Skip to main content
SpringerLink
Log in
Book cover

Gene Expression Analysis pp 121–147Cite as

Transcript Profiling Using Long-Read Sequencing Technologies

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((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.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  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. https://doi.org/10.1038/nrg.2016.49

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1038/nbt.2705

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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. https://doi.org/10.1101/gr.135350.111

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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):1065–1072

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1113/jphysiol.2006.115568

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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. https://doi.org/10.1038/nmeth.2722

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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. https://doi.org/10.1038/nmeth.2714

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  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. 772-763

    PubMed  CAS  Google Scholar 

  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. https://doi.org/10.1371/journal.pone.0157779

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Freeman LA (2013) Cloning full-length transcripts and transcript variants using 5′ and 3′ RACE. Meth Mol Biol (Clifton, NJ) 1027:3–17. https://doi.org/10.1007/978-1-60327-369-5_1

    Article  CAS  Google Scholar 

  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. https://doi.org/10.1038/nbt.2282

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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 2017:16027

    Google Scholar 

  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. https://doi.org/10.1038/srep31602

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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. https://doi.org/10.1038/nmeth.2639

    Article  PubMed  CAS  Google Scholar 

  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. https://doi.org/10.1186/1471-2199-7-3

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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. https://doi.org/10.1128/AEM.71.12.8966-8969.2005

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Wu TD, Watanabe CK (2005) GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21(9):1859–1875. https://doi.org/10.1093/bioinformatics/bti310

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1186/1471-2105-13-238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):e31

    Article  PubMed  PubMed Central  Google Scholar 

  20. Myers TW, Gelfand DH (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry 30(31):7661–7666

    Article  CAS  PubMed  Google Scholar 

  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):2217–2222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1016/j.ab.2006.10.009

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Human Genetics, McGill University and Genome Quebec Innovation Centre, McGill University, Montréal, QC, Canada

    Anthony Bayega, Yu Chang Wang, Spyros Oikonomopoulos, Haig Djambazian, Somayyeh Fahiminiya & Jiannis Ragoussis

  2. Cancer Research Program, The Research Institute of the McGill University Health Centre, Montreal, QC, Canada

    Somayyeh Fahiminiya

  3. Department of Bioengineering, McGill University, Montréal, QC, Canada

    Jiannis Ragoussis

  4. Cancer and Mutagen Unit, King Fahd Center for Medical Research, Department of Biochemistry, Center of Innovation in Personalized Medicine, King Abdulaziz University, Jeddah, Saudi Arabia

    Jiannis Ragoussis

Authors
  1. Anthony Bayega
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yu Chang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Spyros Oikonomopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Haig Djambazian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Somayyeh Fahiminiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jiannis Ragoussis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jiannis Ragoussis .

Editor information

Editors and Affiliations

  1. Division of Geriatrics and Clinical Gerontology, National Institute on Aging, Bethesda, Maryland, USA

    Nalini Raghavachari

  2. Environmental Laboratory, US Army Engineer Research and Development Center, Vicksburg, Mississippi, USA

    Natàlia Garcia-Reyero

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Bayega, A., Wang, Y.C., Oikonomopoulos, S., Djambazian, H., Fahiminiya, S., Ragoussis, J. (2018). Transcript Profiling Using Long-Read Sequencing Technologies. In: Raghavachari, N., Garcia-Reyero, N. (eds) Gene Expression Analysis. Methods in Molecular Biology, vol 1783. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7834-2_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7834-2_6

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7833-5

  • Online ISBN: 978-1-4939-7834-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation