Data Associated with Poecivirus Testing of Individual Birds with Beak Deformities

We opportunistically obtained specimens with grossly deformed beaks from 1999-2016. Birds were either euthanized after capture, shot, or collected after dying in the wild. Specimens were stored frozen at -20°C until processed.

Total RNA was extracted from beak and cloacal tissues. A total of 200 ng of RNA was reverse transcribed in 10 μl reactions containing 100 pmol random hexamer, 1x reaction buffer, 5 mM dithiothreitol, 1.25 mM (each) deoxynucleoside triphosphates (dNTPs), and 100 U Superscript III (Life Technologies, Carlsbad, CA, USA); mixtures were incubated at 25°C for 5 min, 42°C for 60 min, and 70°C for 15 min. cDNA was screened via polymerase chain reaction (PCR) amplification using Poecivirus-specific primers. PCR mixtures contained 1x iProof master mix (Bio-Rad Laboratories, Hercules, CA, USA), 0.5 M primer, and 5µl cDNA. Thermocycler conditions were set at 98°C for 30s, then 40 cycles of 98°C for 10 s, 58-62°C for 10 s, and 72°C for 30 s, then a 5-min elongation step of 72°C. Annealing temperature was optimized for each set of primers using a gradient cycler. Amplicons were visualized on 1.5% agarose gels; those in the correct size range were extracted and purified using a Zymoclean gel DNA recovery kit (Zymo Research, Irvine, CA, USA) and Sanger sequenced (Quintara Biosciences, South San Francisco, CA, USA). Samples were prepped for high-throughput sequencing using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) following the manufacturer’s protocol. Sequencing was conducted on the Illumina HiSeq 4000 using a single sequencing lane and resulted in 135-nucleotide-long paired-end reads. Additional methodological details are available in Zylberberg et al 2021.