DengueSeq: A pan-serotype whole genome amplicon sequencing protocol for dengue virus v2
Authors: Vogels Chantal 
Published: February 2023

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.