACATGGAAGATGGAGAAAGCCTC</td><td>3045–3068</td><td>D ~1.35Kb</td><td></td></tr><tr><td>d2a14-CC</td><td>GCCGTRATTGGTATYGATACWGGA</td><td>4401–4425</td><td></td><td></td></tr><tr><td>d2s8-CC</td><td>ATGGGMGTGACTTAYCTTGCCTCA</td><td>3686–3704</td><td>E ~1.45Kb</td><td>3 ~2.80Kb</td></tr><tr><td>DEN2 CC 3R</td><td>CTTCTCTAACTATGGCTGGAAGG</td><td>5130–5152</td><td></td><td></td></tr><tr><td>DEN2 CC 3F</td><td>TGGTGTTCACAAGGAGTGGAG</td><td>4976–4998</td><td>F ~1.50Kb</td><td></td></tr><tr><td>d2a10-CC</td><td>TACGCCYTTCRCCTGCYTCAG</td><td>6477–6497</td><td></td><td></td></tr><tr><td>d2s13-CC</td><td>GCAGACAGAARGTGGTGTTTTGATG</td><td>6193–6213</td><td>G ~1.15Kb</td><td>4 ~2.30Kb</td></tr><tr><td>DEN2 CC 4R</td><td>CATTAATACTTGAGTCACGCAGAGG</td><td>7350–7374</td><td></td><td></td></tr><tr><td>DEN2 CC 4F</td><td>TTCAAGCAAAAGCAACCAGAGAAGC</td><td>7199–7223</td><td>H ~1.25Kb</td><td></td></tr><tr><td>d2a6-CC</td><td>CATGGTAWGCCAYGTTTTGTATGG</td><td>8468–8488</td><td></td><td></td></tr><tr><td>d2s16-CC</td><td>CAGGAAGTGGATAGAACCYTRGCA</td><td>7669–7692</td><td>I ~1.65Kb</td><td>5 ~3.05Kb</td></tr><tr><td>DEN2 CC 5R</td><td>CATTACTGTGCCTCTTGGTGTTG</td><td>9316–9339</td><td></td><td></td></tr><tr><td>DEN2 CC 5F</td><td>CTGGTTGACAGGGAAAGAAATCTCC</td><td>8870–8893</td><td>J ~1.85Kb</td><td></td></tr><tr><td>d2a5B-CC</td><td>AGAACCTGTTGATTCAACAGCACCAT</td><td>10706–10725</td><td></td><td></td></tr><tr><td rowspan="16">DENV-3</td><td>d3s1C—CC</td><td>AGTTGTTAGTCTACGTGGACCG</td><td>1–22</td><td>A ~1.55Kb</td><td>1 ~2.30Kb</td><td rowspan="16">42 °C</td></tr><tr><td>DEN3 CC 1R</td><td>CATGCTTTGTTCTTCATTGTTAGC</td><td>1524–1547</td><td></td><td></td></tr><tr><td>DEN3 CC 1F</td><td>CAACATAAACACTGACTCAAGATG</td><td>1132–1155</td><td>B ~1.20Kb</td><td></td></tr><tr><td>DEN3 CC BR</td><td>CAAAGTCAAGAGAACTCCTATTCC</td><td>2300–2322</td><td></td><td></td></tr><tr><td>d3s5-CC</td><td>CTGAACCTCCTTTTGGGAAAG</td><td>2035–2053</td><td>C ~1.55Kb</td><td>2 ~2.30Kb</td></tr><tr><td>DEN3 CC 2R</td><td>CTCCATGTTATTTGCCCTGAGA</td><td>3599–3621</td><td></td><td></td></tr><tr><td>DEN3 CC 2F</td><td>AGGTGGAAGATTACGGGTTCG</td><td>2874–2894</td><td>D ~1.45Kb</td><td></td></tr><tr><td>d3a14-CC</td><td>AACTGTGATCATTAAARTTGTGGGA</td><td>4334–4356</td><td></td><td></td></tr><tr><td>DEN3 CC EF</td><td>TTCTCTCCTTAGAAATGATGTACC</td><td>4180–4203</td><td>E ~1.05Kb</td><td>3 ~2.15Kb</td></tr><tr><td>DEN3 CC 3R</td><td>TCAATGCTTCTTCCATCTCAGC</td><td>5201–5222</td><td></td><td></td></tr><tr><td>DEN3 CC 3F</td><td>GAGTGGTTACAAAGAATGGTGG</td><td>4971–4992</td><td>F ~1.35Kb</td><td></td></tr><tr><td>d3a10*</td><td>GCYGCAAARTCCTTGAATTCTC</td><td>6339–6360</td><td></td><td></td></tr><tr><td>d3s13-CC</td><td>CCAGCTCTCTTTGARCCAGAAA</td><td>6032–6053</td><td>G ~1.30Kb</td><td>4 ~2.30Kb</td></tr><tr><td>DEN3 CC 4R</td><td>AGCATAACCTGTCCCAGTTGC</td><td>7318–7338</td><td></td><td></td></tr><tr><td>DEN3 CC 