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RNA-seq03:21

RNA-seq

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Sequencing of mRNA from Whole Blood using Nanopore Sequencing
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In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using Nanopore Sequencing.

Dóra Tombácz1, Balázs Kakuk1, Gábor Torma1

  • 1Department of Medical Biology, Albert Szent-Györgyi Medical School, University of Szeged, Somogyi u. 4, 6720 Szeged, Hungary.

Viruses
|June 24, 2022
PubMed
Summary

This study used direct cDNA sequencing to analyze the bovine alphaherpesvirus type 1 (BoHV-1) transcriptome. Researchers characterized viral gene expression kinetics and identified novel regulatory mechanisms during infection.

Keywords:
bovine alphaherpesvirus type 1direct cDNA sequencingherpesviruseslong-read sequencingnanopore sequencingtranscript isoformstranscription end sitetranscription start sitetranscriptome

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Area of Science:

  • Virology
  • Molecular Biology
  • Genomics

Background:

  • Bovine alphaherpesvirus type 1 (BoHV-1) causes significant disease in cattle.
  • Understanding the BoHV-1 lytic transcriptome is crucial for developing antiviral strategies.
  • Previous transcriptome studies were limited by amplification-based methods causing artifacts.

Purpose of the Study:

  • To kinetically characterize the BoHV-1 lytic transcriptome using an amplification-free long-read sequencing (LRS) method.
  • To investigate transcription start site usage and gene regulation during BoHV-1 infection.
  • To develop a broadly applicable LRS-based method for time-course transcriptome analysis.

Main Methods:

  • Employed direct cDNA sequencing, an amplification-free long-read sequencing (LRS) technique on the Oxford Nanopore MinION platform.
  • Analyzed the poly(A) fraction of the BoHV-1 transcriptome over a 12-hour infection period.
  • Quantified viral gene expression and characterized kinetic profiles.

Main Results:

  • Demonstrated that a single promoter can generate multiple transcription start sites with distinct gene-specific patterns.
  • Revealed that the circ gene exhibits immediate-early (IE) kinetics by co-opting the bicp4 promoter.
  • Observed an overlap between DNA replication initiation and bicp22 gene transcription, suggesting machinery interaction.

Conclusions:

  • Direct cDNA sequencing provides an accurate, amplification-free approach for BoHV-1 transcriptome analysis.
  • Uncovered novel insights into BoHV-1 gene regulation, including promoter usage and kinetic expression patterns.
  • Established a versatile LRS-based methodology applicable to time-course transcriptome studies across various organisms.