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Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen

Publication

Date: 14th February 2019 | Source: Nature Communications

Authors: Daniel P. Depledge, Kalanghad Puthankalam Srinivas, Tomohiko Sadaoka, Devin Bready, Yasuko Mori, Dimitris G. Placantonakis, Ian Mohr, Angus C. Wilson.

Direct RNA sequencing with Oxford Nanopore Technologies avoids bias that can be introduced using library preparation for conventional RNA sequencing, has better coverage of genic regions, and long reads maintain transcript isoform diversity, including splice variants. Depledge et al. performed direct RNA sequencing to characterise the transcriptome of the RNA virus, herpes simplex virus type 1 (HSV-1), during infection of primary cells. 

Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes.

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