Transcript Profiling Using Long-Read Sequencing Technologies

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


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 


  1. 1.
    Goodwin S, McPherson JD, McCombie WR (2016) Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet 17(6):333–351. CrossRefPubMedGoogle Scholar
  2. 2.
    Sharon D, Tilgner H, Grubert F, Snyder M (2013) A single-molecule long-read survey of the human transcriptome. Nat Biotechnol 31(11):1009–1014. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Harrow J, Frankish A, Gonzalez JM, Tapanari E, Diekhans M, Kokocinski F, Aken BL, Barrell D, Zadissa A, Searle S, Barnes I, Bignell A, Boychenko V, Hunt T, Kay M, Mukherjee G, Rajan J, Despacio-Reyes G, Saunders G, Steward C, Harte R, Lin M, Howald C, Tanzer A, Derrien T, Chrast J, Walters N, Balasubramanian S, Pei B, Tress M, Rodriguez JM, Ezkurdia I, van Baren J, Brent M, Haussler D, Kellis M, Valencia A, Reymond A, Gerstein M, Guigo R, Hubbard TJ (2012) GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res 22(9):1760–1774. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Bang ML, Centner T, Fornoff F, Geach AJ, Gotthardt M, McNabb M, Witt CC, Labeit D, Gregorio CC, Granzier H, Labeit S (2001) The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system. Circ Res 89(11):1065–1072CrossRefPubMedGoogle Scholar
  5. 5.
    Gustincich S, Sandelin A, Plessy C, Katayama S, Simone R, Lazarevic D, Hayashizaki Y, Carninci P (2006) The complexity of the mammalian transcriptome. J Physiol 575(Pt 2):321–332. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Engstrom PG, Steijger T, Sipos B, Grant GR, Kahles A, Ratsch G, Goldman N, Hubbard TJ, Harrow J, Guigo R, Bertone P (2013) Systematic evaluation of spliced alignment programs for RNA-seq data. Nat Methods 10(12):1185–1191. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Steijger T, Abril JF, Engstrom PG, Kokocinski F, Hubbard TJ, Guigo R, Harrow J, Bertone P (2013) Assessment of transcript reconstruction methods for RNA-seq. Nat Methods 10(12):1177–1184. CrossRefPubMedGoogle Scholar
  8. 8.
    Hawkins PR, Jin P, Fu GK (2003) Full-length cDNA synthesis for long-distance RT-PCR of large mRNA transcripts. BioTechniques 34(4):768–770. 772-763PubMedGoogle Scholar
  9. 9.
    Cartolano M, Huettel B, Hartwig B, Reinhardt R, Schneeberger K (2016) cDNA library enrichment of full length transcripts for SMRT long read sequencing. PLoS One 11(6):e0157779. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Freeman LA (2013) Cloning full-length transcripts and transcript variants using 5′ and 3′ RACE. Meth Mol Biol (Clifton, NJ) 1027:3–17. CrossRefGoogle Scholar
  11. 11.
    Ramskold D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, Daniels GA, Khrebtukova I, Loring JF, Laurent LC, Schroth GP, Sandberg R (2012) Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol 30(8):777–782. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Byrne A, Beaudin AE, Olsen HE, Jain M, Cole C, Palmer T, DuBois RM, Forsberg EC, Akeson M, Vollmers C (2017) Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells. bioRxiv 2017:16027Google Scholar
  13. 13.
    Oikonomopoulos S, Wang YC, Djambazian H, Badescu D, Ragoussis J (2016) Benchmarking of the Oxford nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations. Sci Rep 6:31602. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Picelli S, Bjorklund AK, Faridani OR, Sagasser S, Winberg G, Sandberg R (2013) Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat Methods 10(11):1096–1098. CrossRefPubMedGoogle Scholar
  15. 15.
    Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7:3–3. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Acinas SG, Sarma-Rupavtarm R, Klepac-Ceraj V, Polz MF (2005) PCR-induced sequence artifacts and bias: insights from comparison of two 16S rRNA clone libraries constructed from the same sample. Appl Environ Microbiol 71(12):8966–8969. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Wu TD, Watanabe CK (2005) GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21(9):1859–1875. CrossRefPubMedGoogle Scholar
  18. 18.
    Chaisson MJ, Tesler G (2012) Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory BMC Bioinformatics 13:238. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Schmidt WM, Mueller MW (1999) CapSelect: a highly sensitive method for 5′ CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs. Nucleic Acids Res 27:21):e31CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Myers TW, Gelfand DH (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30(31):7661–7666CrossRefPubMedGoogle Scholar
  21. 21.
    Boutabout M, Wilhelm M, Wilhelm FX (2001) DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1. Nucleic Acids Res 29(11):2217–2222CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Arezi B, Hogrefe HH (2007) Escherichia coli DNA polymerase III epsilon subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT-PCR procedures. Anal Biochem 360(1):84–91. CrossRefPubMedGoogle Scholar

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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