title: RNA-Seq of Tembusu virus: AHWH
design: the NEBNext Ultra II RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) was used to construct paired-end libraries. After adapter ligation, 10 cycles of PCRamplification were performed to enrich the sequencing target. The libraries were pooled in equimolar amounts, denatured, and diluted to theoptimal concentration prior to sequencing. Sequencing was performed on an Illumina NovaSeq 6000 System (Illumina, San Diego, CA, USA) to generate 150-bp paired-end reads. Raw reads were processed with Fastp (https://github.com/OpenGene/fastp) to remove adapters and low-quality bases (Phred score <20).Host-derived reads (including ribosomal RNAs) were removed using BBMAPv38.51 (https://sourceforge.net/projects/bbmap). De novo genome assembly was performed using SPAdesv3.14.1 (https://github.com/ablab/spades). These extracted assembled scaffolds limited the minimum contig length to 100 bases, with the best BLAST hits to NCBI nucleotide database.