RNA-Seq Parental Type 3 Dearing Reovirus
RNA from viral preparations of T1L, T2J, T3D, r1Reovirus, and r2Reovirus were obtained using an RNeasy RNA purification kit (Qiagen). Ten nanograms of viral RNA was used as input for cDNA synthesis using the Clontech SMARTer Stranded Total RNA-Seq Kit v2 (Pico Input, Mammalian) according to the manufacturers instructions. Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled, and sequenced on an Illumina HiSeq3000 with 100bp paired end reads averaging 13 million reads/sample, yielding an average depth of coverage > 1000 reads. Reads were trimmed of adapter sequence using Trimmomatic (version 0.36, http://www.usadellab.org/cms/?page=trimmomatic) using the TruSeq3-PE-2 paired end adapter reference. Trimmed reads from each sample were aligned to all of the parental strain reference sequences using the Burrows-Wheeler Aligner (BWA version 0.7.10-r789, http://bio-bwa.sourceforge.net/). Deduplication was performed with Picard tools (version 1.74(1243), https://broadinstitute.github.io/picard/), and variation was called, again for each sample against all the parental strain references, using the GATK pipelines (version 3.4, https://software.broadinstitute.org/gatk/) HaplotypeCaller with ploidy set to 1 and other default parameters. The resultant Variant Call Files (.vcf) were examined for sample similarity/variation from the parental reference strains.
SUB6256451
T3D-R1 paired to T3D-R2