Sequencing and phylogenetic analysis
All amplicons were purified using QIAquick Purification Kit (Qiagen,
Germany) according to manufacturer’s instructions. Only PCR products
with a concentration of ≥20 ng/μl were sent to 1st BASE Laboratories in
Singapore for Sanger dideoxy sequencing. Resulting sequences from
forward and reverse primers were analyzed to generate consensus
sequences using MEGA 6 [12]. Consensus sequences were aligned using
MAFFT [13] utilizing default parameters. Resulting alignment was
manually inspected and confirmed using Aliview [14]. Appropriate
model of nucleotide substitution for the dataset was chosen using
jModelTest (Posada, 2008). Phylogenetic analysis was performed using
maximum likelihood method with 1000 bootstrap replicates carried out in
IQ-TREE (Nguyen, Schmidt, von Haeseler, & Minh, 2015) and resulting
phylogenetic tree was visualized and edited using FigTree v.1.4.0
(Rambaut, 2012) and Interactive Tree of Live v.5.