2.5. Sequence-Independent Single-Primer Amplification (SISPA), Rapid Amplification of cDNA Ends (RACE), Next-Generation Sequencing (NGS) and Bioinformatics

Stock virus culture supernatant was added to Trizol-LS (Life Technologies, Waltham, MA, USA) at a ratio of 100 µL supernatant to 300 µL Trizol-LS. RNA was extracted using a column based kit (Direct-Zol RNA kit, Zymo Research, Irvine, CA, USA). To increase sensitivity, rRNA was depleted using the same method as described previously [37]. RNAs were converted to cDNA and amplified using SISPA as described previously with modifications [38]. To enhance coverage of the terminal ends, an oligo containing three rGTP at the 3' end (GCCGGAGCTCTGCAGATATCGGCCATTATGGCCrGrGrG) was added during first-strand cDNA synthesis and the reverse transcriptase was changed to Maxima H Minus (Thermo Scientific, Waltham, MA, USA), which has terminal transferase activity that enables addition of the rGTP containing oligo to the 5' end during cDNA synthesis. Amplicons were sheared and libraries prepared using the Illumina TRuSeq DNA Library preparation kit (Illumina, San Diego, CA, USA). Sequencing was performed either on an Illumina MiSeq (Illumina) or NextSeq 500 (Illumina) using either a 2 × 150 or 2 × 250 version2 kit. Illumina and SISPA adapter sequences were trimmed from the sequencing reads using Cutadapt-1.2.1 [39], quality filtering was conducted with Prinseq-lite (-min len 50-derep 14-lc method dust-lc threshold 3-trim ns left 1-trim ns right 1-trim qual right 15) [40] and reads were assembled into contigs using Ray Meta with kmer length = 25 [41]. Resultant contigs were aligned to the NCBI sequence database using BLAST.