Abstract
RNA-seq using long-read sequencing, such as nanopore and SMRT (Single Molecule, Real-Time) sequencing, enabled the identification of the full-length structure of RNA molecules. Several tools for long-read RNA-seq were developed recently. In this section, we introduce an analytical pipeline of long-read RNA-seq for isoform identification and the estimation of expression levels using minimap2, TranscriptClean, and TALON. We applied this pipeline to the public direct RNA-seq data of the HAP1 and HEK293 cell lines to identify transcript isoforms which can be detected only using long-read RNA-seq data.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Stark R, Grzelak M, Hadfield J (2019) RNA sequencing: the teenage years. Nat Rev Genet 20:631–656. https://doi.org/10.1038/s41576-019-0150-2
Sharon D, Tilgner H, Grubert F, Snyder M (2013) A single-molecule long-read survey of the human transcriptome. Nat Biotechnol 31:1009–1014. https://doi.org/10.1038/nbt.2705
Oikonomopoulos S, Wang YC, Djambazian H et al (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
Gupta I, Collier PG, Haase B et al (2018) Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells. Nat Biotechnol 36:1197–1202. https://doi.org/10.1038/nbt.4259
Byrne A, Beaudin AE, Olsen HE et al (2017) Nanopore long-read RNAseq reveals widespread transcriptional variation among the surface receptors of individual B cells. Nat Commun 8:16027. https://doi.org/10.1038/ncomms16027
Garalde DR, Snell EA, Jachimowicz D et al (2018) Highly parallel direct RN A sequencing on an array of nanopores. Nat Methods 15:201–206. https://doi.org/10.1038/nmeth.4577
Workman RE, Tang AD, Tang PS, et al (2018) Nanopore native RNA sequencing of a human poly(A) transcriptome. bioRxiv 459529. https://doi.org/10.1101/459529
Seki M, Katsumata E, Suzuki A et al (2019) Evaluation and application of RNA-Seq by MinION. DNA Res 26:55–65. https://doi.org/10.1093/dnares/dsy038
Calabrese C, Davidson NR, Demircioğlu D et al (2020) Genomic basis for RNA alterations in cancer. Nature 578:129–136. https://doi.org/10.1038/s41586-020-1970-0
Shiraishi Y, Kataoka K, Chiba K et al (2018) A comprehensive characterization of cis-acting splicing-associated variants in human cancer. Genome Res 28:1111–1125. https://doi.org/10.1101/gr.231951.117
Mitelman F, Johansson B, Mertens F (2004) Fusion genes and rearranged genes as a linear function of chromosome aberrations in cancer. Nat Genet 36:331–334
Li H (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34:3094–3100. https://doi.org/10.1093/bioinformatics/bty191
Liu B, Liu Y, Li J et al (2019) deSALT: fast and accurate long transcriptomic read alignment with de Bruijn graph-based index. Genome Biol 20:274. https://doi.org/10.1186/s13059-019-1895-9
Marić J, Sović I, Križanović K, et al (2019) Graphmap2—splice-aware RNA-seq mapper for long reads. bioRxiv 720458. https://doi.org/10.1101/720458
Frith MC, Kawaguchi R (2015) Split-alignment of genomes finds orthologies more accurately. Genome Biol 16:106. https://doi.org/10.1186/s13059-015-0670-9
Tang AD, Soulette CM, van Baren MJ, et al (2018) Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns. bioRxiv 410183. https://doi.org/10.1101/410183
Wyman D, Balderrama-Gutierrez G, Reese F, et al (2019) A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification bioRxiv 672931. https://doi.org/10.1101/672931
Kovaka S, Zimin AV, Pertea GM et al (2019) Transcriptome assembly from long-read RNA-seq alignments with StringTie2. Genome Biol 20:278. https://doi.org/10.1186/s13059-019-1910-1
Oxford Nanopore Technologies Pinfish. https://github.com/nanoporetech/pinfish. Accessed 24 Feb 2020
Soneson C, Yao Y, Bratus-Neuenschwander A et al (2019) A comprehensive examination of Nanopore native RNA sequencing for characterization of complex transcriptomes. Nat Commun 10:3359. https://doi.org/10.1038/s41467-019-11272-z
Sun Z, Xue S, Xu H et al (2019) Effects of NSUN2 deficiency on the mRNA 5-methylcytosine modification and gene expression profile in HEK293 cells. Epigenomics 11:439–453. https://doi.org/10.2217/epi-2018-0169
Frankish A, Diekhans M, Ferreira A-M et al (2019) GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res 47:D766–D773. https://doi.org/10.1093/nar/gky955
Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192. https://doi.org/10.1093/bib/bbs017
Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079. https://doi.org/10.1093/bioinformatics/btp352
Krueger F Trim Galore! http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/. Accessed 13 Mar 2020
Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17:10. https://doi.org/10.14806/ej.17.1.200
Andrews Simon FastQC a quality control tool for high throughput sequence data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Accessed 13 Mar 2020
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170
Dobin A, Davis CA, Schlesinger F et al (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21. https://doi.org/10.1093/bioinformatics/bts635
Acknowledgments
This work was supported by JSPS KAKENHI Grant Number 16H06279 (PAGS), 19K16108, and 19K16792.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Seki, M., Oka, M., Xu, L., Suzuki, A., Suzuki, Y. (2021). Transcript Identification Through Long-Read Sequencing. In: Picardi, E. (eds) RNA Bioinformatics. Methods in Molecular Biology, vol 2284. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1307-8_29
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1307-8_29
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1306-1
Online ISBN: 978-1-0716-1307-8
eBook Packages: Springer Protocols