5 3 race kit

Meta-transcriptome sequencing was conducted as previously described (Shi et al., 2016 (link)). In brief, after removing ribosomal RNA (rRNA), RNA was fragmented, reverse-transcribed, and adapted, followed by paired-end (150-bp) sequencing on the Illumina Hiseq 2,500 platform. All library preparation and sequencing were performed by Tianjin Novogene Bioinformatics Technology Co., Ltd., Tianjin, China.
Sequencing reads were adaptor- and quality-trimmed using the FASTP program, followed by de novo assemble by the Megahit program, and the resulting contigs were compared against the nr database using the diamond BLASTX program. The confirmed viral contigs with assembly overlaps or from the same scaffold were merged using the SeqMan program (version 7.1, DNAstar, Madison, WI, United States). Reads were mapped to the target contigs using Bowtie 2, and the integrated genomics viewer (IGV) was used to check for assembly faults in order to validate the assembly results. Gaps between the contigs were filled by RT-PCR and Sanger sequencing. The genomic terminus of targeting viruses was determined using the 5′/3′ RACE kits (TaKaRa, Dalian, China). The complete viral genome was confirmed by Sanger sequencing with overlapping primers that covered the entire viral genome.

Sequencing reads were adaptor- and quality-trimmed using the FASTP program [34 (link)] before de novo assembly using the Megahit program [35 (link)], with default parameter settings. The assembled contigs were compared against the database comprising all reference virus proteins using the diamond BLASTX program [36 (link)] with an e-value threshold of 1e–4, and the putative viral contigs were further compared to the non-redundant nucleotide and protein database to eliminate false positives. The confirmed viral contigs with unassembled overlaps or from the same scaffold were merged using the SeqMan program implemented in the Lasergene software package (version 7.1, DNAstar, Madison, WI, USA). To verify the assembly results, reads were mapped back to the target contigs with Bowtie2 [37 (link)], and inspected using integrated genomics viewer (IGV) [38 (link)] for any assembly errors. Gaps between these contigs were filled by RT-PCR and Sanger sequencing. The genome terminal of virus was determined by using 5′/3′ RACE kits (TaKaRa, Dalian, China) as described previously [39 (link)]. The complete viral genome was confirmed by Sanger sequencing with overlapping primers that covered the entire genome, which were designed based on the assembled sequences.

5′ RACE was carried out using the 5′/3′ RLM-RACE kit (Ambion) and 5′/3′ RACE kit (TAKARA) according to manufacture instructions.

For complete genomic sequencing, viral RNA was extracted from 200 µL of viral stocks using TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized using random hexamers (TaKaRa, Dalian, China) following the manufacturer’s instructions. The complete genomic sequences of SCcd17 were amplified with fourteen overlapping fragments by reverse transcription-PCR (RT-PCR) as described previously [32 (link)]. The PCR products were purified and cloned into pMD19-T vector (TaKaRa, Dalian, China) following the manufacturer’s instructions, and then sequenced at least three times by the commercial service (Sangon, Shanghai, China) using Sanger sequencing approach. The 5′ and 3′ ends of the viral genome were amplified using a 5′/3′ RACE kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The sequences of fourteen overlapping fragments from SCcd17 were assembled into full-length genome sequences using the SeqMan program in DNAstar 7.0 software (DNASTAR, Madison, WI, USA).

Total RNA was extracted from the suspensions of PRRSV infected cells using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription (RT) was performed with the PrimeScript^TM RT Reagent Kit (TaKaRa, Dalian, China). Fourteen pairs of primers for PRRSV-2 spanning the entire viral genome were used for amplifying the complete genomes of SCcd16 and SCya17, as previously described [29 (link)]. The PCR products were purified and inserted into a pMD19T-simple vector (TaKaRa, Dalian, China), and at least 3 positive clones of each fragment were sent to the Shanghai Sangon Biological Engineering Technology and Services Co. (Shanghai, China) to determine a consensus sequence (at least 3 times). A deletion was found in the 3’-UTR of SCya17 and confirmed by repeated sequencing (6 times). The 5′/3′ end of the viral genome of each isolate was amplified using a 5′/3′ RACE kit (TaKaRa, Dalian, China).

5′rapid amplification of cDNA ends was performed by smarter RACE 5′/3′ kit (Takara, Otsu, Shiga, Japan) according to the manufacturer’s protocol. The gene-specific primer and sequencing result are shown in Supplementary Table S1.