2-4- Phylogenetic assessment
PCR products underwent purification using High Pure PCR Product
Purification Kit (Roche Diagnostic GmbH, Mannheim, Germany)
corresponding to the manufacturer’s instructions. Also, an ABI 3730 XL
sequencer was used for bidirectional sequencing. Raw data were trimmed
and analyzed using popular bioinformatics software CLC workbench 5, and
Basic Local Alignment Search Tool (BLAST). All sequences were submitted
into GeneBank database (https://www.ncbi.nlm.nih.gov/genbank/) and
released accession numbers were used to draw phylogenetic tree.
SARS-CoV-2 reference sequence (NC_045512.2) and other coronavirus
sequences were obtained from NCBI (https://blast.ncbi.nlm.nih.gov)
database. Moreover, MEGA X software was used for multiple sequence
alignment (MSA) and the phylogenetic trees were drown using 1000
replicate bootstrap method. In addition, the neighbor joining and
maximum likelihood methods were carried out for similarity assessment
and evolution analysis.