Illumina MiSeq Sequencing of Diverse Viral Genomes

Building of a cDNA library and Illumina MiSeq sequencing were performed as described previously [14 (link), 16 (link), 30 (link), 33 (link), 34 (link)]. Briefly, a 200 bp fragment library ligated with bar-coded adapters was constructed for the 14 strains using an NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (New England Biolabs), and NEBNext Multiplex Oligos for Illumina (New England Biolabs) according to the manufacturer’s instructions. The cDNA library was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). After assessing the quality and quantity of the purified cDNA library, nucleotide sequencing was performed on an Illumina MiSeq sequencer (Illumina) using a MiSeq Reagent Kit v2 (Illumina) to generate 151 paired-end reads. Data analysis was performed using CLC Genomics Workbench v8.0.1 (CLC Bio). Contigs were assembled from the obtained sequence reads (trimmed) by de novo assembly. Using the assembled contigs as query sequences, the Basic Local Alignment Search Tool (BLAST) non-redundant nucleotide database was searched to obtain the full-length nucleotide sequence of each gene segment of the 14 strains. The nucleotide sequences were translated into amino acid sequences using GENETYX v11 (GENETYX).