4F</td><td>AGATGTGGACTTGCACCCAGC</td><td>6904–6924</td><td>H ~1.45Kb</td><td></td></tr><tr><td>d3a6-CC2</td><td>CGCATTAACATGTCGAGTTCC</td><td>8342–8361</td><td></td><td></td></tr><tr><td>DEN3 CC IF</td><td>TCCACTTTCACGAAACTCCACG</td><td>8185–8206</td><td>I ~1.25Kb</td><td>5 ~2.50Kb</td></tr><tr><td>DEN3 CC 5R</td><td>TTCAGACCATAAGTTCCTACCTG</td><td>9368–9390</td><td></td><td></td></tr><tr><td>DEN3 CC 5F</td><td>GAAGATGACCTGCACAATGAGG</td><td>9191–9212</td><td>J ~1.50Kb</td><td></td></tr><tr><td>DEN1 CC JR</td><td>CGTTCTGTGCCTGGAATGATGCTG</td><td>10646–10669</td><td></td><td></td></tr><tr><td rowspan="10">DENV-4</td><td>d4s1C—CC</td><td>AGTTGTTAGTCTGTGTGGACCGACAA</td><td>1–26</td><td>A ~1.45Kb</td><td>1 ~2.15Kb</td><td rowspan="10">52 °C</td></tr><tr><td>DEN4 CC 1R</td><td>GTGACCTGGGAGTTATCGTGGCTG</td><td>1420–1443</td><td></td><td></td></tr><tr><td>DEN4 CC 1F</td><td>GACACCCATGCAGTAGGAAATGACAC</td><td>1377–1402</td><td>B ~0.80Kb</td><td></td></tr><tr><td>DEN4 CC BR</td><td>ACTCAAACATCTTGCCAATGGAACTC</td><td>2123–2148</td><td></td><td></td></tr><tr><td>d4s5-CC</td><td>CTCCGTGTAAAGTCCCATAGAGATAA</td><td>1910–1933</td><td>C ~1.75Kb</td><td>2 ~3.05Kb</td></tr><tr><td>DEN4 CC 2R</td><td>CATGATGAGGGCTCGTAGCAAGTCC</td><td>3632–3656</td><td></td><td></td></tr><tr><td>DEN4 CC 2F</td><td>AGAAGATGGGTGCTGGTATGGGATG</td><td>3398–3422</td><td>D ~1.55Kb</td><td></td></tr><tr><td>DEN4 CC DR</td><td>ACTCCATTTCCATAGAGTCCGATGAC</td><td>4959–4984</td><td></td><td></td></tr><tr><td>DEN4 CC EF</td><td>GACTGGGAGATTGGAGCCATCTTG</td><td>4706–4729</td><td>E ~1.50Kb</td><td>3 ~2.60Kb</td></tr></tbody></table>
<table><caption>Table 1 (Continued)</caption><thead><tr><th>Serotype</th><th>Primer Name</th><th>Sequence 5'-3'</th><th>Location</th><th>10 Products</th><th>5 Products</th><th>Annealing Temperature</th></tr></thead><tbody><tr><td>DEN4 CC 3R</td><td>TCTTTGTAAGAAATGCCAGCAGAAGC</td><td>6175–6200</td><td></td><td></td><td></td><td></td></tr><tr><td>DEN4 CC 3F</td><td>CTCAAGCCAGTTATCCTAACAGATGG</td><td>5809–5834</td><td></td><td>F ~1.50Kb</td><td></td><td></td></tr><tr><td>d4a8-CC</td><td>CATGACYTGCCCTAATTGCTTTTCAAA</td><td>7289–7311</td><td></td><td></td><td></td><td></td></tr><tr><td>d4s16-CC</td><td>GAAAAGRACAGCTGCTGGRATCATGA</td><td>7205–7226</td><td></td><td>G ~1.40Kb</td><td>4 ~2.45Kb</td><td></td></tr><tr><td>DEN4 CC 4R</td><td>TCTGTCATGGCTAACTGAGTCACCA</td><td>8573–8597</td><td></td><td></td><td></td><td></td></tr><tr><td>DEN4 CC 4F</td><td>TCACAACAAGGCATAGGAAACCCAC</td><td>8290–8315</td><td></td><td>H ~1.40Kb</td><td></td><td></td></tr><tr><td>d4a3-CC</td><td>TGTGRAARTGGTGGGAGCAAAAAGGA</td><td>9668–9689</td><td></td><td></td><td></td><td></td></tr><tr><td>d4s21-CC</td><td>GAAAGAYATYCCGCAGTGGGAACCA</td><td>9610–9631</td><td></td><td>I ~1.00Kb</td><td>5 ~1.00Kb</td><td></td></tr><tr><td>d4a5B-CC</td><td>AGAACCTGTTGGATCAACAACACCAAT</td><td>10630–10650</td><td></td><td></td><td></td><td></td></tr></tbody></table>

