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).