Kvisgaard, Lise Kirstine1; Hjulsager, Charlotte Kristiane1; Fahnøe, Ulrik4; Breum, Solvej Østergaard5; Ait-Ali, Tahar6; Larsen, Lars Erik1
1 National Veterinary Institute, Technical University of Denmark2 Section for Virology, National Veterinary Institute, Technical University of Denmark3 Section for Public sector service and commercial diagnostics, National Veterinary Institute, Technical University of Denmark4 Molecular Evolution, Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark5 National Food Institute, Technical University of Denmark6 University of Edinburgh
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.
Journal of Virological Methods, 2013, Vol 193, Issue 2, p. 697-705
PRRSV; Full genome sequencing; Next generation sequencing; Illumina HiSeq2000; Roche 454 FLX; Ion Torrent PGM sequencer