Validation of multiplex PCR assays
The multiplex PCR assays were performed over all the 24 positive samples. To validate the reliability of the assays, another five pairs of primer set (Table 2 ) were designed for the clades keeping the probable mutation points within the middle of the amplicons by using Primer3Plus (Untergasser et al., 2012) and Primer-BLAST (Ye et al., 2012). Above mentioned parameter settings were followed to design those (except for the amplicon size ranging from 200-400bp and Tm of 52-54°C). Amplicons were subjected to Sanger sequencing using BigDye™ Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific) to confirm the specific clades (wild type/mutant). The commercial kit protocol was optimized to reduce the cost because of the high cost of this BigDye Terminator reagent (Platt, Woodhall, & George, 2007). 1.0μL (per 10 10μL reaction) undiluted BigDye Terminator v3.1 Ready Reaction mix was used instead of 4μL mentioned in commercial kit protocol. Along with the 1.0 μL BigDye Terminator v3.1 Ready Reaction mix, 1.75 μL 5x sequencing buffer, 1μL primer, 2μL template DNA and 4.25μL nuclease free water was added (to make the final reaction volume 10μL). The cycle sequencing PCR condition was setup accordingly to the kit protocol. The sequences were aligned with and verified by the reference sequence (NC_045512.2_SARS-CoV-2_Wuhan-Hu-1 complete genome) using Molecular Evolutionary Genetics Analysis (MEGA X) software (Kumar, Stecher, Li, Knyaz, & Tamura, 2018). For the most part, the cost of the processes were optimized and compared to our in-house NGS system (Ion torrent; ThermoFisher Scientific, USA).