Department of Medical Biology, Faculty of Medicine, University of Szeged, Somogyi B. u. 4., Szeged, H-6720, Hungary.
Department of Genetics, School of Medicine, Stanford University, 300 Pasteur Dr., Stanford, CA 94305-5120, USA.
Sci Rep. 2017 Mar 3;7:43751. doi: 10.1038/srep43751.
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.
疱疹病毒的基因表达在生产性感染过程中是协调调控和有序进行的。病毒基因可分为三个不同的动力学群：早期基因、早期基因和晚期基因。在这项研究中，一种基于 PacBio 单分子实时测序平台的大规模平行测序技术，用于定量测定在 PK-15 细胞上进行生产性感染的 12 小时内，伪狂犬病病毒 (PRV) 裂解转录组的 poly(A) 分数。其他方法，包括微阵列、实时 RT-PCR 和 Illumina 测序，只能检测特定基因组区域的总转录活性，而不能检测单个疱疹病毒转录本。然而，SMRT 测序允许区分转录本异构体，包括长度和剪接变体，以及重叠的多顺反子 RNA 分子。非扩增的异构体测序（Iso-Seq）方法用于分析裂解 PRV 转录本的动力学特性，并相应地对其进行分类。此外，本研究还证明了长读测序在任何生物体的时间进程分析中对全球基因表达的普遍适用性。
