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

Abstract

Viral genomes exhibit a higher gene density and more diversified transcriptome than the host cell. Coding potential is maximized through the use of multiple reading frames, placement of genes on opposing strands, inefficient or modified use of termination signals, and the deployment of complex alternative splicing patterns. As a consequence, detailed characterization of viral transcriptomes by conventional methods can be challenging. Full length native RNA sequencing (nRNA-seq) using nanopore arrays offers an exciting alternative. Individual transcripts are sequenced directly, without the biases inherent to the recoding or amplification steps included in other sequencing methodologies. nRNA-seq simplifies the detection of variation brought about by RNA splicing, use of alternative transcription initiation and termination sites, and other RNA modifications. Here we use nRNA-seq to profile the herpes simplex virus type 1 transcriptome during early and late stages of productive infection of primary cells. We demonstrate the effectiveness of the approach and identify a novel class of intergenic transcripts, including an mRNA that accumulates late in infection that codes for a novel fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L.

Nanopore sequencing: Review of potential applications in functional genomics

“…Direct RNA sequencing (again, it is unfortunate that true RNA sequencing has to add the word "direct" to differentiate itself from conventional "RNA-Seq"), on the other hand, directly detects the ribonucleobases passing through the pore without RT or PCR amplification, and this approach is free of possible biases or misamplifications introduced during such steps. Such an application is also practical for the study of RNA viral genomes, because the genome has multiple reading frames, anti-sense gene locations, inefficient termination signals, and complex splice forms, and gene annotation is challenging using the conventional RNA-seq method (Depledge et al, 2018). Such an application is also practical for the study of RNA viral genomes, because the genome has multiple reading frames, anti-sense gene locations, inefficient termination signals, and complex splice forms, and gene annotation is challenging using the conventional RNA-seq method (Depledge et al, 2018).…”

“…The gene annotation process is simplified by the direct RNA sequencing, and it has therefore allowed the identification of more complex or novel transcript isoforms genome-wide (Byrne et al, 2017;Krizanovic, Echchiki, Roux, & Sikic, 2018), and the ability to differentiate transcript haplotypes as well as to identify 3′ poly (A) tail lengths (Workman et al, 2018). Such an application is also practical for the study of RNA viral genomes, because the genome has multiple reading frames, anti-sense gene locations, inefficient termination signals, and complex splice forms, and gene annotation is challenging using the conventional RNA-seq method (Depledge et al, 2018).…”

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

“…Furthermore, viral transcriptomes have been investigated using nanopore sequencing of cDNA (Moldován et al , 2018aTombácz et al 2017), being subject to bias from reverse transcription and amplification. Other studies used DRS to study the human poly(A) transcriptome (Workman et al 2018) and the transcriptome of DNA viruses such as HSV (Depledge et al 2018). Furthermore, the genome of influenza A virus has been completely sequenced in its original form using DRS (Keller et al 2018).…”

Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes

“…Long-read RNA-Seq has been used to catalog transcript variation through alternative splicing and to identify novel transcripts or transcript isoforms (41,42). More importantly, when combined with short-read RNA-Seq and/or variant approaches such as CAGE-Seq, it enables fine detailing of viral transcriptomes at a very high resolution (43)(44)(45).…”

“…The accurate mapping of sequence reads first requires error correction, a complex proposition, to accurately map the extreme 5= and 3= ends of transcripts, as well as accurately identify sites of splicing. While aligning reads to a reference genome is relatively simple following the development of MiniMap2 (48), custom pipelines are often required to identify transcription start and RNA cleavage sites (44). A visual inspection of the data is crucial for identifying novel genes or splice variants, and users should be particularly aware of sequencing artefacts (signal loss/interruption) on nanopore platforms that can masquerade as excised introns.…”