Differential mRNA Alternative Splicing

  • Albert Lahat
  • Sushma Nagaraja GrellscheidEmail author


Alternative-splicing is a regulated biological phenomenon where one gene can give rise to a variety of transcript isoforms. Most human genes are alternatively spliced. RNA-seq, in contrast to microarrays, has made it possible to analyse not only gene-level differential expression but also isoform-level switches in gene expression using the same raw data. The wet-lab methodology is the same for both analyses, with the exception that calling splicing events requires a higher depth of sequencing coverage. In order to bioinformatically analyse and study splicing using RNA-seq data, a menagerie of tools exist, which have been summarised here.


RNA-seq Alternative splicing Sequencing depth Isoforms 

Supplementary material


  1. Alamancos GP, Agirre E, Eyras E (2014) Methods to study splicing from high-throughput RNA Sequencing data. Methods Mol Biol 1126:357CrossRefPubMedGoogle Scholar
  2. Alamancos GP, Pagès A, Trincado JL, Bellora N, Eyras E (2015) Leveraging transcript quantification for fast computation of alternative splicing profiles. RNA 21:1521. doi: 10.1101/008763 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Ameur A, Wetterbom A, Feuk L, Gyllensten U (2010) Global and unbiased detection of splice junctions from RNA-seq data. Genome Biol 11(3):R34. doi: 10.1186/gb-2010-11-3-r34 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Anders S, Reyes A, Huber W (2012) Detecting differential usage of exons from RNA-seq data. Genome Res 22(10):2008–2017. doi: 10.1101/gr.133744.111 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Anders S, Pyl PT, Huber W (2015) HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166–169CrossRefPubMedPubMedCentralGoogle Scholar
  6. Aschoff M, Hotz-Wagenblatt A, Glatting K-H, Fischer M, Eils R, König R (2013) SplicingCompass: differential splicing detection using RNA-seq data. Bioinformatics 29(9):1141–1148. doi: 10.1093/bioinformatics/btt101 CrossRefPubMedGoogle Scholar
  7. Au KF, Jiang H, Lin L, Xing Y, Wong WH (2010) Detection of splice junctions from paired-end RNA-seq data by SpliceMap. Nucleic Acids Res 38:4570–4578. doi: 10.1093/nar/gkq211 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Bonizzoni P, Della Vedova G, Pesole G, Picardi E, Pirola Y, Rizzi R (2015) Transcriptome assembly and alternative splicing analysis. Methods Mol Biol 1269:173–188CrossRefPubMedGoogle Scholar
  9. Bryant DW, Shen R, Priest HD, Wong W-K, Mockler TC (2010) Supersplat--spliced RNA-seq alignment. Bioinformatics 26(12):1500–1505. doi: 10.1093/bioinformatics/btq206 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chandramohan R, Wu PY, Phan JH, Wang MD (2013) Benchmarking RNA-seq quantification tools. Conf Proc IEEE Eng Med Biol Soc 2013:647–650PubMedGoogle Scholar
  11. Cooper TA, Wan L, Dreyfuss G (2009) RNA and disease. Cell 136(4):777–793. doi: 10.1016/j.cell.2009.02.011 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Dimon MT, Sorber K, DeRisi JL (2010) HMMSplicer: a tool for efficient and sensitive discovery of known and novel splice junctions in RNA-seq data. PLoS One 5:e13875. doi: 10.1371/journal.pone.0013875 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S et al (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15–21. doi: 10.1093/bioinformatics/bts635 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Emig D, Salomonis N, Baumbach J, Lengauer T, Conklin BR, Albrecht M (2010) AltAnalyze and DomainGraph: analyzing and visualizing exon expression data. Nucleic Acids Res 38(Web Server issue):W755–W762. doi: 10.1093/nar/gkq405 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Engström PG, Steijger T, Sipos B, Grant GR, Kahles A, Rätsch G et al (2013) Systematic evaluation of spliced alignment programs for RNA-seq data. Nat Methods 10(12):1185–1191. doi: 10.1038/nmeth.2722 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Florea L, Song L, Salzberg SL (2013) Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues. F1000Research 2:188, doi: 10.12688/f1000research.2-188.v1PubMedPubMedCentralGoogle Scholar
