title: Full genome of Allantoic fluid: egg
design: Nasal swabs with peak virus titers collected from the indicated contact pigs were analysed by next generation sequencing. Briefly, viral RNA was extracted from swabs with peak virus titers by using QIAamp viral RNA mini kit (Qiagen, Hilden, Germany). RNA was transcribed into cDNA with SuperScriptTM III Reverse Transcriptase (Invitrogen, California, USA). The gene segments were amplified by Q5 High Fidelity DNA polymerase (NEB, Massachusetts, USA) using a pair of primers (454-Tag1-U12:GCC GGA GCT CTG CAG ATA TCA GCR AAA GCA GG; 454-Tag1-U13:GCC GGA GCT CTG CAG ATA TCA GTA GAA ACA AGG). Libraries were prepared based on the Nextera DNA Flex Library Preparation standard protocol51. 300ng PCR products were cleaved and tagged by bead-linked transposome at 55oC, then the tagmented DNA were amplified with a pair of indexes using a 5-cycle PCR program. The amplified libraries were further purified by using 1 volume of sample purification beads. The libraries were finally eluted using 32 L resuspension buffer. The purified libraries were quantified with Agilent fragment analyzer automated CE system with the high sensitivity NGS fragment analysis 474 kit (Agilent, California, USA). 150 pM pool library containing index adapters allowing multiplex sequencing of 4 samples per iSeq 100 i1 Cartridge was run on iseq100 sequencing system (Illumnia, California, USA).