Eco real time pcr system

To perform HRM SNP barcoding, we first quantified the concentration of DNA for all clinical samples, as described above using a Nanodrop. We then diluted the samples in 1X TE buffer to a concentration of total DNA (human and parasite DNA) at 1 ng/μl based on based on OD₂₆₀. For all assays, we included sequenced control samples in each assay plate to identify the reference and alternate SNP temperature melt (T_m) curves. Next, we prepared a master mix (described above) for each assay. We calculated the amount of master mix by multiplying the volume of each component by the number of PCR wells or test samples. We added 7 μl of the master mix to each well in the PCR plate. We gently vortexed and centrifuged the reaction mixture, then added 3 μl of the DNA dilution at 1 ng/μl (final assay concentration 3 ng/μl) to each well for a total reaction volume of 10 μl. We placed a seal on top of the PCR plate with a roller and then centrifuged at 1000 RPM for 1 min. We optimized this protocol to be performed in either a 48-well plate in the Eco Real-Time PCR System or in a 384-well plate in the Applied Biosystems ViiA 7 Real-Time PCR System. We used HRM software on the Eco Real-Time PCR System or the Applied Biosystems ViiA 7 Real-Time PCR System for HRM genotyping analyses. See supplemental materials S1 File for the full barcode assay protocol.