Evaluating an Upper Respiratory Disease Panel on the Portable MinION Sequencer

Wanda J. Lyon; Zachary K. Smith; Brian Grier; James Baldwin; Clarise R. Starr

Standard targeted PCR was done per using primers listed in Table 1 and the SuperScript III OneStep RT-PCR system (Thermo Fisher Scientific) per the manufacturer's instructions. Briefly, 10 ng of DNA-free RNA or RNA-free DNA was used for each 50-μL reaction. All PCR reactions were accomplished with 1.0 ul of each primer at 10 uM. RT-PCR thermocycling parameters were as follows: 30 minutes at 50°C, 2 minutes at 95°C, and then 35 cycles of 30 seconds at 95°C, 30 seconds at 52°C, and 75 seconds at 68°C, followed with a final extension at 68°C for 5 minutes. PCR thermocycling was accomplished with AccuStartII PCR Supermix of the DNA fragments was completed using barcoding kit (EXP-PBC001). Libraries kit used for each of the sequencing libraries are shown in Table 2 and 3, as well as the amount of starting material and flow cells type. Library preparations were done as described by the manufacturer and were quantified using a Qubit 3.0 fluorometer (Life Technologies).