Moldován Norbert, Tombácz Dóra, Szűcs Attila, Csabai Zsolt, Snyder Michael, Boldogkői Zsolt
Department of Medical Biology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
Department of Genetics, School of Medicine, Stanford University, Stanford, CA, United States.
Front Microbiol. 2018 Jan 22;8:2708. doi: 10.3389/fmicb.2017.02708. eCollection 2017.
Third-generation sequencing is an emerging technology that is capable of solving several problems that earlier approaches were not able to, including the identification of transcripts isoforms and overlapping transcripts. In this study, we used long-read sequencing for the analysis of pseudorabies virus (PRV) transcriptome, including Oxford Nanopore Technologies MinION, PacBio RS-II, and Illumina HiScanSQ platforms. We also used data from our previous short-read and long-read sequencing studies for the comparison of the results and in order to confirm the obtained data. Our investigations identified 19 formerly unknown putative protein-coding genes, all of which are 5' truncated forms of earlier annotated longer PRV genes. Additionally, we detected 19 non-coding RNAs, including 5' and 3' truncated transcripts without in-frame ORFs, antisense RNAs, as well as RNA molecules encoded by those parts of the viral genome where no transcription had been detected before. This study has also led to the identification of three complex transcripts and 50 distinct length isoforms, including transcription start and end variants. We also detected 121 novel transcript overlaps, and two transcripts that overlap the replication origins of PRV. Furthermore, analysis revealed 145 upstream ORFs, many of which are located on the longer 5' isoforms of the transcripts.
第三代测序是一项新兴技术，能够解决早期方法无法解决的几个问题，包括转录本异构体和重叠转录本的鉴定。在本研究中，我们使用长读长测序分析伪狂犬病病毒（PRV）转录组，包括牛津纳米孔技术公司的MinION、PacBio RS-II和Illumina HiScanSQ平台。我们还使用了之前短读长和长读长测序研究的数据来比较结果，并确认所获得的数据。我们的研究鉴定出19个以前未知的假定蛋白质编码基因，所有这些基因都是先前注释的较长PRV基因的5'截短形式。此外，我们检测到19个非编码RNA，包括没有可读框的5'和3'截短转录本、反义RNA，以及由病毒基因组中以前未检测到转录的部分编码的RNA分子。本研究还鉴定出三个复杂转录本和50种不同长度的异构体，包括转录起始和终止变体。我们还检测到121个新的转录本重叠，以及两个与PRV复制起点重叠的转录本。此外，分析揭示了145个上游开放阅读框，其中许多位于转录本较长的5'异构体上。