cols NMRCD.2000.0006, NMRCD.2000.0008, NMRCD.2001.0002, NMRCD.2005.0008, NMRCD.2010.0010, NAMRU6.2014.0028, NAMRU6.2015.0010). In two additional instances for DENV1, we also used infected culture supernatants derived from samples. To address the issue of template concentration, DENV genomes were initially quantified using a previously described qRT-PCR protocol that targets a conserved region in all DENV serotypes (McAvin et al., 2005), and thus, can be used to compare DENV-specific RNA concentrations across serotypes. McAvin cycle threshold (Ct) values for the samples tested ranged between 18 and 37. As expected, samples with high concentration of DENV template (Ct values lower than 26) were easily amplified (Fig. 1). In these cases, complete genome coverage was achieved with as few as 5 overlapping amplicons. As the concentration of DENV template decreased (Ct values between 26 and 29), amplification efficiency for large amplicons also decreased. In these cases of medium and low concentration of DENV template, complete genome coverage was achieved by increasing the number of overlapping amplicons up to 10 (Fig. 1). In the case of extremely low concentrations of DENV template (Ct values of 29 and higher), at least some overlapping fragments could be generated using the 10-fragment amplification strategy (not shown). However, we consider that above Ct values of 29, DENV samples are sub-optimal for the purposes of amplicon sequencing by NGS. Although it is possible that further optimization of the PCR conditions could improve the genome coverage in samples of extremely low concentration of DENV template, such efforts were beyond the scope of this work.

