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Transcript Profiling Using Long-Read Sequencing Technologies

  • Anthony Bayega
  • Yu Chang Wang
  • Spyros Oikonomopoulos
  • Haig Djambazian
  • Somayyeh Fahiminiya
  • Jiannis Ragoussis
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1783)

Abstract

RNA sequencing using next-generation sequencing (NGS, RNA-Seq) technologies is currently the standard approach for gene expression profiling, particularly for large-scale high-throughput studies. NGS technologies comprise short-read RNA-Seq (dominated by Illumina) and long-read RNA-Seq technologies provided by Pacific Bioscience (PacBio) and Oxford Nanopore Technologies (ONT). Although short-read sequencing technologies are the most widely used, long-read technologies are increasingly becoming the standard approach for de novo transcriptome assembly and isoform expression quantification due to the complex nature of the transcriptome which consists of variable lengths of transcripts and multiple alternatively spliced isoforms for most genes. In this chapter, we describe experimental procedures for library preparation, sequencing, and associated data analysis approaches for PacBio and ONT with a major focus on full length cDNA synthesis, de novo transcriptome assembly, and isoform quantification.

Key words

RNA-Seq Long read PacBio Nanopore Next-generation sequencing Transcriptome 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Anthony Bayega
    • 1
  • Yu Chang Wang
    • 1
  • Spyros Oikonomopoulos
    • 1
  • Haig Djambazian
    • 1
  • Somayyeh Fahiminiya
    • 1
    • 2
  • Jiannis Ragoussis
    • 1
    • 3
    • 4
  1. 1.Department of Human Genetics, McGill University and Genome Quebec Innovation CentreMcGill UniversityMontréalCanada
  2. 2.Cancer Research ProgramThe Research Institute of the McGill University Health CentreMontrealCanada
  3. 3.Department of BioengineeringMcGill UniversityMontréalCanada
  4. 4.Cancer and Mutagen Unit, King Fahd Center for Medical Research, Department of Biochemistry, Center of Innovation in Personalized MedicineKing Abdulaziz UniversityJeddahSaudi Arabia

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