Optimization of multiplex PCR assays
The singleplex PCRs showed successful annealing at 57°C, however, temperatures for the duplex, triplex and quadruplex assays were needed to be further optimized to 60°C, 57°C and 56°C, respectively (Fig.2 IIA-IIE ). The primer concentrations for duplex were similar (0.2µM of each primer pair for both the SNP variants) to the single-variant specific PCRs, they were adjusted to different strengths for the triplex (for both forward and reverse primers: 0.2µM for 23403 A>G (p.D614G), 0.3µM for 25563 G>T (p.Q57H), and 0.4µM for 28882 G>A (p.R203K)) and the quadruplex (for both forward and reverse primers: 0.4µM for 23403 A>G (p.D614G), 0.3µM for 25563 G>T (p.Q57H), 0.6µM for 28882 G>A (p.R203K) and 0.2µM for 28144 T>C (p.L84S)) to get the maximum possible resolution. The duplex PCR assay simultaneously amplified the desired wild type bands of 568bp and 496bp; the band for 28144 T>C (p.L84S) was faint in the duplex setting comparing to the single run of the variant described before. Besides, the triplex PCR also amplified the products as expected (i.e., 208bp, 279bp and 387bp). One of the subsets of quadruplex that contained the variants of triplex plus 28144C T>C (p.L84S) was able to distinguish the desired bands individually. However, quadruplex (that had 26144 G>T (p.G251V)) and pentaplex arrangement could not discriminate the bands between wild types and mutants (supplementary Fig.s1 ).