Once generated by PCR, amplicons representing the total length of each tested DENV genome were pooled together and sequenced using both Illumina MiSeq and Ion Torrent PGM sequencing platforms at different depths of coverage (Fig. 2). Our targeted amplification strategy results in full-genome coverage of all DENV serotypes for every overlapping fragment in each sample. Sequencing statistics for representative samples are shown in Table 4. Between 93% and 99+% of all reads in each sample corresponded to DENV sequences, indicating this is an effective amplification and sequencing strategy for DENVs that have circulated in South America since the year 2000.

## 4. Discussion

Amplicon sequencing by NGS is a relatively simple technique, at least in theory. In practice however, the generation of overlapping amplicons that cover the complete length of a particular genome of interest can be complicated by a number of factors, including template concentration, sequence diversity, primer specificity, and PCR amplification efficiency. In the current study we report improvements to existing full-genome amplification strategies for DENVs (Christenbury et al., 2010). These changes should benefit those attempting to generate complete DENV genomes in a quick and cost-effective manner, particularly for DENV strains that have circulated in South America since 2000.

The first modification we have implemented is the inclusion of an initial serotype-independent relative quantification of DENV

genome copies using qPCR (McAvin et al., 2005). The advantages of using viral load, as measured by qPCR, to guarantee successful amplification of DENVs from acute serum samples has been previously documented (Rodriguez-Roche et al., 2012). Because this assay targets a conserved region in all DENV genomes it can be used to compare DENV-specific genome copies across serotypes. Our results clearly indicate that DENV positive samples showing Ct values of up to 29 are suitable starting material for this amplification sequencing strategy. In contrast, DENV positive samples with Ct values higher than 29 are not suitable, even for the 10-fragment amplification strategy developed for “low-copy” templates. Despite being DENV positive, such samples contain sub-threshold levels of DENV RNA that render them sub-optimal because they only lead to partial genome amplification. By establishing the relative concentration of starting material at the outset of the amplicon sequencing strategy, the researcher is able to focus on samples that have a high probability of success and avoid potentially costly and time-consuming preparation of samples that are a dead-end. A possible workaround for “low-copy” templates is to passage viruses through an established tissue culture line, like C6/36 cells (Singh and Paul, 1969; Tesh, 1979). However, the drawback to this approach is that viruses may mutate and adapt to the host cell type (Villordo et al., 2015). Thus, it is always preferable to generate amplicon-sequencing data from samples that have not been subjected to laboratory culture (Rodriguez-Roche et al., 2012). This is particularly important in downstream applications that assess viral evolution, since by avoiding passage through a laboratory culture one also avoids the introduction of mutations that are not representative of viruses circulating in the wild. In this study, we have tested a total of 46 samples, representing all four DENV serotypes. All samples reported here were human serum samples, which is significant for two reasons. First, serum samples contain viruses that have not been subject to passage through laboratory cell lines, and thus, they represent wild-type viruses that have actually circulated in nature. Second, serum samples tend to have much lower viral RNA concentrations that those found of cultured samples, thus a methodology that can successfully generate full genomes from wild-type viruses in serum samples is robust. In some instances we have also sequenced cultured samples, indicating the primer sets described can be used both for “easy-to-sequence” cultured samples and for “high-value” wild-type samples.

