Dual isoform sequencing reveals complex transcriptomic and epitranscriptomic landscapes of a prototype baculovirus

In this study, two long-read sequencing (LRS) techniques, MinION from Oxford Nanopore Technologies and Sequel from the Pacific Biosciences, were used for the transcriptional characterization of a prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus. LRS is able to read full-length RNA molecules, and thereby distinguish between transcript isoforms, mono- and polycistronic RNAs, and overlapping transcripts. Altogether, we detected 875 transcript species, of which 759 were novel and 116 were annotated previously. These RNA molecules include 41 novel putative protein coding transcripts [each containing 5′-truncated in-frame open reading frames (ORFs), 14 monocistronic transcripts, 99 polygenic RNAs, 101 non-coding RNAs, and 504 untranslated region isoforms. This work also identified novel replication origin-associated transcripts, upstream ORFs, cis-regulatory sequences and poly(A) sites. We also detected RNA methylation in 99 viral genes and RNA hyper-editing in the longer 5′-UTR transcript isoform of the canonical ORF 19 transcript.