Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: Identification of a candidate etiologic agent

Kistler, Amy L; Gancz, Ady; Clubb, Susan; Skewes-Cox, Peter; Fischer, Kael; Sorber, Katherine; Chiu, Charles Y; Lublin, Avishai; Mechani, Sara; Farnoushi, Yigal; Greninger, Alexander; Wen, Christopher C; Karlene, Scott B; Ganem, Don; DeRisi, Joseph L

Sample preparation and sequencing 500 ng of total RNA derived from one of the PDD case specimens was linearly amplified via modification of the MesssageAmp aRNA kit (Applied Biosystems/Ambion, Austin, TX). To ensure the amplification of both mRNA and vRNA present in the specimen, T7-tailed random nonamer was mixed in an equimolar ratio with the manufacturer-provided T7-oligo(dT) primer during the 1 st strand synthesis step. The resulting aRNA was next used as input for a modified version of Genomic DNA sample preparation protocol for ultra high-throughput Solexa sequencing (Illumina, Hayward, CA). 400 ng of the input aRNA was reverse-transcribed with reverse transcriptase (Clontech Laboratories, Inc., Mountain View, CA) using a random nonamer tailed with 19 bp of the Solexa Long (5'-CACGACGCTCTTCCGATCTNNNNNNNNN-3') primer sequence (Illumina, Hayward CA). Following termination of reaction, first strand cDNA products were purified from the reaction with Qiagen MinElute spin column (Qiagen USA, Valencia CA). To ensure stringent separation from primers, the MinElute eluate was then filtered through a Microcon YM30 centrifugal filter (Millipore Corp., Billerica, MA). The resulting eluate served as template for 2 nd strand synthesis in a standard Sequenase 2.0 (USB, Cleveland, OH) reaction primed with a random nonamer tailed with 22 bp (5'-GGCATACGA GCTCTTC-CGATCTNNNNNNNNN-3') of the Solexa Short primer sequence (Illumina, Hayward CA). Double-stranded DNA products were separated from primers and very short products through a second Qiagen MinElute spin column run followed by a Microcon YM50 centrifugal filter. This eluate was used as template for 10 cycles of PCR amplification with the full length Solexa L and S primers using KlenTaq LA DNA polymerase mix (Sigma-Aldrich, St. Louis, MO). PCR product was purified from the reaction with a MinElute spin column. Following cluster generation, Solexa sequencing primer was annealed to the flow cell, and 36 cycles of single base pair extensions were performed with image capture using a 1G Genome Analyzer (Illumina, Hayward, CA). The Solexa Pipeline software suite version 0.2.2.6 (Illumina, Hayward, CA) was utilized for base calling from these images. Using software default quality filters, cycles 4-36 were deemed high quality, resulting in a total of 1.4 million 33 mer reads for downstream sequence analyses.