Abstract
Herpesvirus gene expression is co-ordinately regulated and sequentially ordered during productive infection. The viral genes can be classified into three distinct kinetic groups: immediate-early, early, and late classes. In this study, a massively parallel sequencing technique that is based on PacBio Single Molecule Real-time sequencing platform, was used for quantifying the poly(A) fraction of the lytic transcriptome of pseudorabies virus (PRV) throughout a 12-hour interval of productive infection on PK-15 cells. Other approaches, including microarray, real-time RT-PCR and Illumina sequencing are capable of detecting only the aggregate transcriptional activity of particular genomic regions, but not individual herpesvirus transcripts. However, SMRT sequencing allows for a distinction between transcript isoforms, including length- and splice variants, as well as between overlapping polycistronic RNA molecules. The non-amplified Isoform Sequencing (Iso-Seq) method was used to analyse the kinetic properties of the lytic PRV transcripts and to then classify them accordingly. Additionally, the present study demonstrates the general utility of long-read sequencing for the time-course analysis of global gene expression in practically any organism.
Highlights
The pseudorabies virus (PRV), a neurotropicherpesvirus is an important pathogen of swine and is considered a causative agent of Aujeszky’s disease[1]
Illumina sequencing and real-time RT-PCR are not efficient in the identification of full-length and complex transcripts, and these techniques are only capable of monitoring the gross activity of genes or of particular genomic regions, but are incapable of conveying which isoforms are expressed at a given time, or whether a certain gene is expressed as a monocistronic transcript or as a part of a polycistronic RNA molecule
A PacBio RSII-based polyadenylation sequencing (PA-seq) method was employed to monitor the dynamic profile of the PRV transcriptome on productively infected PK-15 cells
Summary
The pseudorabies virus (PRV), a neurotropic (alpha-)herpesvirus is an important pathogen of swine and is considered a causative agent of Aujeszky’s disease[1]. The aim of this work was twofold: first, we aimed to characterise and classify the PRV transcripts based on their kinetic properties using a novel method, and second, we strived to demonstrate the utility of long-read sequencing for the quantitative analysis of global transcription, which included the profiling of time-varying gene expression on a genome-wide scale. The most significant advantage of long-read sequencing is its capacity for easy identification of length- and splice transcript isoforms This method is capable of distinguishing the various overlapping mono- and polycistronic RNA molecules. Illumina sequencing and real-time RT-PCR are not efficient in the identification of full-length and complex transcripts, and these techniques are only capable of monitoring the gross activity of genes or of particular genomic regions, but are incapable of conveying which isoforms are expressed at a given time, or whether a certain gene is expressed as a monocistronic transcript or as a part of a polycistronic RNA molecule
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