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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Genet. | doi: 10.3389/fgene.2019.00834

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

 Dóra Tombácz1, Zsolt Balázs1,  Gábor Gulyás1, Miklós Boldogkői1,  Zsolt Csabai1, Michael Snyder2 and  Zsolt Boldogkői3*
  • 1Department of Medical Biology, Faculty of Medicine, University of Szeged, Hungary
  • 2Departments of Genetics, School of Medicine, Stanford University, United States
  • 3Department of Medical Biology, University of Szeged, Hungary

Long-read sequencing (LRS) has become increasingly important in RNA research due to its strength in resolving complex transcriptomic architectures. In this regard, currently two LRS platforms have demonstrated adequate performance: the Single Molecule Real-Time Sequencing by Pacific Biosciences (PacBio) and the nanopore sequencing by Oxford Nanopore Technologies (ONT). Even though these techniques produce lower coverage and are more error prone than short-read sequencing, they continue to be more successful in identifying polycistronic RNAs, transcript isoforms including splice and transcript end variants, as well as transcript overlaps. Recent reports have successfully applied LRS for the investigation of the transcriptome of viruses belonging to various families. These studies have substantially increased the number of previously known viral RNA molecules. In this work, we used the Sequel and MinION technique from PacBio and ONT, respectively, to characterize the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). In most samples, we analyzed the poly(A) fraction of the transcriptome, but we also performed random oligonucleotide-based sequencing. Besides cDNA sequencing, we also carried out native RNA sequencing. Our investigations identified more than 2,300 previously undetected transcripts, including coding and non-coding RNAs, multi-splice transcripts, as well as polycistronic and complex transcripts. Furthermore, we found previously unsubstantiated transcriptional start sites, polyadenylation sites, and splice sites. A large number of novel transcriptional overlaps were also detected. Random-primed sequencing revealed that each convergent gene pair produces non-polyadenylated read-through RNAs overlapping the partner genes. Furthermore, we identified novel replication-associated transcripts overlapping the HSV-1 replication origins, and novel LAT variants with very long 5’ regions, which are co-terminal with the LAT-0.7kb transcript. Overall, our results demonstrated that the HSV-1 transcripts form an extremely complex pattern of overlaps, and that entire viral genome is transcriptionally active. In most viral genes, if not in all, both DNA strands are expressed.

Keywords: Herpes Simplex Virus, transcript isoforms, direct RNA sequencing, Pacific BioSciences, Oxford Nanopore Technologies, Isoform sequencing

Received: 31 Jan 2019; Accepted: 13 Aug 2019.

Copyright: © 2019 Tombácz, Balázs, Gulyás, Boldogkői, Csabai, Snyder and Boldogkői. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Zsolt Boldogkői, University of Szeged, Department of Medical Biology, Szeged, 6720, Hungary, boldogkoi.zsolt@med.u-szeged.hu