A Novel and Rapid Selective Viral Genome Amplification and Sequencing Method for African Swine Fever Virus

African swine fever virus (ASFV) is the etiological agent of African swine fever, a highly contagious hemorrhagic disease affecting both wild boars and domestic pigs with lethality rates up to 100%. Until now, the most effective measure to prevent an outbreak of ASFV was early detection. In this situation, whole genome sequencing (WGS) allows the gathering of detailed information about the identity and epidemiology of the virus. However, due to the large genome size and complex genome ends, WGS is challenging. Current WGS workflows require either elaborate enrichment methods or are based on tiled PCR approaches, which are susceptible to genetic differences between ASFV strains. To overcome this, we developed a novel approach for WGS of ASFV, using the Phi29 DNA polymerase-based multiple displacement amplification in combination with only seven primers. Furthermore, we applied an alkaline-based DNA denaturation step to significantly increase the number of viral reads, which resolves the near-full genome of ASFV. This novel isothermal WGS approach can be used in authorized laboratories for the genomic epidemiological analysis of ASFV outbreaks caused by different genotypes.