Nextera xt v2 dna library prep kit

Samples submitted for sequencing were confirmed positive for detectable APPV RNA via qRT-PCR at the KSVDL. Three total samples were submitted for APPV E2 gene sequencing -one serum sample from a piglet with cycle threshold (Ct) value of 26.25 from the farrow-to-finish farm, one semen dose intended for artificial insemination of gilts or sows with Ct value of 30.46, and one serum sample from a replacement gilt housed at the isolation nursery to be moved to the farrow-to-finish farm with Ct value of 27.48. Except for the semen dose, which generated a partial E2 sequence, two samples generated complete E2 sequences. Viral RNA was extracted with the MagMax viral RNA Isolation kit (Thermo Fisher) on a Kingfisher platform. Amplicons were generated from viral RNA using Superscript III One-Step RT-PCR System with Platinum Taq and specific primers, using a 133°F annealing temperature and 30 s extension time. Amplicons were purified using the HighPrep PCR Clean-up System, library prepped by Nextera XT v2 DNA Library prep kit and sequenced on an Illumina Iseq (300-cycle cartridge), as specified by the manufacturer. Raw reads were trimmed for quality and mapped to the closest Genbank reference (UNL082017 #MK728876). Consensus sequences were extracted from the mapping and used for subsequent analysis. All bioinformatics was performed in CLC workbench v20 using default parameters.