Abstract
Viral genomes have high gene densities and complex transcription strategies rendering transcriptome analysis through short-read RNA-seq approaches problematic. Adenovirus transcription and splicing is especially complex. We used long-read direct RNA sequencing to study adenovirus transcription and splicing during infection. This revealed a previously unappreciated complexity of alternative splicing and potential for secondary initiating codon usage. Moreover, we find that most viral transcripts tend to shorten polyadenylation lengths as infection progresses. Development of an open reading frame centric bioinformatics analysis pipeline provided a deeper quantitative and qualitative understanding of adenovirus’s genetic potential. Across the viral genome adenovirus makes multiple distinctly spliced transcripts that code for the same protein. Over 11,000 different splicing patterns were recorded across the viral genome, most occurring at low levels. This low-level use of alternative splicing patterns potentially enables the virus to maximise its coding potential over evolutionary timescales.
Highlights
Viral genomes have high gene densities and complex transcription strategies rendering transcriptome analysis through short-read RNA-seq approaches problematic
Adenoviruses use of alternative splicing combined with a modest number of promoters and polyadenylation sites makes for a complex transcriptome
Part of this challenge is the large volume of data generated by dRNA-seq
Summary
Viral genomes have high gene densities and complex transcription strategies rendering transcriptome analysis through short-read RNA-seq approaches problematic. We used long-read direct RNA sequencing to study adenovirus transcription and splicing during infection This revealed a previously unappreciated complexity of alternative splicing and potential for secondary initiating codon usage. Nanopore-based direct RNA sequencing (dRNA-seq) avoids these issues because individual mRNA molecules are introduced, poly-A tail first, into the nanopore with their sequences read as they pass through the pore This provides a direct and complete record of the exons present in any given mRNA without reverse transcription or amplification steps. We provide the raw data, software tools and processed data enabling other researchers to interrogate this rich dataset This analysis cements adenovirus’s reputation as probably the most transcriptionally complex virus examined to date, with respect to splice and polyadenylation site usage
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