Skip to main content
SpringerLink
Log in

Transcriptomics Using Next Generation Sequencing Technologies

  • Protocol
  • First Online:
Xenopus Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 917))

Abstract

Next generation sequencing technologies may now be applied to the study of transcriptomics. RNA-Seq or RNA sequencing employs high-throughput sequencing of complementary DNA fragments delivering a transcriptional profile. In this chapter, we aim to provide a starting point for Xenopus researchers planning on starting an RNA-Seq transcriptomics study. We begin by providing a section on template isolation and library preparation. The next section comprises the main bioinformatics procedures that need to be performed for raw data processing, normalization, and differential gene expression. Finally, we have included a section on studying deep sequencing results in Xenopus, which offers general guidance as to what can be done in this model.

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

Access this chapter

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 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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

Institutional subscriptions

References

  1. Metzker ML (2010) Sequencing technologies–the next generation. Nat Rev Genet 11:31–46

    Article  PubMed  CAS  Google Scholar 

  2. Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M, Snyder M (2008) The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320:1344–1349

    Article  PubMed  CAS  Google Scholar 

  3. Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, Boutell JM, Bryant J, Carter RJ, Keira Cheetham R, Cox AJ, Ellis DJ, Flatbush MR, Gormley NA, Humphray SJ, Irving LJ, Karbelashvili MS, Kirk SM, Li H, Liu X, Maisinger KS, Murray LJ, Obradovic B, Ost T, Parkinson ML, Pratt MR, Rasolonjatovo IM, Reed MT, Rigatti R, Rodighiero C, Ross MT, Sabot A, Sankar SV, Scally A, Schroth GP, Smith ME, Smith VP, Spiridou A, Torrance PE, Tzonev SS, Vermaas EH, Walter K, Wu X, Zhang L, Alam MD, Anastasi C, Aniebo IC, Bailey DM, Bancarz IR, Banerjee S, Barbour SG, Baybayan PA, Benoit VA, Benson KF, Bevis C, Black PJ, Boodhun A, Brennan JS, Bridgham JA, Brown RC, Brown AA, Buermann DH, Bundu AA, Burrows JC, Carter NP, Castillo N, Chiara ECM, Chang S, Neil Cooley R, Crake NR, Dada OO, Diakoumakos KD, Dominguez-Fernandez B, Earnshaw DJ, Egbujor UC, Elmore DW, Etchin SS, Ewan MR, Fedurco M, Fraser LJ, Fuentes Fajardo KV, Scott Furey W, George D, Gietzen KJ, Goddard CP, Golda GS, Granieri PA, Green DE, Gustafson DL, Hansen NF, Harnish K, Haudenschild CD, Heyer NI, Hims MM, Ho JT, Horgan AM, Hoschler K, Hurwitz S, Ivanov DV, Johnson MQ, James T, Huw Jones TA, Kang GD, Kerelska TH, Kersey AD, Khrebtukova I, Kindwall AP, Kingsbury Z, Kokko-Gonzales PI, Kumar A, Laurent MA, Lawley CT, Lee SE, Lee X, Liao AK, Loch JA, Lok M, Luo S, Mammen RM, Martin JW, McCauley PG, McNitt P, Mehta P, Moon KW, Mullens JW, Newington T, Ning Z, Ling Ng B, Novo SM, O’Neill MJ, Osborne MA, Osnowski A, Ostadan O, Paraschos LL, Pickering L, Pike AC, Chris Pinkard D, Pliskin DP, Podhasky J, Quijano VJ, Raczy C, Rae VH, Rawlings SR, Chiva Rodriguez A, Roe PM, Rogers J, Rogert Bacigalupo MC, Romanov N, Romieu A, Roth RK, Rourke NJ, Ruediger ST, Rusman E, Sanches-Kuiper RM, Schenker MR, Seoane JM, Shaw RJ, Shiver MK, Short SW, Sizto NL, Sluis JP, Smith MA, Ernest Sohna Sohna J, Spence EJ, Stevens K, Sutton N, Szajkowski L, Tregidgo CL, Turcatti G, Vandevondele S, Verhovsky Y, Virk SM, Wakelin S, Walcott GC, Wang J, Worsley GJ, Yan J, Yau L, Zuerlein M, Mullikin JC, Hurles ME, McCooke NJ, West JS, Oaks FL, Lundberg PL, Klenerman D, Durbin R, Smith AJ (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456:53–59

