Bubbles: Alternative Splicing Events of Arbitrary Dimension in Splicing Graphs

  • Michael Sammeth
  • Gabriel Valiente
  • Roderic Guigó
Part of the Lecture Notes in Computer Science book series (LNCS, volume 4955)


Eukaryotic splicing structures are known to involve a high degree of alternative forms derived from a premature transcript by alternative splicing (AS). With the advent of new sequencing technologies, evidence for new splice forms becomes more and more easily available—bit by bit revealing that the true splicing diversity of “AS events” often comprises more than two alternatives and therefore cannot be sufficiently described by pairwise comparisons as conducted in analyzes hitherto. Further challenges emerge from the richness of data (millions of transcripts) and artifacts introduced during the technical process of obtaining transcript sequences (noise)—especially when dealing with single-read sequences known as expressed sequence tags (ESTs). We describe a novel method to efficiently predict AS events in different resolutions (i.e., dimensions) from transcript annotations that allows for combination of fragmented EST data with full-length cDNAs and can cope with large datasets containing noise. Applying this method to estimate the real complexity of alternative splicing, we found in human thousands of novel AS events that either have been disregarded or mischaracterized in earlier works. In fact, the majority of exons that are observed as “mutually exclusive” in pairwise comparisons truly involve at least one other alternative splice form that disagrees with their mutual exclusion. We identified four major classes that contain such “optional” neighboring exons and show that they clearly differ from each other in characteristics, especially in the length distribution of the middle intron.

General Terms: Alternative Splicing, ESTs, New Sequencing Technologies, Algorithms, Graph Theory.


exon-intron structure splicing variation alternative splicing event expressed sequence tags high-throughput sequencing parallel sequencing directed acyclic graph galled network blob bubble 