The second modification we have implemented is a dual approach for high- and low-copy templates. High-copy templates (with Ct values as high as 26) can be easily covered in full by as few as 5 overlapping amplicons, whereas low-copy templates (with Ct values between 26 and 29) can be covered by no more than 10. Given that the relative concentration of starting template in the sample is determined before amplicon sequencing takes place, the researcher again has an added level of control that informs strategy and enables selection of the most appropriate amplification strategy. Perhaps this flexibility is not very significant in the context of a handful of genomes, but when hundreds or thousands of full genomes need to be generated accurately, quickly, and consistently, tailored strategies that increase the possibilities of success
<caption>**Table 2**</caption>

DENV complete genome sequences used as references for primer design.
Sequences were carefully selected to maximize diversity in terms of serotype, year of isolation, and country of origin.

<table><thead><tr><th>Serotype</th><th>Accession #</th><th>Year</th><th>Location</th><th>Length (bp)</th></tr></thead><tbody><tr><td rowspan="20">DENV-1</td><td>FJ850070</td><td>2000</td><td>Brazil</td><td>10677</td></tr><tr><td>GU131832</td><td>2000</td><td>Venezuela</td><td>10460</td></tr><tr><td>FJ850073</td><td>2001</td><td>Brazil</td><td>10692</td></tr><tr><td>GU131948</td><td>2001</td><td>Colombia</td><td>10627</td></tr><tr><td>GU131834</td><td>2001</td><td>Venezuela</td><td>10468</td></tr><tr><td>FJ850075</td><td>2002</td><td>Brazil</td><td>10683</td></tr><tr><td>FJ850077</td><td>2003</td><td>Brazil</td><td>10684</td></tr><tr><td>FJ850081</td><td>2004</td><td>Brazil</td><td>10688</td></tr><tr><td>FJ639796</td><td>2004</td><td>Venezuela</td><td>10676</td></tr><tr><td>FJ850084</td><td>2005</td><td>Brazil</td><td>10686</td></tr><tr><td>KJ189304</td><td>2005</td><td>Colombia</td><td>10447</td></tr><tr><td>FJ639812</td><td>2005</td><td>Venezuela</td><td>10640</td></tr><tr><td>FJ850087</td><td>2006</td><td>Brazil</td><td>10690</td></tr><tr><td>GQ868563</td><td>2006</td><td>Colombia</td><td>10661</td></tr><tr><td>HQ332182</td><td>2006</td><td>Venezuela</td><td>10735</td></tr><tr><td>FJ850090</td><td>2007</td><td>Brazil</td><td>10673</td></tr><tr><td>GQ868567</td><td>2007</td><td>Colombia</td><td>10690</td></tr><tr><td>HQ332183</td><td>2007</td><td>Venezuela</td><td>10735</td></tr><tr><td>FJ850093</td><td>2008</td><td>Brazil</td><td>10690</td></tr><tr><td>GQ868570</td><td>2008</td><td>Colombia</td><td>10674</td></tr><tr><td>FJ850104</td><td>2008</td><td>Venezuela</td><td>10690</td></tr><tr><td>KC692513</td><td>2010</td><td>Argentina</td><td>10735</td></tr><tr><td>JX669462</td><td>2010</td><td>Brazil</td><td>10759</td></tr><tr><td>FJ898466</td><td>2000</td><td>Venezuela</td><td>10679</td></tr><tr><td>FJ850074</td><td>2001</td><td>Brazil</td><td>10678</td></tr><tr><td>FJ850076</td><td>2002</td><td>Brazil</td><td>10663</td></tr><tr><td>KC294222</td><td>2002</td><td>Peru</td><td>10723</td></tr><tr><td>FJ850078</td><td>2003</td><td>Brazil</td><td>10667</td></tr><tr><td>FJ850082</td><td>2004</td><td>Brazil</td><td>10679</td></tr><tr><td>FJ639788</td><td>2004</td><td>Venezuela</td><td>10678</td></tr><tr><td>FJ850085</td><td>2005</td><td>Brazil</td><td>10677</td></tr><tr><td>FJ850088</td><td>2006</td><td>Brazil</td><td>10667</td></tr><tr><td>FJ850091</td><td>2007</td><td>Brazil</td><td>10678</td></tr><tr><td>GQ868558</td><td>2007</td><td>Colombia</td><td>10679</td></tr><tr><td>KC294200</td><td>2007</td><td>Peru</td><td>10724</td></tr><tr><td>EU482607</td><td>2007</td><td>Venezuela</td><td>10679</td></tr><tr><td>GQ199890</td><td>2008</td><td>Brazil</td><td>10679</td></tr><tr><td>FJ850107</td><td>2008</td><td>Venezuela</td><td>10667</td></tr><tr><td>KC294201</td><td>2009</td><td>Peru</td><td>10723</td></tr><tr><td>KC294223</td><td>2010</td><td>Peru</td><td>10723</td></tr><tr><td>KC294218</td><td>2011</td><td>Peru</td><td>10723</td></tr><tr><td>FJ898457</td><td>2000</td><td>Ecuador</td><td>10663</td></tr><tr><td>FJ898468</td><td>2000</td><td>Venezuela</td><td>10663</td></tr><tr><td>FJ913015</td><td>2001</td><td>Brazil</td><td>10663</td></tr><tr><td>GU131950</td><td>2001</td><td>Colombia</td><td>10616</td></tr><tr><td>GQ252678</td><td>2001</td><td>Venezuela</td><td>10641</td></tr><tr><td>GQ868571</td><td>2002</td><td>Colombia</td><td>10663</td></tr><tr><td>FJ898458</td><td>2002</td><td>Peru</td><td>10663</td></tr><tr><td>FJ898471</td><td>2002</td><td>Venezuela</td><td>10663</td></tr><tr><td>FJ898447</td><td>2003</td><td>Brazil</td><td>10663</td></tr><tr><td>GU131952</td><td>2003</td><td>Colombia</td><td>10638</td></tr><tr><td>FJ898473</td><td>2003</td><td>Venezuela</td><td>10663</td></tr><tr><td>FJ850083</td><td>2004</td><td>Brazil</td><td>10663</td></tr><tr><td>GU131953</td><td>2004</td><td>Colombia</td><td>10588</td></tr><tr><td>KJ189266</td><td>2004</td><td>Peru</td><td>10696</td></tr><tr><td>JX669501</td><td>2005</td><td>Brazil</td><td>10707</td></tr><tr><td>KJ189299</td><td>2005</td><td>Peru</td><td>10692</td></tr><tr><td>GU131865</td><td>2006</td><td>Brazil</td><td>10501</td></tr><tr><td>GU131954</td><td>2006</td><td>Colombia</td><td>10525</td></tr><tr><td>KJ189297</td><td>2006</td><td>Peru</td><td>10692</td></tr><tr><td>FJ639825</td><td>2006</td><td>Venezuela</td><td>10648</td></tr><tr><td>FJ850092</td><td>2007</td><td>Brazil</td><td>10648</td></tr><tr><td>GQ868578</td><td>2007</td><td>Colombia</td><td>10659</td></tr><tr><td>HQ235027</td><td>2007</td><td>Paraguay</td><td>10707</td></tr><tr><td>KJ643590</td><td>2007</td><td>Peru</td><td>10692</td></tr><tr><td>FJ850111</td><td>2007</td><td>Venezuela</td><td>10663</td></tr><tr><td>FJ850094</td><td>2008</td><td>Brazil</td><td>10662</td></tr><tr><td>KJ189301</td><td>2008</td><td>Peru</td><td>10693</td></tr><tr><td>FJ639827</td><td>2008</td><td>Venezuela</td><td>10635</td></tr><tr><td>JF808120</td><td>2009</td><td>Brazil</td><td>10707</td></tr><tr><td>FJ639748</td><td>2000</td><td>Venezuela</td><td>10551</td></tr><tr><td>GQ868582</td><td>2001</td><td>Colombia</td><td>10606</td></tr><tr><td>FJ639773</td><td>2001</td><td>Venezuela</td><td>10606</td></tr><tr><td>GQ868584</td><td>2004</td><td>Colombia</td><td>10552</td></tr><tr><td>GQ868585</td><td>2005</td><td>Colombia</td><td>10606</td></tr></tbody></table>
<table><caption>Table 2 (Continued)</caption><thead><tr><th>Serotype</th><th>Accession #</th><th>Year</th><th>Location</th><th>Length (bp)</th></tr></thead><tbody><tr><td></td><td>JN819406</td><td>2006</td><td>Venezuela</td><td>10357</td></tr><tr><td></td><td>GQ868643</td><td>2007</td><td>Venezuela</td><td>10593</td></tr><tr><td></td><td>FJ882592</td><td>2008</td><td>Venezuela</td><td>10606</td></tr><tr><td></td><td>JN983813</td><td>2010</td><td>Brazil</td><td>10649</td></tr><tr><td></td><td>JQ513335</td><td>2011</td><td>Brazil</td><td>10604</td></tr><tr><td></td><td>KJ596671</td><td>2012</td><td>Brazil</td><td>10649</td></tr></tbody></table>

**Table 3**

Samples used for protocol validation.

Most samples are human serum containing wild-type DENVs. In two instances for DENV1, supernatants from cultured serum samples were also used.

