ANASFV: a workflow for African swine fever virus whole-genome analysis

African swine fever virus (ASFV) is highly transmissible and can cause up to 100% mortality in pigs. The virus has spread across most regions of Asia and Europe, resulting in the deaths of millions of pigs. A deep understanding of the genetic diversity and evolutionary dynamics of ASFV is necessary to effectively manage outbreaks. Genetic analysis of the ASFV requires sequencing and assembling its genome. Nanopore sequencing is increasingly used for ASFV analysis due to advantages such as long reads and portability. However, applying nanopore sequencing to ASFV genomes faces challenges, notably a higher error rate. Additionally, there is a lack of standardized methods for evaluating genome quality. Furthermore, an increasing number of recombinant isolates between genotypes I and II have been observed, complicating phylogenetic analysis. To overcome these obstacles, we developed ANASFV (analysis of an ASFV whole genome), a comprehensive pipeline that accomplishes four main tasks. First, the pipeline introduces an amplicon sequencing approach that significantly improves genomic coverage, enabling reliable genome assembly, and employs reference-aided polishing techniques to correct small indels caused by nanopore sequencing errors. Second, it establishes a system to provide a relative reference for assessing genome quality in terms of both completeness and accuracy of the assembled genomes. We found that almost all ASFV genomes based solely on nanopore sequencing in the NCBI were of poor quality, which improved significantly after reference-aided polishing. Third, the pipeline introduces a method to rapidly analyse whether an isolate is a recombinant between genotypes I and II, as well as to determine the pattern of recombination based on gene similarity. We identified 11 recombinant ASFV genotypes I and II in the NCBI. Lastly, a comprehensive phylogenetic analysis based on coding sequences was conducted, allowing researchers to generate a refined phylogenetic tree that includes all known ASFV genomes. The ANASFV pipeline facilitates ASFV whole-genome sequencing using the nanopore platform and supports robust downstream bioinformatic analyses by assessing gene completeness and conducting detailed phylogenetic studies based on whole-genome data. © 2025 The Authors.