Kapa hifi hotstart mastermix

Two steps were used for amplification of the target amplicon. 11 μl of cDNA was combined with 0.75 μl of 10 nM forward primer (R1_5’_WNV_for), 0.75 μl of 10 nM forward primer (5’_ID_Primer_Rev), and 12.5 μl of 2x KAPA HiFi HotStart mastermix (VWR). PCR conditions were 95°C for 3 min, 98°C for 20 s, 72°C for 45 s, 72°C for 1 min with 35 cycles. Samples were purified using Agencourt XP beads (Beckman Coulter) at 0.6X concentration and eluted in 20 μl of nuclease-free water.

The second round of PCR amplification served to add barcodes and adapters. 2 μl of purified PCR product from PCR amplification step 1 was combined with 9 μl of nuclease-free water, 0.75μl of 10 nM forward primer (illumina index i5), 0.75 μl of 10 nM forward primer (illumina index i7), 12.5 μl of 2x KAPA HiFi HotStart mastermix (VWR) and the same PCR conditions were followed as in PCR step 1, repeated for 10 cycles. Samples were purified using AMPure XP beads at 0.6x concentration with elution into 22 μl of nuclease-free water.
Samples were pooled at a volume of 5 μl each and concentrated using AMPure XP beads at 1.5x concentration. Pooled samples were quantified using Qubit and size distribution was verified by Tapestation. Additional size selection was performed using AMPure XP beads according to manufacturer’s guidelines. Libraries were quantified using the NEB library quantification kit.