<table><thead><tr><th>Serotype</th><th>Sample Code</th><th>Year</th><th>Location</th><th>Sample Type</th><th>Ct value</th></tr></thead><tbody><tr><td rowspan="10">DENV-1</td><td>OBS9842</td><td>2000</td><td>Peru</td><td>culture</td><td>22.03</td></tr><tr><td>FSB2837</td><td>2009</td><td>Bolivia</td><td>culture</td><td>12.63</td></tr><tr><td>FCB0983</td><td>2009</td><td>Bolivia</td><td>serum</td><td>37.27</td></tr><tr><td>FSE7098</td><td>2010</td><td>Ecuador</td><td>serum</td><td>32.39</td></tr><tr><td>FPA2544</td><td>2010</td><td>Paraguay</td><td>serum</td><td>31.59</td></tr><tr><td>FSP1646</td><td>2010</td><td>Peru</td><td>serum</td><td>26.44</td></tr><tr><td>FYA00055</td><td>2011</td><td>Paraguay</td><td>serum</td><td>28.61</td></tr><tr><td>FEG00002</td><td>2012</td><td>Ecuador</td><td>serum</td><td>35.07</td></tr><tr><td>FPJ00740</td><td>2014</td><td>Peru</td><td>serum</td><td>24.68</td></tr><tr><td>FYC00721</td><td>2015</td><td>Paraguay</td><td>serum</td><td>22.93</td></tr><tr><td rowspan="15">DENV-2</td><td>FPJ00811</td><td>2015</td><td>Peru</td><td>serum</td><td>21.45</td></tr><tr><td>FVB2118</td><td>2011</td><td>Bolivia</td><td>serum</td><td>20.95</td></tr><tr><td>FVB2121</td><td>2011</td><td>Bolivia</td><td>serum</td><td>26.11</td></tr><tr><td>FPI00403</td><td>2011</td><td>Peru</td><td>serum</td><td>22.1</td></tr><tr><td>FEG00005</td><td>2012</td><td>Ecuador</td><td>serum</td><td>32.34</td></tr><tr><td>FPI04256</td><td>2012</td><td>Peru</td><td>serum</td><td>23.30</td></tr><tr><td>FYA00328</td><td>2013</td><td>Paraguay</td><td>serum</td><td>23.61</td></tr><tr><td>FPI06921</td><td>2013</td><td>Peru</td><td>serum</td><td>22.60</td></tr><tr><td>FPY00498</td><td>2014</td><td>Peru</td><td>serum</td><td>20.69</td></tr><tr><td>FPU00873</td><td>2014</td><td>Peru</td><td>serum</td><td>32.32</td></tr><tr><td>FPI10982</td><td>2014</td><td>Peru</td><td>serum</td><td>25.54</td></tr><tr><td>FPM00818</td><td>2014</td><td>Peru</td><td>serum</td><td>28.64</td></tr><tr><td>FPI13322</td><td>2015</td><td>Peru</td><td>serum</td><td>22.41</td></tr><tr><td>FPP02381</td><td>2015</td><td>Peru</td><td>serum</td><td>23.43</td></tr><tr><td>FVM00186</td><td>2015</td><td>Venezuela</td><td>serum</td><td>26.17</td></tr><tr><td rowspan="12">DENV-3</td><td>IQD3765</td><td>2002</td><td>Peru</td><td>serum</td><td>20.99</td></tr><tr><td>FSB0417</td><td>2003</td><td>Bolivia</td><td>serum</td><td>25.28</td></tr><tr><td>FSL1212</td><td>2003</td><td>Peru</td><td>serum</td><td>19.35</td></tr><tr><td>FSE1036</td><td>2004</td><td>Ecuador</td><td>serum</td><td>22.36</td></tr><tr><td>FSB0879</td><td>2005</td><td>Bolivia</td><td>serum</td><td>28.56</td></tr><tr><td>FSB0931</td><td>2005</td><td>Bolivia</td><td>serum</td><td>22.87</td></tr><tr><td>IQE2562</td><td>2005</td><td>Peru</td><td>serum</td><td>18.64</td></tr><tr><td>IQE3021</td><td>2006</td><td>Peru</td><td>serum</td><td>18.53</td></tr><tr><td>FCB0589</td><td>2007</td><td>Bolivia</td><td>serum</td><td>23.82</td></tr><tr><td>FPA0171</td><td>2007</td><td>Paraguay</td><td>serum</td><td>28.58</td></tr><tr><td>FPA0313</td><td>2007</td><td>Paraguay</td><td>serum</td><td>23.16</td></tr><tr><td>FPI13521</td><td>2015</td><td>Peru</td><td>serum</td><td>20.89</td></tr><tr><td>FPP02377</td><td>2015</td><td>Peru</td><td>serum</td><td>18.36</td></tr><tr><td rowspan="8">DENV-4</td><td>FSL3797</td><td>2008</td><td>Peru</td><td>serum</td><td>26.73</td></tr><tr><td>FSL3887</td><td>2009</td><td>Peru</td><td>serum</td><td>26.37</td></tr><tr><td>FSP1830</td><td>2010</td><td>Peru</td><td>serum</td><td>27.81</td></tr><tr><td>FMD3542</td><td>2010</td><td>Peru</td><td>serum</td><td>27.32</td></tr><tr><td>FPM00691</td><td>2013</td><td>Peru</td><td>serum</td><td>28.24</td></tr><tr><td>FPM00697</td><td>2013</td><td>Peru</td><td>serum</td><td>28.03</td></tr><tr><td>FVM00212</td><td>2015</td><td>Venezuela</td><td>serum</td><td>21.55</td></tr></tbody></table>