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  1. 1.
    The human sequencing consortium. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001)Google Scholar
  2. 2.
    Smith, C.W., Valcarcel, J.: Alternative pre-mrna splicing: the logic of combinatorial control. Annu. Rev. Genet. 25, 381–388 (2000)Google Scholar
  3. 3.
    Lopez, A.J.: Alternative splicing of pre-mrna: developmental consequences and mechanisms of regulation. Annu. Rev. Genet. 32, 279–305 (1998)CrossRefGoogle Scholar
  4. 4.
    Kuyumcu-Martinez, N.M., Cooper, T.A.: Mis-regulation of alternative splicing causes pathogenesis in myotonic dystrophy. Prog. Mol. Subcell. Biol. 44, 133–159 (2006)CrossRefGoogle Scholar
  5. 5.
    Stamm, S., Riethoven, J.J., Le Texier, V., Gopalakrishnan, C., Kumanduri, V., Tang, Y., Barbosa-Morais, N.L., Thanaraj, T.A.: ASD: A bioinformatics resource on alternative splicing. Nucleic Acids Res. 34, D46–55 (2006)CrossRefGoogle Scholar
  6. 6.
    Le Texier, V., Riethoven, J.J., Kumanduri, V., Gopalakrishnan, C., Lopez, F., Gautheret, D., Thanaraj, T.A.: AltTrans: Transcript pattern variants annotated for both alternative splicing and alternative polyadenylation. BMC Bioinformatics 7, 169 (2006)CrossRefGoogle Scholar
  7. 7.
    Dralyuk, I., Brudno, M., Gelfand, M.S., Zorn, M., Dubchak, I.: ASDB: Database of alternatively spliced genes. BMC Bioinformatics 28, 296–297 (2000)Google Scholar
  8. 8.
    Holste, D., Huo, G., Tung, V., Burge, C.B.: HOLLYWOOD: a comparative relational database of alternative splicing. Nucleic Acids Res. 34, D56–62 (2006)CrossRefGoogle Scholar
  9. 9.
    Zhou, Y., Zhou, C., Ye, L., Dong, J., Xu, H., Cai, L., Zhang, L., Wei, L.: Database and analyses of known alternatively spliced genes in plants. Genomics 82, 584–595 (2003)CrossRefGoogle Scholar
  10. 10.
    Coward, E., Haas, S., Vingron, M.: SpliceNest: visualizing gene structure and alternative splicing based on EST clusters. Trends in Genetics 18, 53–55 (2002)CrossRefGoogle Scholar
  11. 11.
    Huang, Y.H., Chen, Y.T., Lai, J.J., Yang, S.T., Yang, U.C.: PALS dbç: Putative alternative splicing database. Nucleic Acids Res. 30, 186–190 (2002)CrossRefGoogle Scholar
  12. 12.
    Burset, M., Seledtsov, I.A., Solovyev, V.V.: SpliceDB: database of canonical and non-canonical mammalian splice sites. Nucleic Acids Res. 29, 255–259 (2001)CrossRefGoogle Scholar
  13. 13.
    Ji, H., Zhou, Q., Wen, F., Xia, H., Lu, X., Li, Y.: AsMamDB: An alternative splice database of mammals. Nucleic Acids Res. 29, 260–263 (2001)CrossRefGoogle Scholar
  14. 14.
    Modrek, B., Resch, A., Grasso, C., Lee, C.: Genome-wide analysis of alternative splicing using human expressed sequence data. Nucleic Acids Res. 29, 2850–2859 (2001)CrossRefGoogle Scholar
  15. 15.
    Huang, H.D., Horng, J.T., Lee, C.C., Liu, B.J.: Prosplicer: A database of putative alternative splicing information derived from protein, mrna and expressed sequence tag sequence data. Genome Biol. 4, R29 (2003)CrossRefGoogle Scholar
  16. 16.
    Bhasi, A., Pandey, R.V., Utharasamy, S.P., Senapathy, P.: ASD: a bioinformatics resource on alternative splicing. Boinformatics 23, 1815–1823 (2007)CrossRefGoogle Scholar
  17. 17.
    Nagasaki, H., Arita, M., Nishizawa, T., Suwa, M., Gotoh, O.: Species-specific variation of alternative splicing and transcriptional initiation in six eukaryotes. Gene 364, 53–62 (2005)CrossRefGoogle Scholar
  18. 18.
    Kim, E., Magen, A., Ast, G.: Different levels of alternative splicing among eukaryotes. Nucleic Acids Res. 35, 125–131 (2007)CrossRefGoogle Scholar
  19. 19.
    Yandell, M., Mungall, C.J., Smith, C., Prochnik, S., Kaminker, J., Hartzell, G., Lewis, G.M., Rubin, S.: Large-scale trends in the evolution of gene structures within 11 animal genomes. PLoS Comput. Biol., vol. 2, p. 15 (2006)Google Scholar
  20. 20.
    Grasso, C., Modrek, B., Xing, Y., Lee, C.: Genome-wide detection of alternative splicing in expressed sequences using partial order multiple sequence alignment graphs. In: Pac. Symp. Biocomput., pp. 29–41 (2004)Google Scholar
  21. 21.
    Zavolan, M., van Nimwegen, E.: The types and prevalence of alternative splice forms. Curr. Opin. Struct. Biol. 16, 1–6 (2006)CrossRefGoogle Scholar
  22. 22.
    Florea, L., Hartzell, G., Zhang, Z., Rubin, G.M., Miller, W.: A computer program for aligning a cDNA sequence with a genomic DNA sequence. Genome Res. 8, 967–974 (1998)Google Scholar
