基于扩增子的牛结节性皮肤病病毒全基因组Nanopore测序方法的建立OA

To establish a simple and reliable whole-genome sequencing(WGS)method for lumpy skin disease virus(LSDV)and to facilitate timely surveillance of circulating strains,we designed and optimized 23 pairs of amplicon primers targeting the central genomic regions of LSDV,together with one pair of primers spanning the 5′ and 3′ termini,to ensure full genome coverage.PCR amplicons were verified by 1%agarose gel electrophoresis and subsequently sequenced using Nanopore sequencing.Sequencing reads were assembled and analyzed with bioinformatics tools,and the accuracy of the Nanopore sequencing approach was validated by second-generation sequencing.To infer the ancestry of the LSDV strains obtained from four samples and to elucidate their phylogenetic relationships,the assembled whole genomes were aligned with 49 representative LSDV genomes,and a WGS-based phylogenetic tree was constructed.The results showed that an amplicon-based Nanopore WGS method for LSDV was successfully established.Genome coverage for the four LSDV-positive samples exceeded 96.00%,with concordance reaching 99.99%compared with second-generation sequencing.The method enabled successful genome assembly even at relatively low sequencing depth and demonstrated clear advantages in resolving repetitive regions and structural variations.Whole-genome phylogenetic analysis indicated that all four isolates belonged to Clade 2.5.In conclusion,an amplicon-based Nanopore WGS method for LSDV was successfully developed.This approach is simple to operate and yields reliable results,providing technical support for the prevention and control of lumpy skin disease(LSD)and for molecular epidemiological studies in China.