Illumina MiSeq Sequencing of Strain SKT-27

Preparation of a cDNA library and Illumina MiSeq sequencing were carried out as described previously [30 (link), 33 (link)]. Briefly, a 200 bp fragment library ligated with bar-coded adapters was constructed for strain SKT-27 using an NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (New England Biolabs) according to the manufacturer’s instructions. Library purification was performed using Agencourt AMPure XP magnetic beads (Beckman Coulter). The quality of the purified cDNA library was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). 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 carried out using CLC Genomics Workbench v8.0.1 (CLC Bio). Contigs were assembled from the obtained sequence reads 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 strain SKT-27. The nucleotide sequences were translated into amino acid sequences using GENETYX v11 (GENETYX).