  23. 23.
    Kent, W.J.: BLAT - the blast-like alignment tool. Genome Res. 12, 656–664 (2002)CrossRefMathSciNetGoogle Scholar
  24. 24.
    Bonizzoni, P., Rizzi, R., Pesole, G.: ASPIC: a novel method to predict the exon-intron structure of a gene that is optimally compatible to a set of transcript sequences. BMC Bioinformatics 6, 244 (2005)CrossRefGoogle Scholar
  25. 25.
    Pruitt, K.D., Tatusova, T., Maglott, D.R.: NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 35, D61–D65 (2007)CrossRefGoogle Scholar
  26. 26.
    Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J., Wheeler, D.L.: GenBank. Nucleic Acids Res. 35, D21–D25 (2007)CrossRefGoogle Scholar
  27. 27.
    Weber, A.P., Weber, K.L., Carr, K., C.,, Wilkerson, O.J.B.: Sampling the arabidopsis transcriptome with massively parallel pyrosequencing. Plant Physiol. 144, 32–42 (2007)CrossRefGoogle Scholar
  28. 28.
    Ruan, Y., Ooi, H.S., Choo, S.W., Chiu, K.P., Zhao, X.D., Srinivasan, K.G., Yao, F., Choo, C.Y., Liu, J., Ariyaratne, P., Bin, W.G.W., Kuznetsov, V.A., Shahab, A., Sung, W.-K., Bourque, G., Palanisamy, N., Wei, C.-L.: Fusion transcripts and transcribed retrotransposed loci discovered through comprehensive transcriptome analysis using paired-end ditags (pets). Genome Res. 17, 828–838 (2007)CrossRefGoogle Scholar
  29. 29.
    Sugnet, C.W., Kent, W.J., Ares, M., Haussler, D.: Transcriptome and genome conservation of alternative splicing events in humans and mice. In: Pac. Symp. Biocomput., pp. 66–77 (2004)Google Scholar
  30. 30.
    Heber, S., Alekseyev, M., Sing-Hoi, S., Pevzner, P.: Splicng graphs and EST assembly problem. Bioinformatics 18, 181–188 (2002)Google Scholar
  31. 31.
    Gusfield, D., Bansal, V.: A fundamental decomposition theorem for phylogenetic networks and incompatible characters. In: Miyano, S., Mesirov, J., Kasif, S., Istrail, S., Pevzner, P.A., Waterman, M. (eds.) RECOMB 2005. LNCS (LNBI), vol. 3500, pp. 217–232. Springer, Heidelberg (2005)Google Scholar
  32. 32.
    Gusfield, D., Eddhu, S., Langley, C.: Optimal, efficient reconstruction of phylogenetic networks with constrained recombination. J. Bioinformatics and Computational Biology 2, 173–213 (2004)CrossRefGoogle Scholar
  33. 33.
    University of California Santa Cruz (UCSC) Genome Browser,
  34. 34.
    Boguski, M.S., Lowe, T.M., Tolstoshev, C.M.: dbEST–database for ”expressed sequence tags. Nat. Genet. 4, 332–333 (1993)CrossRefGoogle Scholar
  35. 35.
    Human Genome Sequencing Consortium,
  36. 36.
    Mouse Genome Sequencing Consortium,
  37. 37.
    R Development Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria (2007) ISBN 3-900051-07-0Google Scholar
  38. 38.
    Smith, C.W., Nadal-Ginard, B.: Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing. Cell 56, 749–758 (1989)CrossRefGoogle Scholar
  39. 39.
    Zhuang, Y., Leung, H., Weiner, A.M.: The natural 5’ splice site of simian virus 40 large t antigen can be improved by increasing the base complementarity to u1 rna. Mol. Cell Biol. 7, 3018–3020 (1987)Google Scholar
  40. 40.
    Kuo, H.C., Nasim, F.H., Grabowski, P.J.: Control of alternative splicing by the differential binding of u1 small nuclear ribonucleoprotein particle. Science 251, 1045–1050 (1991)CrossRefGoogle Scholar
  41. 41.
    Mullen, M.P., Smith, C.W.J., Patton, J.G., Nadal-Girnard, B.: α-tropomyosin mutually exclusive exon selection: competition between branchpoint/polypyrimidine tracts determines default exon choice. Genes Dev. 5, 642–655 (1991)CrossRefGoogle Scholar
  42. 42.
    Fu, X.Y., Ge, H., Manley, J.L.: In vitro splicing of mutually exclusive exons from the chicken β-tropomyosin gene: role of the branch point location and very long pyrimidine stretch. EMBO J. 7, 809–817 (1988)Google Scholar
  43. 43.
    Noble, J.C., Pan, Z.Q., Prives, C., Manley, J.L.: Splicing of sv40 early pre-mrna to large t and small t mrnas utilizes different patterns of lariat branch sites. Cell 27, 227–236 (1987)CrossRefGoogle Scholar
  44. 44.
    Noble, J.C., Prives, C., Manley, J.L.: Alternative splicing of sv40 early pre-mrna is determined by branch site selection. Genes Dev. 2, 1460–1475 (1988)CrossRefGoogle Scholar
  45. 45.
