Bigdye xterminator kit

For sequencing, selected samples were prepared by PCR. Reaction mixture filled with water to a final volume of 50 μl per sample was composed using GoTaq® Green Master Mix (2×) (Promega, Madison, USA), 5 μl of 10 μM primers mix and 5 μl DNA. Reaction conditions were as follows: an initial denaturation at 95 °C for 3 min, 45 amplification cycles of denaturation at 95 °C for 30 s, annealing at specific temperature depending on the primers used for 30 s and extension at 72 °C for 45 s, followed by final extension at 72 °C for 5 min (list of primers for exon sequencing is shown in Table 1). Afterwards, PCR products were purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany). DNA concentration and size of PCR product was determined by agarose electrophoresis of 2 μl of each sample. Based on the intensity of bands the samples were diluted from 5 to 50 times and applied for sequencing reaction using BigDye Terminator V3.1 (Applied Biosystems, USA) according to the manufacturer's protocol. The PCR-sequencing product was purified using BigDye X-Terminator kit following the manufacturer's protocol (Applied Biosystems, USA). Further, 30 μl of each purified sample was applied to 96 wells of titration plates and analyzed in 3500 Genetic Analyzer (Applied Biosystems, USA).

Total RNA was extracted from the infected cell monolayer using the High Pure Viral Nucleic Acid Kit (Roche). The full-length cDNA of Seg-7, coding VP7 protein, was obtained by retrotranscription with Superscript II RTase (Invitrogen) using random primers and amplified using Pfu turbo polymerase (Stratagene). Primer used for cDNA amplification were designed from reference sequence of BTV serotype 2 (Genbank accession number JN255868.1), including restriction site sequences specific for KpnI and XhoI (Roche):
FwKpnIvp7_2_5′-ATATGGTACCACGACACTATCGCGGCAAGAG-3′.
RwXhoIvp7_2_5′-ACACCTCGAGTTACTACACATAAGCGGCGC-3′.
The Seg-7 cDNA was cloned into pCR-XL-TOPO (Invitrogen) and sub-cloned into pENTR1A Dual Selection (Invitrogen) according manufacturer instructions. We obtained the pENTR1A-BTV2-VP7 that was verified by restriction analysis and sequenced using the BigDye X Terminator kit and ABI PRISM 3100 sequencer (Applied Biosystems). pENTR1A BTV-2-VP7 was subsequently used to perform homologous recombination reaction to transfer the gene of interest into the N-terminal BaculoDirect™ Vector (Invitrogen), obtaining the recombinant Autographa californica multiple nucleopolyhedrovirus (rAcMNPV_BTV-2_VP7) containing V5 and His tag in N-terminal on BTV-2 VP7, according manufacturer instructions.

All the identified variants were confirmed by Sanger sequencing. We designed primers using Primer3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi) to perform a direct sequencing. The PCR products were purified using AMPure (Agencourt-Beckmann Coulter, Inc., Brea, CA, USA), then, sequenced in both directions using a Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems Foster City, CA, USA). Sequencing products were purified using a Big Dye X-Terminator Kit (Applied Biosystems Foster City, CA, USA) and run on an ABI 3730 Genetic Analyzer (Applied Biosystems Foster City, CA, USA). Amplicon Sequences were compared with the reference sequence (hg19) using Sequencer 5.0 Software (Gene Codes). Primers are available on request. The validated variants were reported in the results depending on whether they were previously reported in the ALS literature, in the non-ALS literature, in population databases (including those reported only in dbSNP), or never reported (novel variants).