A Serotype-Specific and Multiplex PCR Method for Whole-Genome Sequencing of Dengue Virus Directly from Clinical Samples

Abstract

Dengue virus (DENV) is the most globally prevalent member of the genus Flavivirus in the family Flaviviridae, which can be classified into four serotypes. Historically, molecular epidemiological studies of DENV depended on E gene sequencing. The development of next-generation sequencing (NGS) allowed its application to viral whole-genome sequencing (WGS). In this study, we report the improvement of the existing WGS process for DENV by optimizing the primer design procedure, designing serotype-specific primer panels and reducing the sizes of amplicons. A total of 31 DENV-positive serum samples belonging to 4 serotypes and 9 genotypes of DENV were involved in the validation of the primer panels. The threshold cycle (C_T) values of these samples ranged from 23.91 to 35.11. The validation results showed that the length of consensus sequences generated at a coverage depth of 20× or more ranged from 10,370 to 10,672 bp, with 100.00% coverage of the open reading frames and 97.34% to 99.52% coverage of the DENV genome. The amplification efficiency varied across amplicons, genotypes, and serotypes of DENVs. These results indicate that the serotype-specific primer panels allow users to obtain the whole genome of DENV directly from clinical samples, providing a universal, rapid, and effective tool for the integration of genomics with dengue surveillance. IMPORTANCE Dengue virus (DENV) is becoming the most globally prevalent arbovirus. The number of people living under the threat of DENV is increasing year by year. With the development of next-generation sequencing (NGS) technology, whole-genome sequencing (WGS) has been more and more widely used in infectious disease surveillance and molecular epidemiological studies. DENV population sequencing by NGS can increase our understanding of the changing epidemiology and evolution of the DENV genome at the molecular level, which demands universal primer panels and combination with NGS platforms. Multiplex PCR with a short-amplicon approach proved superior for amplifying viral genomes from clinical samples, particularly when the viral RNA was present at low concentrations. Additionally, DENV are known for their genetic diversity within serotype groups and geographical regions, so the primer panels we designed focused on universality, which would be useful in future local DENV outbreaks.

Keywords: dengue; dengue virus; multiplex PCR; next-generation sequencing; whole-genome sequencing.

FIG 1

Imbricated design of the multiplex PCR primer panels for DENV-1 through DENV-4. Odd-numbered and even-numbered primers were divided into primer pools 1 and 2, respectively. The genome positions of DENV-1 through DENV-4 were based on NCBI reference sequences (GenBank accession numbers NC_001477, NC_001474, NC_001475, and NC_002640, respectively). Primers in the same rows belong to the same pool. The length of the horizontal red lines represents the approximate size of the imbricated amplicons. The intended overlap regions between pools are also presented. UTR, untranslated region.

FIG 2

Strategy for improvement by manual modification of primers. (A) Coverage depth of a DENV-4 isolate (GenBank accession number MW881530) at position 2300 to 2750 before manual modification of primer D4#6R; (B) coverage depth of a DENV-4 isolate (MW881530) at position 2300 to 2750 after manual modification of primer D4#6R; (C) manual modification of primer D4#6R at the fifth site by replacing the “C” with a “Y”; (D) manual modification of primer D4#18R on the 5′ end lead to the change of hairpin T_m.

FIG 3

Sequencing coverage of DENV-positive RNA control samples. Downstream data generated using the Illumina MiniSeq platform were analyzed using CLC Genomics Workbench. Short (below 60 bp) and low-quality (below Q30) reads were discarded. The reference sequences used for mapping can be found under GenBank accession numbers NC_001477 (DENV-1), NC_001474 (DENV-2), NC_001475 (DENV-3), and NC_002640 (DENV-4).

FIG 4

Coverage depth of the downstream data mapping results from the 31 DENV-positive clinical samples. Amplification efficiency varied across amplicons, genotypes, and serotypes of DENVs. The downstream data analysis was performed using CLC Genomics Workbench. The specific mapping results are shown in Table 1.

FIG 5

Genome coverage of the validation results for the 31 DENV-positive clinical samples. The downstream data were generated using the Illumina MiniSeq platform and analyzed using CLC Genomics Workbench. The consensus sequences were generated with a coverage depth of 20× or more with a process including primer trimming, mapping refinement, and manual checking to match the design length.

FIG 6

Complete 10-step workflow in this study and the key points in each step.