title: A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2.
abstract: PRRSV is a positive-sense RNA virus with a high degree of genetic variability among isolates. For diagnostic sensitivity and vaccine design it is essential to monitor PRRSV genetic diversity. However, to date only a few full genome sequences of PRRSV isolates have been made publicly available. In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally.
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark. Electronic address: likik@vet.dtu.dk.
authors.forename: Lise K
authors.initials: LK
authors.lastname: Kvisgaard
authors.forename: Charlotte K
authors.initials: CK
authors.lastname: Hjulsager
authors.forename: Ulrik
authors.initials: U
authors.lastname: Fahnøe
authors.forename: Solvej Ø
authors.initials: SØ
authors.lastname: Breum
authors.forename: Tahar
authors.initials: T
authors.lastname: Ait-Ali
authors.forename: Lars E
authors.initials: LE
authors.lastname: Larsen
mesh_terms: D000818:Animals; D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000821:virology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013552:Swine; D014773:Virology / Q000379:methods*
chemical_list: D012367:RNA, Viral
keywords: Full genome sequencing; Illumina HiSeq2000; Ion Torrent PGM sequencer; Next generation sequencing; PRRSV; Roche 454 FLX