Sequel binding kit 2

After bead purification with Agencourt AMPure XP (Beckman Coulter, USA), sequencing libraries were built using the SMRTbell Template Prep Kit 1.0‐SPv3 following the guidelines in the amplicon template protocol (Pacific Biosciences, USA). DNA damage repair, end-repair and ligation of hairpin adapters were performed according to the manufacturer’s instruction. DNA template libraries were bound to the Sequel polymerase 2.0 using the Sequel Binding Kit 2.0 (Pacific Biosciences, USA). The data collection per sample was done in a single Sequel SMRT Cell 1M v2 with 600 min movie time on the Sequel system (Pacific Biosciences, USA). We used a 5 pM on-plate loading concentration using Diffusion Loading mode and the Sequel Sequencing Plate 2.0 (Pacific Biosciences, USA).

Purified genomic DNA (gDNA) was used for library construction with the SMRTbell Express Template Prep Kit (Pacific Biosciences, Cat. No. 101-357-000). In brief, gDNA was mechanically sheared to an average size of 20 kb using a Covaris g-TUBE device (Part No. 520079). In total, 5 μg of sheared gDNA was damage-repaired and end-repaired using polishing enzymes. Blunt-end adapter ligation was used to create the SMRTbell template. Adapter dimers and contaminants were removed using the AMPure XP bead purification system (Beckman Coulter, Cat. No. A63882). A BluePippin size selection system (Sage Science, Cat. No. BLU0001) was used to size select the SMRTbell template and enrich for fragments > 15 kb. Sequencing primer v4 was annealed to the SMRTbell template, and a DNA polymerase/template complex was created using the Sequel Binding Kit 2.1 (Pacific Biosciences, Cat. No. 101-365-900). An additional AMPure XP purification step was performed to remove excess primer and polymerase prior to sequencing. The library was sequenced on a Sequel instrument using SMRT Cell 1M v2 (Pacific Biosciences), taking one movie of 10 hours per cell with the Sequel Sequencing Kit 2.0 (Pacific Biosciences).

The extracted PRV FJ DNA samples were sent to Wuhan BaiYi biotechnology company for complete genome sequencing. After the samples were qualified, the database was built with the PRV HLJ8 strain (National Center of Biotechnology Information [NCBI] accession number: KT824771.1) as the reference sequence. Third- and second-generation high-throughput sequencing was carried out using a PacBio RS II sequencing system and a MGISEQ-2000 sequencing system, respectively. For the PacBio RS II system, the Sequel Binding Kit 2.1, the Sequel Sequencing Kit 2.1, and the Sequel SMRT Cell 1mv2 (Pacific Biosciences, Menlo Park, CA, USA) were used for sequencing. The data were processed with the SMRT LINK 6.0 software. The read quality value in the original data was filtered. Based on the complete genome sequencing using the PacBio equipment, the obtained sequence was corrected using the MGISEQ-2000 s-generation sequencing platform. Finally, the complete PRV FJ genome sequence was assembled and annotated.