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.