Li, K.; Han, X.; Shao, Y.; Zhao, X.; Johnson, E. W.; Li, R.

African Swine Fever Virus (ASFV) poses an ongoing threat with widespread outbreaks affecting both domestic and wild pig populations globally. Effective management of ASFV outbreaks necessitates a deep understanding of its genetic diversity and evolutionary dynamics. Despite the advantages of nanopore sequencing for genome analysis, its application to ASFV genomes encounters specific challenges, such as high host DNA contamination that reduces viral genome coverage and an inherently higher error rate producing small insertions and deletions (indels). To overcome these obstacles, we developed the ANASFV (analysis of an ASFV) pipeline, which enhances both laboratory and bioinformatics workflows for ASFV genome sequencing. This study introduces an amplicon sequencing approach that significantly improves genomic coverage, enabling reliable genome assembly. We also implemented reference-aided polishing techniques to correct small indels caused by nanopore sequencing errors. Additionally, we established a genome quality assessment system, akin to the Benchmarking Universal Single-Copy Orthologue (BUSCO), to evaluate the completeness and accuracy of the assembled genomes. A comprehensive phylogenetic analysis based on coding sequences (CDS) was conducted, allowing us to generate a refined phylogenetic tree that includes all known ASFV genomes. The ANASFV pipeline will facilitate ASFV full genome sequencing using the nanopore platform. It will also support robust downstream bioinformatics analyses to assess genome integrity and conduct detailed phylogenetic studies based on whole-genome data.