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Next-generation Sequencing03:00

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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
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Updated: Mar 9, 2026

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing
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Characterizing HIV-1 Splicing by Using Next-Generation Sequencing.

Ann Emery1, Shuntai Zhou2, Elizabeth Pollom3

  • 1Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Journal of Virology
|January 13, 2017
PubMed
Summary

Researchers developed a novel Primer ID-tagged deep sequencing assay to quantify human immunodeficiency virus type 1 (HIV-1) splicing. This assay reveals conserved but variable splicing patterns and identifies new regulatory mechanisms for HIV-1 RNA variants.

Keywords:
HIV-1RNA splicingnext-generation sequencingprimer IDsimian immunodeficiency virus

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Area of Science:

  • Virology
  • Molecular Biology
  • Genomics

Background:

  • Human immunodeficiency virus type 1 (HIV-1) generates over 50 spliced RNA variants from its full-length RNA genome, which serves as both the genome and mRNA.
  • These transcripts exist in two size classes (1.8 kb and 4 kb) and are crucial for viral replication and gene expression.

Purpose of the Study:

  • To develop and validate a Primer ID-tagged deep sequencing assay for precise quantification of HIV-1 RNA splicing.
  • To investigate the patterns, regulation, and conservation of HIV-1 splicing across different viral strains and subtypes.

Main Methods:

  • Development of a Primer ID-tagged deep sequencing assay to quantify HIV-1 splicing events.
  • Analysis of splicing patterns in the lab strain NL4-3, subtype B transmitted/founder viruses, a subtype C isolate, and a simian immunodeficiency virus (SIV) isolate.
  • Investigation of temperature sensitivity and structural element disruption on splicing to specific acceptors.

Main Results:

  • The assay identified A5 (env/nef) as the most frequently used splice acceptor (~50%) and A3 (tat) as the least used (~3%) in NL4-3.
  • High usage of donors D2 and D3 significantly reduced vif and vpr transcript levels.
  • Distinct temperature sensitivities were observed for splicing to acceptors A1 and A2, and disruption of a conserved structure near A1 caused a 10-fold decrease in A1-utilized transcripts.
  • Splicing patterns were largely conserved across subtype B viruses but showed variability; subtype C was similar, while SIV differed significantly.
  • Trans-splicing events were detected at low frequency (0.3%), and splicing suppression was observed when the env intron was retained.

Conclusions:

  • The Primer ID-tagged deep sequencing assay is a powerful tool for quantifying HIV-1 splicing with high depth, enabling monitoring of low-frequency splice variants.
  • New mechanisms of HIV-1 splicing regulation have been identified, including the impact of specific donor/acceptor usage, temperature sensitivity, and structural elements.
  • The assay can be utilized to further explore HIV-1 splicing regulation and screen for factors that modulate these conserved splicing patterns.