    Article  PubMed  CAS  Google Scholar 

  4. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, Di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652

    Article  PubMed  CAS  Google Scholar 

  5. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63

    Article  PubMed  CAS  Google Scholar 

  6. Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y (2008) RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res 18:1509–1517

    Article  PubMed  CAS  Google Scholar 

  7. Li P, Ponnala L, Gandotra N, Wang L, Si Y, Tausta SL, Kebrom TH, Provart N, Patel R, Myers CR, Reidel EJ, Turgeon R, Liu P, Sun Q, Nelson T, Brutnell TP (2010) The developmental dynamics of the maize leaf transcriptome. Nat Genet 42:1060–1067

    Article  PubMed  CAS  Google Scholar 

  8. Velculescu VE, Zhang L, Vogelstein B, Kinzler KW (1995) Serial analysis of gene expression. Science 270:484–487

    Article  PubMed  CAS  Google Scholar 

  9. Lu C, Meyers BC, Green PJ (2007) Construction of small RNA cDNA libraries for deep sequencing. Methods 43:110–117

    Article  PubMed  Google Scholar 

  10. Mortazavi A, Williams BA, Mccue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628

    Article  PubMed  CAS  Google Scholar 

  11. Morin R, Bainbridge M, Fejes A, Hirst M, Krzywinski M, Pugh T, McDonald H, Varhol R, Jones S, Marra M (2008) Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. Biotechniques 45:81–94

    Article  PubMed  CAS  Google Scholar 

  12. Akkers RC, van Heeringen SJ, Jacobi UG, Janssen-Megens EM, Françoijs K-J, Stunnenberg HG, Veenstra GJC (2009) A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial gene regulation in Xenopus embryos. Dev Cell 17:425–434

    Article  PubMed  CAS  Google Scholar 

  13. Armisen J, Gilchrist MJ, Wilczynska A, Standart N, Miska EA (2009) Abundant and dynamically expressed miRNAs, piRNAs, and other small RNAs in the vertebrate Xenopus tropicalis. Genome Res 19:1766–1775

    Article  PubMed  CAS  Google Scholar 

  14. Lau NC, Ohsumi T, Borowsky M, Kingston RE, Blower MD (2009) Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi. EMBO J 28:2945–2958

    Article  PubMed  CAS  Google Scholar 

  15. Robine N, Lau NC, Balla S, Jin Z, Okamura K, Kuramochi-Miyagawa S, Blower MD, Lai EC (2009) A broadly conserved pathway generates 3’UTR-directed primary piRNAs. Curr Biol 19:2066–2076

    Article  PubMed  CAS  Google Scholar 

  16. Faunes, F., Sanchez, N., Moreno, M., Olivares, G. H., Lee-Liu, D., Almonacid, L., Slater, A. W., Norambuena, T., Taft, R. J., Mattick, J. S., Melo, F., and Larrain, J. (2011) Expression of transposable elements in neural tissues during Xenopus development, PLoS ONE 6, e22569

    Google Scholar 

  17. Hellsten U, Harland RM, Gilchrist MJ, Hendrix D, Jurka J, Kapitonov V, Ovcharenko I, Putnam NH, Shu S, Taher L, Blitz IL, Blumberg B, Dichmann DS, Dubchak I, Amaya E, Detter JC, Fletcher R, Gerhard DS, Goodstein D, Graves T, Grigoriev IV, Grimwood J, Kawashima T, Lindquist E, Lucas SM, Mead PE, Mitros T, Ogino H, Ohta Y, Poliakov AV, Pollet N, Robert J, Salamov A, Sater AK, Schmutz J, Terry A, Vize PD, Warren WC, Wells D, Wills A, Wilson RK, Zimmerman LB, Zorn AM, Grainger R, Grammer T, Khokha MK, Richardson PM, Rokhsar DS (2010) The genome of the Western clawed frog Xenopus tropicalis. Science 328:633–636

    Article  PubMed  CAS  Google Scholar 

  18. Gilchrist MJ, Zorn AM, Voigt J, Smith JC, Papalopulu N, Amaya E (2004) Defining a large set of full-length clones from a Xenopus tropicalis EST project. Dev Biol 271:498–516

