Multiplex PCR for SEOV Genome Sequencing

We designed multiplex PCR primers for SEOV L, M, and S segments and amplified cDNA by using primers (Technical Appendix 1) and primer mixtures and Solg 2× Uh-Taq PCR Smart Mix (Solgent, Daejeon, South Korea), according to the manufacturer’s instructions. We performed the first and second enrichments in a 25-μL reaction mixture containing 12.5 μL of 2× Uh pre-mix, 1 μL of cDNA template, 10 μL of primer mixture, and 1.5 μL of distilled water. Initial denaturation was at 95°C for 15 min, followed by 40 cycles or 25 cycles at 95°C for 20 s, 50°C for 40 s, and 72°C for 1 min, and a final elongation at 72°C for 3 min.
We prepared multiplex PCR products by using the TruSeq Nano DNA LT Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. We mechanically sheared samples by using an M220 focused ultrasonicator (Covaris, Woburn, MA, USA). The cDNA amplicon was size-selected, A-tailed, ligated with indexes and adaptors, and enriched. We sequenced libraries by using the MiSeq benchtop sequencer (Illumina) with 2 × 150 bp and a MiSeq reagent V2 (Illumina). We imported and analyzed Illumina FASTQ files by using EDGE (21 (link)).