  17. Foissac S, Sammeth M (2007) ASTALAVISTA: dynamic and flexible analysis of alternative splicing events in custom gene datasets. Nucleic Acids Res 35(Web Server issue):W297–W299. doi: 10.1093/nar/gkm311 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Glaus P, Honkela A, Rattray M (2012) Identifying differentially expressed transcripts from RNA-seq data with biological variation. Bioinformatics 28(13):1721–1728. doi: 10.1093/bioinformatics/bts260 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Gonzalez-Porta, M., & Brazma, A. (2014). Identification, annotation and visualisation of extreme changes in splicing from RNA-seq experiments with SwitchSeq. bioRxiv. Cold Spring Harbor Labs Journals. doi: 10.1101/005967
  20. Gonzalez-Porta M, Frankish A, Rung J, Harrow J, Brazma A (2013) Transcriptome analysis of human tissues and cell lines reveals one dominant transcript per gene. Genome Biol 14(7):R70. doi: 10.1186/gb-2013-14-7-r70 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 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(7):644–652. doi: 10.1038/nbt.1883 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Grant GR, Farkas MH, Pizarro AD, Lahens NF, Schug J, Brunk BP et al (2011) Comparative analysis of RNA-seq alignment algorithms and the RNA-seq unified mapper (RUM). Bioinformatics 27:2518–2528. doi: 10.1093/bioinformatics/btr427 PubMedPubMedCentralGoogle Scholar
  23. Gulledge AA, Vora H, Patel K, Loraine AE (2014) A protocol for visual analysis of alternative splicing in RNA-seq data using integrated genome browser. Methods Mol Biol 1158:123–137. doi: 10.1007/978-1-4939-0700-7_8 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hu Y, Huang Y, Du Y, Orellana CF, Singh D, Johnson AR et al (2013) DiffSplice: the genome-wide detection of differential splicing events with RNA-seq. Nucleic Acids Res 41(2):e39. doi: 10.1093/nar/gks1026 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kanitz A, Gypas F, Gruber AJ, Gruber AR, Martin G, Zavolan M (2015) Comparative assessment of methods for the computational inference of transcript isoform abundance from RNA-seq data. Genome Biol 16(1):150. doi: 10.1186/s13059-015-0702-5 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Katz Y, Wang ET, Airoldi EM, Burge CB (2010) Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat Methods 7(12):1009–1015. doi: 10.1038/nmeth.1528 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 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. doi: 10.1186/gb-2009-10-3-r25 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Leng N, Dawson JA, Thomson JA, Ruotti V, Rissman AI, Smits BM, Haag JD, Gould MN, Stewart RM, Kendziorski C (2013) EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments. Bioinformatics 29(8):1035–1043. doi: 10.1093/bioinformatics/btt087 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome. BMC Bioinformatics 12:323. doi: 10.1186/1471-2105-12-323 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Li B, Fillmore N, Bai Y, Collins M, Thomson JA, Stewart R, Dewey CN (2014) Evaluation of de novo transcriptome assemblies from RNA-seq data. Genome Biol 15(12):553. doi: 10.1186/s13059-014-0553-5 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Lindner R, Friedel CC (2012) A comprehensive evaluation of alignment algorithms in the context of RNA-seq. PLoS One 7(12):e52403. doi: 10.1371/journal.pone.0052403 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Liu R, Loraine AE, Dickerson JA (2014) Comparisons of computational methods for differential alternative splicing detection using RNA-seq in plant systems. BMC Bioinformatics 15(1):364CrossRefPubMedPubMedCentralGoogle Scholar
