DENV Genome Sequencing and Analysis

The nucleotide sequence of the genome region encoding the envelope was determined by Sanger sequencing using an ABI sequencer. The sequencing reactions were prepared using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit, version 3.1 (Applied Biosystems, Vilnius, Lithuania). Briefly, 2 μL of DNA template was mixed with 2 μL of reaction mix, 1 μL of 5× sequencing buffer, 1.6 μL of specific primer (Table S1), and 3.4 μL of nuclease-free water. The amplification cycle conditions were programmed according to the manufacturer’s protocol.
The whole genome sequences of DENV isolates obtained in the present study were determined by next-generation sequencing (NGS) on a Miseq platform (Illumina, San Diego, CA, USA). Preparation of the NGS library using NexteraXT (Illumina, San Diego, CA, USA) was described previously [8 (link)]. The FASTQ results were examined using CLC Genomics Workbench software, version 21 (CLC Bio, QIAGEN, Valencia, CA, USA). Whole-genome assembly was conducted using Map read reference command (DENV-1 Mochizuki AB074760.1 and DENV-2 16681 NC_001474.2 were used as reference strains).
All newly obtained sequences of the envelope and the whole genome were deposited in GenBank with accession numbers OQ832560-OQ832594 and OQ832609-OQ832627, respectively.