Whole-Genome Sequencing of Bovine Polyomavirus

For BoPyV whole-genome sequencing, conducted at UdelaR, viral genomic DNA was purified from the whole genomic DNA from kidney by extracting the 4–6 kb region of a 1% agarose gel. DNA purity, integrity, and concentration were assessed with a Qubit device (Thermo Fisher Scientific). The sequencing library was prepared with the ligation sequencing kit (SQK-LSK109) following the manufacturer’s instructions and directly sequenced on a FLO-MIN106 flow cell in a MinION device (Oxford Nanopore Technologies^®, Oxford, UK) for 24 h. High-accuracy basecalling was performed with Guppy v3.6.0, and reads were trimmed and filtered with Nanofilt and Nanoplot [37 (link)]. Reads with quality over 10 were used in further analyses. A host filtering step was executed by mapping clean reads to the Bos taurus reference genome (GCF_002263795.1_ARS-UCD1.2_genomic.fna) using Minimap2 [38 (link)]. Unmapped reads were then mapped against the BoPyV-1 reference genome (NC_001442) and the consensus sequence was obtained using SAMtools [39 (link)]. The obtained complete genome sequence was deposited in GenBank. Genome annotation was performed using the BoPyV-1 reference genome (NC_001442).