Genomic sequencing of influenza virus: PA_H3N2
Viral RNA was extracted from samples using the QIAamp Viral RNA Mini kit (Qiagen), according to the manufacturers instructions. The PA gene was amplified using the One-Step SuperScript III RT-PCR kit (Invitrogen) and PA segment-specific primers (Supplementary Table 12 of the paper contains the primer pairs). PCR amplicons were cleaned by 0.45X of Agencourt AMPure XP Magnetic Beads (Beckman Coulter) according to manufacturers protocol. The concentration of purified amplicons was measured using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). After samples were normalized to a concentration of 0.2 ng/l, adapters were added by tagmentation using the Nextera XT DNA library preparation kit (Illumina). Samples were purified using 0.6X of Agencourt AMPure XP Magnetic Beads and fragment size distributions were analyzed on a Bioanalyzer using the High Sensitivity DNA kit (Agilent). After bead-based normalization (Illumina) according to the manufacturer's protocols, sequence-ready libraries were sequenced in a paired-end run using the MiSeq v2, 300 cycle reagent kit (Illumina).