<p>Oligonucleotide primers used in this study.</p>

ASFV transcription is particularly complex because of its unique host-independent transcription system. Using nanopore sequencing technology, we accurately identified the viral transcription start and termination sites.

Combining direct RNA sequencing with experimental verification strongly supports the presence of long multicistronic RNAs. By combining this information with the viral genome annotation, we identified six novel transcriptional genes with potential functions. Furthermore, we observed transcriptional “read-through” events, in which transcription initiated at a common start site continues beyond typical termination sites, producing multiple RNA transcripts of different lengths.