    Article  PubMed  Google Scholar 

  19. Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Church DM, DiCuccio M, Edgar R, Federhen S, Helmberg W, Kenton DL, Khovayko O, Lipman DJ, Madden TL, Maglott DR, Ostell J, Pontius JU, Pruitt KD, Schuler GD, Schriml LM, Sequeira E, Sherry ST, Sirotkin K, Starchenko G, Suzek TO, Tatusov R, Tatusova TA, Wagner L, Yaschenko E (2005) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 33:D39–D45

    Article  PubMed  CAS  Google Scholar 

  20. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25

    Article  PubMed  Google Scholar 

  21. Haider S, Ballester B, Smedley D, Zhang J, Rice P, Kasprzyk A (2009) BioMart Central Portal–unified access to biological data. Nucleic Acids Res 37:W23–W27

    Article  PubMed  CAS  Google Scholar 

  22. Roe BA, Ma DP, Wilson RK, Wong JF (1985) The complete nucleotide sequence of the Xenopus laevis mitochondrial genome. J Biol Chem 260:9759–9774

    PubMed  CAS  Google Scholar 

  23. Schuler GD (1997) Pieces of the puzzle: expressed sequence tags and the catalog of human genes. J Mol Med (Berl) 75:694–698

    Article  CAS  Google Scholar 

  24. Sayers EW, Barrett T, Benson DA, Bolton E, Bryant SH, Canese K, Chetvernin V, Church DM, DiCuccio M, Federhen S, Feolo M, Fingerman IM, Geer LY, Helmberg W, Kapustin Y, Landsman D, Lipman DJ, Lu Z, Madden TL, Madej T, Maglott DR, Marchler-Bauer A, Miller V, Mizrachi I, Ostell J, Panchenko A, Phan L, Pruitt KD, Schuler GD, Sequeira E, Sherry ST, Shumway M, Sirotkin K, Slotta D, Souvorov A, Starchenko G, Tatusova TA, Wagner L, Wang Y, Wilbur WJ, Yaschenko E, Ye J (2011) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 39:D38–D51

    Article  PubMed  Google Scholar 

  25. Bowes JB, Snyder KA, Segerdell E, Gibb R, Jarabek C, Noumen E, Pollet N, Vize PD (2008) Xenbase: a Xenopus biology and genomics resource. Nucleic Acids Res 36:D761–D767

    Article  PubMed  CAS  Google Scholar 

  26. McCormick KP, Willmann MR, Meyers BC (2011) Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments. Silence 2:2

    Article  PubMed  CAS  Google Scholar 

  27. Autio R, Kilpinen S, Saarela M, Kallioniemi O, Hautaniemi S, Astola J (2009) Comparison of Affymetrix data normalization methods using 6,926 experiments across five array generations. BMC Bioinformatics 10(Suppl 1):S24

    Article  PubMed  Google Scholar 

  28. Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19:185–193

    Article  PubMed  CAS  Google Scholar 

  29. Irizarry RA, Wu Z, Jaffee HA (2006) Comparison of Affymetrix GeneChip expression measures. Bioinformatics 22:789–794

    Article  PubMed  CAS  Google Scholar 

  30. Barbacioru CC, Wang Y, Canales RD, Sun YA, Keys DN, Chan F, Poulter KA, Samaha RR (2006) Effect of various normalization methods on Applied Biosystems expression array system data. BMC Bioinformatics 7:533

    Article  PubMed  Google Scholar 

  31. Binder H, Preibisch S, Berger H (2010) Calibration of microarray gene-expression data. Methods Mol Biol 576:375–407

    Article  PubMed  CAS  Google Scholar 

  32. Harr B, Schlotterer C (2006) Comparison of algorithms for the analysis of Affymetrix microarray data as evaluated by co-expression of genes in known operons. Nucleic Acids Res 34:e8

    Article  PubMed  Google Scholar 

  33. Millenaar FF, Okyere J, May ST, van Zanten M, Voesenek LA, Peeters AJ (2006) How to decide? Different methods of calculating gene expression from short oligonucleotide array data will give different results. BMC Bioinformatics 7:137

    Article  PubMed  Google Scholar 

  34. Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11:R25

    Article  PubMed  Google Scholar 

  35. Bullard JH, Purdom E, Hansen KD, Dudoit S (2010) Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinformatics 11:94

    Article  PubMed  Google Scholar 

  36. Ro S, Park C, Jin J, Sanders KM, Yan W (2006) A PCR-based method for detection and quantification of small RNAs. Biochem Biophys Res Commun 351:756–763

