Characterization of the Dynamic Transcriptome of a Herpesvirus with Long-read Single Molecule Real-Time Sequencing

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.

Pervasive differential splicing in Marek’s Disease Virus can discriminate CVI-988 vaccine strain from RB-1B virulent strain in chicken embryonic fibroblasts

“…A number of studies based on high-throughput RNA-sequencing conducted with a number of technologies – including Illumina, Pacific Biosciences and Oxford Nanopore – have been published so far for all subfamilies of herpesviruses: the alphaherpesviruses herpes simplex virus (11, 12) and pseudorabies virus (27, 28); the betaherpesvirus cytomegalovirus (CMV); the gammaherpesvirus Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (13, 14, 29). For several of those, the viral transcriptome has even been characterised for several tissues and biological conditions.…”

“…A much better vantage point might be represented by the viral transcriptome – for one thing, it has already been shown in the past that a number of herpesviruses have an unsuspectedly rich repertoire of RNAs (11–14). Detailed knowledge of the viral transcriptomes can also potentially shed a much better light on the different mechanisms taking place during virus-host interaction.…”

Analysis of Alternative Splicing and Alternative Polyadenylation in Populus alba var. pyramidalis by Single-Molecular Long-Read Sequencing

“…Single-molecule real-time (SMRT) sequencing, also called the third-generation sequencing technology developed by Pacific Biosciences (PacBio), allows direct sequencing of full-length complementary DNA (cDNA) sequences and avoids the transcriptome assembly that is required for short-read sequencing (Sharon et al, 2013;Peng et al, 2016;Tombacz et al, 2017). Use of SMRT sequencing permits efficient analysis of exon-intron structure and accurate identification of full-length splice isoforms and APA sites, thus facilitating a complete understanding of the transcriptome diversity.…”

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

“…The PacBio approach is based on single-molecule real-time (SMRT) technology, while the ONT platform utilizes the nanopore sequencing concept. Both techniques have already been applied for the transcriptomic analysis of various organisms (Byrne et al, 2017;Chen et al, 2017;Cheng et al, 2017;Li et al, 2018;Nudelman et al, 2018;Wen et al, 2018;Zhang et al, 2018), including viruses, such as herpesviruses Depledge et al, 2018;Moldován et al, 2017b;O'Grady et al, 2016;Tombácz et al, 2015Tombácz et al, , 2016Tombácz et al, , 2017bTombácz et al, , 2017aTombácz et al, , 2018d, poxviruses (Tombácz et al, 2018c), baculoviruses , retroviruses and circoviruses .…”