Sequencing Six Ontario Virus Genomes

The complete nucleotide sequences of 6 viruses were determined using the 454 GS Junior Titanium platform (Roche Applied Science, Indianapolis, IN, USA) as described by Grgic et al. [21 (link)]. Briefly, RNA of all 6 Ontario 2012 viruses was fragmented by ZnCl₂ into fragments between 500 bp and 1500 bp. Sheared RNA was used for first and second strand cDNA synthesis and for fragment end repair. Double-stranded cDNA purification was performed using AMPure beads and Magnetic Particle Concentrator (MPC). Once the End Repair program was completed the 454 rapid library multiplex identifier (RL MID) was ligated to the fragments according to the GS Junior Titanium cDNA rapid library preparation method (Roche). Quality assessment of the RNA samples was performed using the FlashGel system. Sequence assembly was done with the Newbler (version 2.5p1) de novo sequence assembly software (Roche).