Abstract
In Metazoa, the diversity of transcripts produced by the RNA Polymerase II is generated essentially through post-transcriptional processing of the nascent transcripts. The regulation of exon inclusion by alternative splicing is one of the main sources of this diversity, which leads to the expansion of the proteome. The portfolio of alternative transcripts remains largely underestimated. Improvement of the sequencing technologies has enhanced the characterization of RNA isoforms and led to the perpetual incrementation of gene expression diversity. Here, we describe a high throughput approach to assess in-depth the splicing regulation of target gene(s) using the third-generation sequencing (TGS) technologies.
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References
Berget SM, Moore C, Sharp PA (1977) Spliced segments at the 5’ terminus of adenovirus 2 late MRNA. Proc Natl Acad Sci U S A 74:3171–3175
Chow LT, Gelinas RE, Broker TR, Roberts RJ (1977) An amazing sequence arrangement at the 5’ ends of adenovirus 2 messenger RNA. Cell 12:1–8
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467
Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470–476
Sun W, You X, Gogol-Döring A, He H, Kise Y, Sohn M, Chen T, Klebes A, Schmucker D, Chen W (2013) Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing. EMBO J 32:2029–2038
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:E1291–E1299
Schreiner D, Nguyen T-M, Russo G, Heber S, Patrignani A, Ahrné E, Scheiffele P (2014) Targeted combinatorial alternative splicing generates brain region-specific repertoires of neurexins. Neuron 84:386–398
Clark MB, Wrzesinski T, Garcia AB, Hall NAL, Kleinman JE, Hyde T, Weinberger DR, Harrison PJ, Haerty W, Tunbridge EM (2020) Long-read sequencing reveals the complex splicing profile of the psychiatric risk gene CACNA1C in human brain. Mol Psychiatry 25:37–47
Ray TA, Cochran K, Kozlowski C, Wang J, Alexander G, Cady MA, Spencer WJ, Ruzycki PA, Clark BS, Laeremans A et al (2020) Comprehensive identification of MRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease. Nat Commun 11:3328
Bolisetty MT, Rajadinakaran G, Graveley BR (2015) Determining exon connectivity in complex MRNAs by nanopore sequencing. Genome Biol 16:204
Sahlin K, Medvedev P (2020) De novo clustering of long-read transcriptome data using a greedy, quality value-based algorithm. J Comput Biol 27:472–484
Tang AD, Soulette CM, van Baren MJ, Hart K, Hrabeta-Robinson E, Wu CJ, Brooks AN (2020) Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns. Nat Commun 11:1438
Al Kadi M, Jung N, Ito S, Kameoka S, Hishida T, Motooka D, Nakamura S, Iida T, Okuzaki D (2020) UNAGI: an automated pipeline for nanopore full-length CDNA sequencing uncovers novel transcripts and isoforms in yeast. Funct Integr Genomics 20:523–536
Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc 1:581–585
Wick RR, Judd LM, Holt KE (2018) Deepbinner: demultiplexing barcoded Oxford nanopore reads with deep convolutional neural networks. PLoS Comput Biol 14:e1006583
Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-Seq aligner. Bioinforma Oxf Engl 29:15–21
Shabardina V, Kischka T, Manske F, Grundmann N, Frith MC, Suzuki Y, Makałowski W (2019) NanoPipe-a web server for nanopore MinION sequencing data analysis. GigaScience 8:giy169
Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinforma Oxf Engl 26:841–842
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421
Slater GSC, Birney E (2005) Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics 6:31
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We thank Svetlana Dokudovskaya for comments on the manuscript.
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Allemand, E., Ango, F. (2022). Analysis of Splicing Regulation by Third-Generation Sequencing. In: Scheiffele, P., Mauger, O. (eds) Alternative Splicing. Methods in Molecular Biology, vol 2537. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2521-7_6
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DOI: https://doi.org/10.1007/978-1-0716-2521-7_6
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