title: Vaccine immune pressure influences viral population complexity of avian influenza virus during infection
design: Total RNA was isolated using Nucleospin RNA kit (Macherey-Nagel, Duren, Germany). Viral RNA encoding the HA gene segment was retro-transcribed and amplified using SuperScript III one-step reverse transcription-PCR (RT-PCR) system with PlatinumTaq High Fidelity (Invitrogen, Carlsbad, CA) using H5 specific primers. Sequencing libraries were prepared using Nextera XT DNA Sample preparation kit (Illumina) and processed as described by Monne et al. (2014) on an Illumina Miseq desktop sequencer.