  33. Luo R, Liu B, Xie Y, Li Z, Huang W, Yuan J, He G, Chen Y, Pan Q, Liu Y, Tang J, Wu G, Zhang H, Shi Y, Liu Y, Yu C, Wang B, Lu Y, Han C, Cheung DW, Yiu SM, Peng S, Xiaoqian Z, Liu G, Liao X, Li Y, Yang H, Wang J, Lam TW, Wang J (2012) SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. Gigascience 1(1):18. doi: 10.1186/2047-217X-1-18 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Majoros WH, Lebeck N, Ohler U, Li S (2014) Improved transcript isoform discovery using ORF graphs. Bioinformatics 30(14):1958–1964. doi: 10.1093/bioinformatics/btu160 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Mazin P, Xiong J, Liu X, Yan Z, Zhang X, Li M et al (2013) Widespread splicing changes in human brain development and aging. Mol Syst Biol 9(1):633. doi: 10.1038/msb.2012.67 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Nariai N, Kojima K, Mimori T, Sato Y, Kawai Y, Yamaguchi-Kabata Y, Nagasaki M (2014) TIGAR2: sensitive and accurate estimation of transcript isoform expression with longer RNA-Seq reads. BMC Genomics 15(Suppl 10):S5. doi: 10.1186/1471-2164-15-S10-S5 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Nicol JW, Helt GA, Blanchard SG, Raja A, Loraine AE (2009) The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics 25(20):2730–2731. doi: 10.1093/bioinformatics/btp472 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Nicolae M, Mangul S, Măndoiu II, Zelikovsky A (2011) Estimation of alternative splicing isoform frequencies from RNA-seq data. Algorithms Mol Biol 6(1):9. doi: 10.1186/1748-7188-6-9 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Pachter L (2011) Models for transcript quantification from RNA-seq. Genomics; Methodology. Available from
  40. Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40(12):1413–1415. doi: 10.1038/ng.259 CrossRefPubMedGoogle Scholar
  41. Patro R, Mount SM, Kingsford C (2014) Sailfish enables alignment-free isoform quantification from RNA-seq reads using lightweight algorithms. Nat Biotechnol 32(5):462–464. doi: 10.1038/nbt.2862 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Raney BJ, Dreszer TR, Barber GP, Clawson H, Fujita PA, Wang T, Nguyen N, Paten B, Zweig AS, Karolchik D, Kent WJ (2014) Track data hubs enable visualization of user-defined genome-wide annotations on the UCSC Genome Browser. Bioinformatics 30(7):1003–1005. doi: 10.1093/bioinformatics/btt637 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Roberts A, Pachter L (2013) Streaming fragment assignment for real-time analysis of sequencing experiments. Nat Methods 10(1):71–73. doi: 10.1038/nmeth.2251 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Robertson G, Schein J, Chiu R, Corbett R, Field M, Jackman SD, Mungall K, Lee S, Okada HM, Qian JQ, Griffith M, Raymond A, Thiessen N, Cezard T, Butterfield YS, Newsome R, Chan SK, She R, Varhol R, Kamoh B, Prabhu AL, Tam A, Zhao Y, Moore RA, Hirst M, Marra MA, Jones SJ, Hoodless PA, Birol I (2010) De novo assembly and analysis of RNA-seq data. Nat Methods 7(11):909–912. doi: 10.1038/nmeth.1517 CrossRefPubMedGoogle Scholar
  45. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24. doi: 10.1038/nbt.1754 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Ryan MC, Cleland J, Kim R, Wong WC, Weinstein JN (2012) SpliceSeq: a resource for analysis and visualization of RNA-seq data on alternative splicing and its functional impacts. Bioinformatics 28(18):2385–2387. doi: 10.1093/bioinformatics/bts452 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Shen S, Park JW, Huang J, Dittmar KA, Lu Z, Zhou Q et al (2012) MATS: a Bayesian framework for flexible detection of differential alternative splicing from RNA-seq data. Nucleic Acids Res 40(8):e61. doi: 10.1093/nar/gkr1291 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Steijger T, Abril JF, Engström PG, Kokocinski F, Hubbard TJ, Guigó R, Harrow J, Bertone P, RGASP Consortium (2013) Assessment of transcript reconstruction methods for RNA-seq. Nat Methods 10(12):1177–1184CrossRefPubMedGoogle Scholar
