Spri te library system

The extracted RNA was used as a template for cDNA synthesis with the cDNA Synthesis System (Roche, Mannheim, Germany), and fragmented with a Covaris M220 Focused-ultrasonicator (Covaris, Brighton, UK) applying a target size of 500 bp. The fragmented cDNA was then transformed to barcoded libraries using Illumina compatible adapters (Bioo Scientific Corp., Austin, TX, USA) on a SPRI-TE library system (Beckman Coulter) with SPRIworks Fragment Library Cartridge II (for Roche FLX DNA sequencer; Beckman Coulter) without size selection. Upper and lower size selection was done manually with Agencourt AMPure XP magnetic beads (Beckman Coulter), for a target peak size of 670 bp. The obtained libraries were quality checked with a Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany) and quantified using the Kapa Library Quantification Kit for Illumina (Kapa Biosystems, Cape Town, South Africa) on a Bio-Rad CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA). Sequencing was done with an Illumina MiSeq instrument with MiSeq reagent Kit v3 in 2x300bp PE mode (Illumina, San Diego, CA, USA).

For whole genome sequencing, viral RNA served as a template for double-stranded cDNA synthesis using the cDNA Synthesis System (Roche, Mannheim, Germany). In summary, RNA was hybridized with random hexanucleotides and subsequently, first strand and second strand syntheses were performed according to the manufacturer’s instructions (Roche, Mannheim, Germany). The Covaris M220 ultrasonicator was used for DNA fragmentation of 0.5-1 µg of double-stranded DNA to an average size of about 300 base-pairs. For library preparation of the fragmented DNA, Illumina adaptors (Biooscientific, Austin, USA) and SPRIworks Fragment Library Cartridge II (Beckman Coulter, USA) were used on a SPRI-TE library system (Beckman Coulter, Fullerton, USA) with manual size selection afterwards. Upper and lower size selection was performed using AMPure XP magnetic beads (Beckmann Coulter). The quality of the library was checked on a Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany) using a High Sensitivity DNA Chip and corresponding reagents. Quantity was determined via qPCR with Kapa Library Quantification Kit (Kapa Biosystems, Cape Town, South Africa). Paired-end sequencing was performed on an Illumina MiSeq using MiSeq reagent kit v3 (Illumina, San Diego, USA). Raw sequence data was analyzed and assembled using the Genome Sequencer software suite (v. 2.8, Roche).

Full-length sequences of the PCR products obtained from each cloned cDNA were determined using Ion Plus fragment library kit (Life technologies) and sequenced by the Ion PGM platform (Life technologies) or by using a Miseq instrument (Illumina) with libraries constructed by the SPRI-TE library system (Beckman Coulter) using Illumina indices on a SPRIworks Fragment Library Cartridge II (Beckman Coulter). Sequencing data were assembled by the Newbler de novo assembler (Roche) and mapped to the CSFV Roesrath reference sequence (GU233734) by the BWA aligner using the BWASW algorithm [18 (link)] and processed by Samtools [19 (link)]. Consensus sequences of all clones were aligned using the MAFFT algorithm in Geneious R7.

cDNA was generated from total RNA with the cDNA synthesis system kit (Roche, Mannheim, Germany) and random hexamer primers (Roche) according to the Genome Sequencer RNA rapid library preparation manual (Roche). The resulting cDNA was fragmented to a target size of 300 bp using a Covaris M220 instrument (Covaris, Brighton, United Kingdom) and subsequently transformed to barcoded sequencing libraries using Illumina compatible adapters (Bioo Scientific Corp., Austin, USA) on a SPRI-TE library system (Beckman Coulter) with SPRIworks Fragment Library Cartridge II (Beckman Coulter) without size selection. After manual size selection with Agencourt AMPure XP magnetic beads (Beckman Coulter) for a target peak size of 350 bp, library quality was assessed using a Bioanalyzer 2100 instrument (Agilent Technologies, Böblingen, Germany) with a High Sensitivity DNA kit (Agilent Technologies). Finally, the libraries were quantified with the KAPA Library Quantification Kit for Illumina (Kapa Biosystems, Cape Town, South Africa) on a CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, USA) and sequenced using the Illumina MiSeq instrument with MiSeq reagent kit v2 (Illumina, San Diego, USA) in 2 x 250 bp mode.