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Although adenoviral promoters, splice junctions, and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and RNA cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts that meet stringent criteria for expression. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORFs), six novel ORF-containing transcripts, and 15 transcripts encoding for messages that could alter protein functions through truncation or fusion of canonical ORFs. In addition, we detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking distinct gene transcription units. Among these chimeric proteins we detected an evolutionarily conserved protein containing the N-terminus of E4orf6 fused to the downstream DBP\/E2A ORF. Loss of this novel protein, E4orf6\/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies combined with mass spectrometry can reveal further complexity within viral transcriptomes and resulting proteomes.<\/jats:p>","DOI":"10.1371\/journal.ppat.1010797","type":"journal-article","created":{"date-parts":[[2022,9,12]],"date-time":"2022-09-12T17:47:03Z","timestamp":1663004823000},"page":"e1010797","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":32,"title":["Novel viral splicing events and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts"],"prefix":"10.1371","volume":"18","author":[{"given":"Alexander M.","family":"Price","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert 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