Long-read sequencing uncovers a complex transcriptome topology in varicella zoster virus

Abstract

BackgroundVaricella zoster virus (VZV) is a human pathogenic alphaherpesvirus harboring a relatively large DNA molecule. The VZV transcriptome has already been analyzed by microarray and short-read sequencing analyses. However, both approaches have substantial limitations when used for structural characterization of transcript isoforms, even if supplemented with primer extension or other techniques. Among others, they are inefficient in distinguishing between embedded RNA molecules, transcript isoforms, including splice and length variants, as well as between alternative polycistronic transcripts. It has been demonstrated in several studies that long-read sequencing is able to circumvent these problems.ResultsIn this work, we report the analysis of the VZV lytic transcriptome using the Oxford Nanopore Technologies sequencing platform. These investigations have led to the identification of 114 novel transcripts, including mRNAs, non-coding RNAs, polycistronic RNAs and complex transcripts, as well as 10 novel spliced transcripts and 25 novel transcription start site isoforms and transcription end site isoforms. A novel class of transcripts, the nroRNAs are described in this study. These transcripts are encoded by the genomic region located in close vicinity to the viral replication origin. We also show that the ORF63 exhibits a complex structural variation encompassing the splice sites of VZV latency transcripts. Additionally, we have detected RNA editing in a novel non-coding RNA molecule.ConclusionsOur investigations disclosed a composite transcriptomic architecture of VZV, including the discovery of novel RNA molecules and transcript isoforms, as well as a complex meshwork of transcriptional read-throughs and overlaps. The results represent a substantial advance in the annotation of the VZV transcriptome and in understanding the molecular biology of the herpesviruses in general.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5267-8) contains supplementary material, which is available to authorized users.

Multiple Long-read Sequencing Survey of Herpes Simplex Virus Lytic Transcriptome

“…Both raRNAs are long TSS isoform produced the neighboring genes, us1 for OriS, and ul30 for OriL. Similar transcripts have also been recently described in other viruses Prazsák et al, 2018;Tombácz et al, 2015Tombácz et al, , 2018c. Intriguingly, since the replication origin is located at different genomic regions, the sequences of raRNAs are nonhomologous within the subfamily of Alphaherpesvirinae.…”

“…A diverse collection of methods and approaches has already been employed for the investigation of the herpesvirus transcriptomes, including in silico detection of open reading frames (ORFs) and cisregulatory motifs, Northern-blot analysis (Costa et al, 1984;Sedlackova et al, 2008), S1 nuclease mapping (McKnight, 1980;Rixon and Clements, 1982), primer extension (Naito et al, 2005;Perng et al, 2002), real-time reverse transcription-PCR (RT 2 -PCR) analysis (Tombácz et al, 2009), microarrays (Stingley et al, 2000), Illumina sequencing (Harkness et al, 2014;Oláh et al, 2015), PacBio RS II (O'Grady et al, 2016;Tombácz et al, 2016Tombácz et al, , 2017b and Sequel sequencing, as well as ONT MinION sequencing Prazsák et al, 2018).…”

Combined short and long-read sequencing reveals a complex transcriptomic architecture of African swine fever virus

“…RaRNAs have been identified in every examined virus [36]. In herpesviruses, these transcripts can be non-coding or alternatively, the longer TSS or TES variants of mRNAs [37]. In ASFV raRNAs we detected 6 low-abundance raRNA molecules at late infection times (12 to 20 hours), which are encoded at the repeat region ( Fig.…”

Long-read RNA sequencing analysis of the lytic human cytomegalovirus transcriptome

“…Long-read RNA sequencing has effectively characterized the transcriptome of several organisms before (Abdel-Ghany et al, 2016;Moldován et al, 2018;O'Grady et al, 2016;Prazsák et al, 2018;Sharon et al, 2013;Tombácz et al, 2016Tombácz et al, , 2017bWang et al, 2016;Workman et al, 2018). In all of the cases, long-read sequencing has revealed countless novel transcripts and transcript isoforms (Tombácz et al, 2018).…”

“…The source code is open and available online (Balázs, 2018). This toolkit was also used in the analysis of the varicella zoster transcriptome (Prazsák et al, 2018). In order to present a complete picture of the long-read RNA sequencing experiments of our group, all HCMV data have been processed by the LoRTIA software.…”