RT-PCR WGS of linfuenza A virus:oropharyngeal/clocalswab
We used the Superscript III high-fidelity RT-PCR kit (ThermoFisher, U.S.A.) and the Opti1 primer set that targets the 12 bp conserved regions on the 5 and 3 ends of each gene segment. The primer set includes primers Opti1-F1 (5-GTTACGCGCCAGCAAAAGCAGG), Opti1-F2 (5-GTTACGCGCCAGCGAAAGCAGG), and Opti1-R1 (5-GTTACGCGCCAGTAGAAACAAGG). Amplification of the swab RNA occurred in 50 L reactions and contained 25 L 2x rt-PCR reaction buffer, 0.2 M of both Opti1-F1 and Opti-F2, 0.4 M of Opti1-R1, 2 uL of the RT/Taq enzyme and 10 L of RNA template. The PCR thermocycler program for whole genome amplification was as follows: 55C for 2 minutes (m), 42C for 60 m, 94C for 2 m, 5 cycles of 94C for 30 seconds (s)/ 44C for 30 s/ 68C for 3.5 min, 30 cycles of 94C for 30 s/ 57C for 30 s / 68C for 3.5 min, and a final extension of 68C for 10 min. Whole genome amplification success was evaluated by running a subset of samples on a 1% agarose gel stained with ethidium bromide. PCR products, and all library preparation reactions, were purified with TotalPure NGS beads (Omega Biotek, U.S.A.). We prepared libraries using the Nextera XT DNA library kit (Illumina, U.S.A.). Libraries were pooled then evaluated and quantified on a Qiaxcel (Qiagen, Germany) using the DNA High Resolution Kit and with qPCR using the KAPA Library Quantification Kit for Illumina Platforms (KAPA Biosystems, U.S.A), respectively. Libraries were sequenced with a Miseq reagent kit V2 500-cycle (Illumina, U.S.A).