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Updated: Dec 13, 2025

Amplicon Sequencing using the Long-Read Sequencing Technologies
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Methodologies for Transcript Profiling Using Long-Read Technologies.

Spyros Oikonomopoulos1, Anthony Bayega1, Somayyeh Fahiminiya1

  • 1McGill Genome Centre, Department of Human Genetics, McGill University, Montréal, QC, Canada.

Frontiers in Genetics
|August 1, 2020
PubMed
Summary

Long-read RNA sequencing offers advanced transcriptome analysis, enabling detailed study of gene expression and RNA modifications. Despite challenges, these single-molecule technologies provide high-resolution insights into gene function and biological systems.

Keywords:
PacBioRNA-Seqlong readnanoporenext-generation sequencingtranscriptome

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Area of Science:

  • Genomics and Molecular Biology
  • Transcriptomics
  • Next-Generation Sequencing

Background:

  • Next-generation sequencing (NGS) is standard for gene expression profiling.
  • Short-read RNA sequencing has limitations in analyzing complex transcriptomes.
  • Emerging single-molecule, long-read technologies offer new transcriptome analysis capabilities.

Purpose of the Study:

  • To review single-molecule sequencing technologies for high-resolution transcriptome analysis.
  • To highlight advancements in studying RNA species, gene function, and the epi-transcriptome.
  • To discuss current challenges and future directions in long-read RNA sequencing.

Main Methods:

  • Focus on single-molecule, long-read RNA sequencing technologies (PacBio, ONT).
  • Integration of single-cell technologies with perturbation tools for high-resolution RNA analysis.
  • Direct RNA sequencing for epi-transcriptome analysis.

Main Results:

  • Long-read technologies facilitate de novo transcriptome assembly and isoform quantification.
  • Enables in-depth analysis of complete RNA species, including non-poly-A transcripts.
  • Provides new avenues for understanding gene function at various biological levels.

Conclusions:

  • Single-molecule, long-read RNA sequencing is transforming transcriptome characterization.
  • Advancements allow detailed analysis of RNA modifications and their biological impact.
  • Ongoing challenges include library preparation and bioinformatics for accurate transcript identification.