    Article  PubMed  CAS  Google Scholar 

  37. Ro S, Yan W (2010) Detection and quantitative analysis of small RNAs by PCR. Methods Mol Biol 629:295–305

    PubMed  Google Scholar 

  38. Martello G, Zacchigna L, Inui M, Montagner M, Adorno M, Mamidi A, Morsut L, Soligo S, Tran U, Dupont S, Cordenonsi M, Wessely O, Piccolo S (2007) MicroRNA control of Nodal signalling. Nature 449:183–188

    Article  PubMed  CAS  Google Scholar 

  39. Sive HL, Grainger RM, Harland RM (2000) Early development of Xenopus laevis. A laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  40. Agrawal R, Tran U, Wessely O (2009) The miR-30 miRNA family regulates Xenopus pronephros development and targets the transcription factor Xlim1/Lhx1. Development 136:3927–3936

    Article  PubMed  CAS  Google Scholar 

  41. Watanabe T, Imai H, Minami N (2008) Identification and expression analysis of small RNAs during development. Methods Mol Biol 442:173–185

    Article  PubMed  CAS  Google Scholar 

  42. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760

    Article  PubMed  CAS  Google Scholar 

  43. Li R, Yu C, Li Y, Lam TW, Yiu SM, Kristiansen K, Wang J (2009) SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics 25:1966–1967

    Article  PubMed  CAS  Google Scholar 

  44. Li H, Ruan J, Durbin R (2008) Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 18:1851–1858

    Article  PubMed  CAS  Google Scholar 

  45. Lin H, Zhang Z, Zhang MQ, Ma B, Li M (2008) ZOOM!Zillions of oligos mapped. Bioinformatics 24:2431–2437

    Article  PubMed  CAS  Google Scholar 

  46. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079

    Article  PubMed  Google Scholar 

  47. Szymanski M, Erdmann VA, Barciszewski J (2007) Noncoding RNAs database (ncRNAdb). Nucleic Acids Res 35:D162–D164

    Article  PubMed  CAS  Google Scholar 

  48. Griffiths-Jones S, Moxon S, Marshall M, Khanna A, Eddy SR, Bateman A (2005) Rfam: annotating non-coding RNAs in complete genomes. Nucleic Acids Res 33:D121–D124

    Article  PubMed  CAS  Google Scholar 

  49. He S, Liu C, Skogerbo G, Zhao H, Wang J, Liu T, Bai B, Zhao Y, Chen R (2008) NONCODE v2.0: decoding the non-coding. Nucleic Acids Res 36:D170–D172

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by research grants from FONDECYT (No. 1110400), ICM (No. P09-016-F) (LIA and FM), Center for Aging and Regeneration (CARE), and Millennium Nucleus in Regenerative Biology (MINREB) (DLL, FF, JL). We thank Dr. Mauricio Moreno for providing information on RNA yield from Xenopus embryos.

Author information

Authors and Affiliations

  1. Center for Aging and Regeneration and Millennium Nucleus in Regenerative Biology, Pontificia Universidad Catolica de Chile, Santiago, Chile

    Dasfne Lee-Liu, Fernando Faunes & Juan Larrain

  2. Molecular Bioinformatics Laboratory, Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Catolica de Chile, Santiago, Chile

    Leonardo I. Almonacid

  3. Molecular Bioinformatics Laboratory, Millennium Institute on Immunology and Immunotheraphy, Pontificia Universidad Catolica de Chile, Santiago, Chile

    Francisco Melo

Authors
  1. Dasfne Lee-Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leonardo I. Almonacid
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fernando Faunes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Francisco Melo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juan Larrain
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Juan Larrain .

Editor information

Editors and Affiliations

  1. , Institute of Medical Sciences, University of Aberdeen, FORESTERHILL, ABERDEEN, AB25 2ZD, United Kingdom

    STEFAN HOPPLER

  2. , Department of Biological Science, University of Calgary, University Drive NW 2500, Calgary, T2N 1N4, Canada

    Peter D Vize

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Lee-Liu, D., Almonacid, L.I., Faunes, F., Melo, F., Larrain, J. (2012). Transcriptomics Using Next Generation Sequencing Technologies. In: HOPPLER, S., Vize, P. (eds) Xenopus Protocols. Methods in Molecular Biology, vol 917. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-992-1_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-992-1_18

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-991-4

  • Online ISBN: 978-1-61779-992-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation