Prediction of Primer dimer formation and RNA secondary structure
We carried out RNA secondary structure prediction of the ORF3a or NS3
RNA using the Mfold web server (Zuker, 2003). The full NS3 sequence was
extracted from the SARS-CoV-2 reference sequence of NCBI GenBank. The
default parameters were used in generating structure. Besides, we used
oligoanalyzer v3.1 of integrated DNA technologies (IDT) to examine
possible primer duplexes and calculate the primer-dimer (both self and
hetero) formation energy in case of the primers, NS3_26144_F,
NS3_26144_wR, and NS3_26144_mR, targeting ORF3a multiplexed
amplicons. The possibility of dimers and energy values for the targeted
primers were checked against other twelve primer sets.