2.4. Nanopore Sequencing and Bioinformatics

All samples were sequenced in multiplex on a nanopore MinION Mk1B device in veterinary labs in Ukraine (NSC IECVM in Kharkiv; SSRILDVSE and NAAS IVM in Kyiv), using an end-ligation (SQK-LSK109) with a native barcoding (EXP-NBD104) protocol according to the manufacturer’s instructions (ONT: Oxford Nanopore Technologies, Oxford, UK). We basecalled and demultiplexed the raw sequence reads using Guppy version 3.6 (ONT). To generate consensus sequences from the raw reads, and discover mixed genotype co-infections, we took a competitive reference-based assembly approach. We filtered, binned, and competitively mapped ORF2 reads to a subset of 66 PCV2 ORF2 sequences, with at least one known references from genotypes a, b, d, f, and g ORF2 sequences, with minimap2 v2.22 [34] to generate consensus genomes. For additional detailed description of bioinformatics methods for consensus genome assembly, see Supplementary Materials.