Long amplicon sequencing for improved genetic characterization of African swine fever virus

African Swine Fever Virus (ASFV) causes a transmissible and fatal disease in pigs that is currently devastating global swine production. Efficient and economical collection of genetic data from ASFV field isolates is essential for bio-surveillance, to limit and control its spread, and to better understand ASF disease ecology. Standard genotyping and subtyping of ASFV field isolates is currently limited to a few variable regions within the ASFV genome. However, more extensive sequencing is necessary to better understand ASFV molecular evolution and identify regions relevant to genetic diversity. In this study, we developed a method for rapid and efficient next generation sequencing of approximately 40% of the ASFV genome using long PCR amplification of six different genomic regions. The amplified regions contain all segments currently used for genotyping and additional genes predicted to contribute to ASFV diversity. The primers used for amplification are broadly compatible with published ASFV genomes, permitting their use on relevant ASFV isolates. This methodology provides the enhanced depth of coverage of amplicon-based sequencing while mitigating complications associated with ASFV whole-genome sequencing. Implementation of this methodology could substantially increase the scale of ASFV genetic data collection, which is necessary to effectively monitor and combat this critical agricultural disease.