  49. Sturgill D, Malone JH, Sun X, Smith HE, Rabinow L, Samson M-L, Oliver B (2013) Design of RNA splicing analysis null models for post hoc filtering of Drosophila head RNA-seq data with the splicing analysis kit (Spanki). BMC Bioinformatics 14(1):320. doi: 10.1186/1471-2105-14-320 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14(2):178–192. doi: 10.1093/bib/bbs017 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Tilgner H, Jahanbani F, Blauwkamp T, Moshrefi A, Jaeger E, Chen F et al (2015) Comprehensive transcriptome analysis using synthetic long-read sequencing reveals molecular co-association of distant splicing events. Nat Biotechnol 33(7):736–742. doi: 10.1038/nbt.3242 CrossRefPubMedPubMedCentralGoogle Scholar
  52. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25:1105–1111. doi: 10.1093/bioinformatics/btp120 CrossRefPubMedPubMedCentralGoogle Scholar
  53. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28(5):511–515. doi: 10.1038/nbt.1621 CrossRefPubMedPubMedCentralGoogle Scholar
  54. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7(3):562–578. doi: 10.1038/nprot.2012.016 CrossRefPubMedPubMedCentralGoogle Scholar
  55. Treutlein B, Gokce O, Quake SR, Südhof TC (2014) Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing. Proc Natl Acad Sci U S A 111(13):E1291–E1299. doi: 10.1073/pnas.1403244111 CrossRefPubMedPubMedCentralGoogle Scholar
  56. Turro E, Su SY, Gonçalves Â, Coin LJ, Richardson S, Lewin A (2011) Haplotype and isoform specific expression estimation using multi-mapping RNA-seq reads. Genome Biol 12(2):R13. doi: 10.1186/gb-2011-12-2-r13 CrossRefPubMedPubMedCentralGoogle Scholar
  57. Vitting-Seerup K, Porse BT, Sandelin A, Waage J (2014) spliceR: an R package for classification of alternative splicing and prediction of coding potential from RNA-seq data. BMC Bioinformatics 15(1):81. doi: 10.1186/1471-2105-15-81 CrossRefPubMedPubMedCentralGoogle Scholar
  58. Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C et al (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456(7221):470–476. doi: 10.1038/nature07509 CrossRefPubMedPubMedCentralGoogle Scholar
  59. Wang K, Singh D, Zeng Z, Coleman SJ, Huang Y, Savich GL et al (2010) MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res 38:e178. doi: 10.1093/nar/gkq622 CrossRefPubMedPubMedCentralGoogle Scholar
  60. Wang W, Qin Z, Feng Z, Wang X, Zhang X (2013) Identifying differentially spliced genes from two groups of RNA-seq samples. Gene 518(1):164–170. doi: 10.1016/j.gene.2012.11.045 CrossRefPubMedGoogle Scholar
  61. Wu TD, Nacu S (2010) Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26(7):873–881. doi: 10.1093/bioinformatics/btq057 CrossRefPubMedPubMedCentralGoogle Scholar
  62. Wu J, Akerman M, Sun S, McCombie WR, Krainer AR, Zhang MQ (2011) SpliceTrap: a method to quantify alternative splicing under single cellular conditions. Bioinformatics 27(21):3010–3016. doi: 10.1093/bioinformatics/btr508 CrossRefPubMedPubMedCentralGoogle Scholar
  63. Wu E, Nance T, Montgomery SB (2014) SplicePlot: a utility for visualizing splicing quantitative trait loci. Bioinformatics 30:1025–1026. doi: 10.1093/bioinformatics/btt733 CrossRefPubMedPubMedCentralGoogle Scholar
  64. Xie Y, Wu G, Tang J, Luo R, Patterson J, Liu S, Huang W, He G, Gu S, Li S, Zhou X, Lam TW, Li Y, Xu X, Wong GK, Wang J (2014) SOAPdenovo-Trans: de novo transcriptome assembly with short RNA-seq reads. Bioinformatics 30(12):1660–1666. doi: 10.1093/bioinformatics/btu077 CrossRefPubMedGoogle Scholar
  65. Zhao Q-Y, Wang Y, Kong Y-M, Luo D, Li X, Hao P (2011) Optimizing de novo transcriptome assembly from short-read RNA-seq data: a comparative study. BMC Bioinformatics 12(Suppl 14):S2. doi: 10.1186/1471-2105-12-S14-S2 CrossRefGoogle Scholar
  66. Zhou A, Breese MR, Hao Y, Edenberg HJ, Li L, Skaar TC, Liu Y (2012) Alt Event Finder: a tool for extracting alternative splicing events from RNA-seq data. BMC Genomics 13(Suppl 8):S10. doi: 10.1186/1471-2164-13-S8-S10 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.School of Biological and Biomedical SciencesDurham UniversityDurhamUK

Personalised recommendations