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Multiple Long-Read Sequencing Survey of Herpes Simplex Virus Dynamic Transcriptome.

Dóra Tombácz1, Norbert Moldován1, Zsolt Balázs1

  • 1Department of Medical Biology, Faculty of Medicine, University of Szeged, Szeged, Hungary.

Frontiers in Genetics
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Long-read sequencing revealed over 2,300 new herpes simplex virus type 1 (HSV-1) transcripts, uncovering a complex viral transcriptome with extensive overlaps and full genome activity. This advanced RNA research tool enhances viral gene expression understanding.

Keywords:
Oxford Nanopore TechnologiesPacific Biosciencesdirect RNA sequencingherpes simplex virusherpesviruseslong-read sequencingtranscript isoforms

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Area of Science:

  • Genomics
  • Molecular Biology
  • Virology

Background:

  • Long-read sequencing (LRS) excels at resolving complex transcriptomic architectures, surpassing short-read sequencing for identifying polycistronic RNAs, isoforms, and overlaps.
  • LRS has proven effective in viral transcriptome studies, significantly expanding the known repertoire of viral RNA molecules.
  • Herpes Simplex Virus Type 1 (HSV-1) research benefits from advanced sequencing to elucidate its intricate gene expression.

Purpose of the Study:

  • To characterize the lytic transcriptome of herpes simplex virus type 1 (HSV-1) using long-read sequencing technologies.
  • To identify novel viral transcripts, including coding and non-coding RNAs, and to map previously unknown transcriptional features.
  • To comprehensively analyze the complexity and transcriptional activity of the HSV-1 genome.

Main Methods:

  • Utilized Single Molecule Real-Time Sequencing (PacBio Sequel) and nanopore sequencing (ONT MinION) for LRS.
  • Performed both poly(A)-selected and random-primed sequencing, alongside native RNA sequencing.
  • Analyzed cDNA and native RNA samples to capture a comprehensive view of the HSV-1 transcriptome.

Main Results:

  • Identified over 2,300 previously undetected HSV-1 transcripts, encompassing coding, non-coding, multi-splice, polycistronic, and complex forms.
  • Discovered novel transcriptional start sites, polyadenylation sites, and splice sites, alongside a high number of transcriptional overlaps.
  • Revealed that convergent gene pairs produce overlapping read-through RNAs and identified novel replication-associated transcripts and LAT variants.

Conclusions:

  • The HSV-1 transcriptome exhibits an exceptionally complex pattern of overlapping transcripts, indicating extensive transcriptional activity across the entire viral genome.
  • Both DNA strands of most viral genes are expressed, contributing to the intricate transcriptomic landscape.
  • LRS technologies are crucial for uncovering the full complexity of viral transcriptomes, significantly advancing our understanding of viral biology.