**Table 4**

Sequencing statistics for sequenced DENV samples.

Following PCR amplification, overlapping fragments were pooled in roughly equimolar amounts in order to cover the length of each genome. Pools were prepared into both Ion and Illumina libraries and sequenced using PGM and MiSeq platforms, respectively, at different depths of coverage.

<table><thead><tr><th rowspan="2">Sample</th><th rowspan="2">Serotype</th><th colspan="4">Ion Torrent Data</th><th colspan="4">MiSeq Data</th></tr><tr><th>Total Reads</th><th>Total Bases</th><th>DENV Reads</th><th>% DENV Reads</th><th>Total Reads</th><th>Total Bases</th><th>DENV Reads</th><th>%DENV Reads</th></tr></thead><tbody><tr><td>1</td><td>DENV-1</td><td>581</td><td>90,570</td><td>548</td><td>94.32%</td><td>582,328</td><td>174,440,938</td><td>581,361</td><td>99.83%</td></tr><tr><td>2</td><td>DENV-1</td><td>4,136</td><td>702,875</td><td>3,960</td><td>95.74%</td><td>841,056</td><td>252,934,514</td><td>815,656</td><td>96.98%</td></tr><tr><td>3</td><td>DENV-2</td><td>500</td><td>54,731</td><td>466</td><td>93.20%</td><td>444,294</td><td>293,192,662</td><td>424,307</td><td>95.50%</td></tr><tr><td>4</td><td>DENV-2</td><td>6,179</td><td>908,554</td><td>5,944</td><td>96.20%</td><td>1,353,824</td><td>405,714,890</td><td>1,343,717</td><td>99.25%</td></tr><tr><td>5</td><td>DENV-2</td><td>82,997</td><td>11,132,821</td><td>79,689</td><td>96.01%</td><td>946,264</td><td>283,873,702</td><td>938,124</td><td>99.14%</td></tr><tr><td>6</td><td>DENV-3</td><td>3,603</td><td>560,731</td><td>3,545</td><td>98.39%</td><td>926,635</td><td>277,773,636</td><td>909,683</td><td>98.17%</td></tr><tr><td>7</td><td>DENV-4</td><td>3,592</td><td>568,281</td><td>3,555</td><td>98.97%</td><td>901,131</td><td>269,778,474</td><td>898,848</td><td>99.75%</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>Fig. 2. Representative depth of coverage plots for DENV1-4.</figcaption></figure>

DENV1 and 3 were sequenced on the Ion Torrent platform at a low depth of coverage (50X on average), whereas DENV2 and 4 were sequenced on the MiSeq platform at a high depth of coverage (1,500X on average). DENV1, 2 and 4 coverage plots were derived from 5 "long" overlapping fragments (shown in red with labels 1–5), whereas the DENV3 coverage plot was derived from 10 "short" overlapping fragments (labels A–J). In the case of DENV4, the last overlapping fragment is the same in both the high and low copy amplification strategies (label 5/I).

while reducing costs, time and handling, can have a very significant positive impact.

The final modification we have implemented is a complete re-
design of primer sequences with the dual purpose of ensuring
successful amplification of DENV strains that have circulated in
South America, and of normalizing annealing temperatures for all
primers within each serotype set. South American DENV strains are
often poorly amplified, if amplified at all, by existing primer sets
(Christenbury et al., 2010; Rodriguez-Roche et al., 2016) because
those primers have been developed for DENV strains that have
primarily circulated in Asia. In addition, some of these primer
sets have been developed using clinical isolates where specific
serotypes predominated over others (introducing bias towards the
over-represented serotype(s)), or using viruses that have been
passaged through laboratory cell lines (introducing bias towards
laboratory-adjusted strains). The primer sets described here have
been validated using balanced samples sets composed of diverse
wild-type DENV genomes derived from human serum samples rep-
resenting all four DENV serotypes and a variety of geographical
regions in South America over a 15-year period. These primer sets
have the added benefit of sharing annealing temperatures, which
should streamline handling by enabling interchangeable use at a
single annealing temperature within each set. Once again, the aim
has been to simplify workflows as much as possible in order to
increase processing output while maintaining or improving PCR
amplification efficiency for South American strains that were pre-
viously difficult or impossible to sequence. This added efficiency
should be particularly relevant during outbreaks, when a labora-
tory may be required to process increased numbers of samples in
a short period of time.