    Gattoni, R., Schmitt, P., Stevenin, J.: In vitro splicing of adenovirus e1a transcripts: characterization of novel reactions and of multiple branch points abnormally far from the 3’ splice site. Nucleic Acids Res. 16, 2389–2409 (1988)CrossRefGoogle Scholar
  46. 46.
    Helfman, D.M., Ricci, W.M.: Branch point selection in alternative splicing of tropomyosin pre-mrnas. Nucleic Acids Res. 17, 5633–5650 (1989)CrossRefGoogle Scholar
  47. 47.
    Goux-Pelletan, M., Libri, D., d’Aubenton-Carafa, Y., Fiszman, M., Brody, E., Marie, J.: In vitro splicing of mutually exclusive exons from the chicken β-tropomyosin gene: role of the branch point location and very long pyrimidine stretch. EMBO J. 9, 241–249 (1990)Google Scholar
  48. 48.
    Helfman, D.M., Roscigno, R.F., Mulligan, G.J., Finn, L.A., Weber, K.S.: Identification of two distinct intron elements involved in alternative splicing of the β-tropomyosin pre-mRNA. Genes Dev. 4, 98–110 (1990)CrossRefGoogle Scholar
  49. 49.
    Reed, R., Maniatis, T.: The role of the mammalian branchpoint sequence in pre-mrna splicing. Genes Dev. 2, 1268–1276 (1988)CrossRefGoogle Scholar
  50. 50.
    Zhuang, Y.A., Goldstein, A.M., Weiner, A.M.: Uacuaac is the preferred branch site for mammalian mrna splicing. Proc. Natl. Acad. Sci. USA 86, 2752–2756 (1989)CrossRefGoogle Scholar
  51. 51.
    Libri, D., Goux-Pelletan, M., Brody, E., Fiszman, M.Y.: Exon as well as intron sequences are cis-regulating elements for the mutually exclusive alternative splicing of the β tropomyosin gene. Mol. Cell Biol. 10, 5036–5046 (1990)Google Scholar
  52. 52.
    Reed, R., Maniatis, T.: A role for exon sequences and splice-site proximity in splice-site selection. Cell 46, 681–690 (1986)CrossRefGoogle Scholar
  53. 53.
    Mardon, H.J., Sebastio, G., Baralle, F.E.: A role for exon sequences in alternative splicing of the human fibronectin gene. Nucleic Acids Res. 15, 7725–7733 (1987)CrossRefGoogle Scholar
  54. 54.
    Somasekhar, M.B., Mertz, J.E.: Exon mutations that affect the choice of splice sites used in processing the sv40 late transcripts. Nucleic Acids Res. 13, 5591–5609 (1985)CrossRefGoogle Scholar
  55. 55.
    Helfman, D.M., Ricci, W.M., Finn, L.A.: Alternative splicing of tropomyosin pre-mrnas in vitro and in vivo. Genes Dev. 2, 1627–1638 (1988)CrossRefGoogle Scholar
  56. 56.
    Cooper, T.A., Ordahl, C.P.: Nucleotide substitutions within the cardiac troponin t alternative exon disrupt pre-mrna alternative splicing. Nucleic Acids Res. 17, 7905–7921 (1989)CrossRefGoogle Scholar
  57. 57.
    Hampson, R.K., La Follette, L., Rottman, F.M.: Alternative processing of bovine growth hormone mRNA is influenced by downstream exon sequences. Mol. Cell Biol. 9, 1604–1610 (1989)Google Scholar
  58. 58.
    Streuli, M., Saito, H.: Regulation of tissue-specific alternative splicing: exon-specific cis-elements govern the splicing of leukocyte common antigen pre-mRNA. EMBO J. 8, 787–796 (1989)Google Scholar
  59. 59.
    Black, D.L.: Does steric interference between splice sites block the splicing of a short c-src neuron-specific exon in non-neuronal cells? Genes Dev. 5, 389–402 (1991)CrossRefGoogle Scholar
  60. 60.
    Libri, D., Piseri, A., Fiszman, M.Y.: Exon as well as intron sequences are cis-regulating elements for the mutually exclusive alternative splicing of the β tropomyosin gene. Science 252, 1842–1845 (1991)CrossRefGoogle Scholar
  61. 61.
    Ge, H., Manley, J.L.: A protein factor, asf, controls cell-specific alternative splicing of sv40 early pre-mrna in vitro. cell 13, 25–34 (1990)CrossRefGoogle Scholar
  62. 62.
    Krainer, A.R., Conway, G.C., Kozak, D.: The essential pre-mrna splicing factor sf2 influences 5’ splice site selection by activating proximal sites. Cell 13, 35–42 (1990)CrossRefGoogle Scholar
  63. 63.
    Foissac, S., Sammeth, M.: Astalavista: dynamic and flexible analysis of alternative splicing events in custom gene datasets. Nucleic Acids Res. 35, W297–W299 (2007)CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2008

Authors and Affiliations

  • Michael Sammeth
    • 1
  • Gabriel Valiente
    • 2
  • Roderic Guigó
    • 1
  1. 1.Genome Bioinformatics LabCenter for Genomic RegulationBarcelona 
  2. 2.Algorithms, Bioinformatics, Complexity and Formal Methods Research GroupTechnical University of CataloniaBarcelona 

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