DENVs are known for their genetic variation within serotype
groups and geographical regions (Rico-Hesse, 2003; Rodriguez-
Roche et al., 2012; Weaver and Vasilakis, 2009). Our targeted
amplicon-sequencing strategy aims to normalize and facilitate
workflows that can be cumbersome, expensive, and time-
consuming, particularly if they are attempted on samples with
sub-optimal concentrations of viral genomes that fail to yield
complete genome sequences. We have shown that the modifica-
tions implemented result in successful generation of overlapping
amplicons that can be processed using different library con-
structions approaches and NGS platforms. Specifically, we have
processed a diverse group of wild-type DENV sequences derived
from serum using both mechanical and enzymatic library construc-
tion approaches, two separate sequencing platforms (Ion Torrent
and MiSeq), and two different depths of coverage (DENV1 and
DENV3 at a low depth of 50X on average, and DENV2 and DENV4 at a
high depth of 1,500X on average). Nevertheless, we have avoided a
detailed discussion of sequencing statistics, including depth of cov-
erage and error rates for example, in order to highlight the flexibility
of this approach, which allows the researcher to control different
aspects of sequencing in order to suit a variety of applications that
may require dramatically different levels of depth of coverage. The
main advantage of this sequencing strategy is that the majority of
sequencing reads obtained (97% on average) correspond to DENVs.
This has the added benefit of simplifying downstream bioinformat-
ics processing because a reduced number of "contaminating" reads
is an advantage over non-targeted methods like shotgun sequenc-
ing, where a large number of host-specific reads often impede
adequate representation of the complete viral genome of interest
(Yozwiak et al., 2012). We believe this methodology has a number of
downstream applications, including epidemiological surveillance,
studies of quasi-species and dominant genomes, and studies of
DENV evolution where measurements of intra-host diversity can
be used to identify evolutionary forces (e.g.: natural selection vs.
genetic drift) that act on viral populations, particularly in the case
of DENV strains like the ones that have circulated in South America
over the last 15 years.

**5. Disclaimers**

The views expressed in this article are those of the authors
and do not reflect the official policy or position of the Depart-
ment of the Navy, Department of Defense, nor the U.S. Government.
We are employees of the U.S. Government. The work was pre-
pared as part of our official duties. Title 17 U.S.C. §105 provides
that 'Copyright protection under this title is not available for any
work of the United States Government.' Title 17 U.S.C. §101 defines
U.S. Government work as a work prepared by military service
members or employees of the U.S. Government as part of that
person's official duties. This work was supported by an award
to ML (work unit number 847705.82000.25GB.B0016, Promis ID#
MLeguia-PO166-14, for FY2014) from the Global Emerging Infec-
tions Surveillance and Response System (GEIS) of the Armed
Forces Health Surveillance Center (AFHSC). LL received support
from the French Government's Investissement d'Avenir program
Laboratoire d'Excellence Integrative Biology of Emerging Infec-
tious Diseases grant ANR-10-LABX-62-IBEID, the City of Paris
Emergence(s) program in Biomedical Research, and National Insti-
tutes of Health grant 1P01AI098670-01A1. The study protocol was
approved by NAMRU-6's IRB in compliance with all applicable Fed-
eral regulations governing the protection of human subjects.

**Conflicts of interest**

The authors declare no conflict of interest.

**Acknowledgements**

We are deeply grateful to Dr. Julia Sonia Ampuero for
enabling access to DENV-positive human serum samples gathered
throughout Latin America. We also thank local and international
collaborators who generously provide samples as part of joint,
ongoing surveillance efforts, in particular the Instituto Nacional de
Salud (INS) – Ministerio de Salud del Perú; Dr. Yelin Roca Sánchez
at the Centro Nacional de Enfermedades Tropicales (CENETROP)
in Bolivia; Dr. Nicolás Aguayo at ONG Rayos de Sol – Ministerio
de Salud Pública y Binenestar Social del Paraguay; Dr. Guillermo
Comach at Laboratorio Regional de Diagnóstico e Investigación
del Dengue y otras Enfermedades Virales (LARDIDEV) – Insti-
tuto de Investigaciones Biomédicas de la Universidad de Carabobo
(BIOMED-UC) in Venezuela. Finally, we are grateful to Dr. Ros-
mari Rodríguez-Roche for providing some of the modified primer
sequences and Sebastian Lequime for assistance with primer
redesign.

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