Abstract
High-throughput mRNA sequencing (RNA-seq) uses massively parallel sequencing to allow an unbiased analysis of both genome-wide transcription levels and mutation status of a tumor. In the RNA-seq method, complementary DNA (cDNA) is used to generate short sequence reads by immobilizing millions of amplified DNA fragments onto a solid surface and performing the sequence reaction. The resulting sequences are aligned to a reference genome or transcript database to create a comprehensive description of the analyzed transcriptome. This chapter describes a protocol to perform RNA-seq using the Illumina sequencing platform, presents sequencing data quality metrics and outlines a bioinformatic pipeline for sequence alignment, digital gene expression, and mutation discovery.
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
References
Morin RD, Mendez-Lago M, Mungall AJ et al (2011) Frequent mutation of histone-Âmodifying genes in non-Hodgkin lymphoma. Nature 476:298–303
Pasqualucci L, Trifonov V, Fabbri G et al (2011) Analysis of the coding genome of diffuse large B-cell lymphoma. Nat Genet 43:830–837
Ngo VN, Young RM, Schmitz R et al (2011) Oncogenically active MYD88 mutations in human lymphoma. Nature 470:115–119
Puente XS, Pinyol M, Quesada V et al (2011) Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature 475:101–105
Kridel R, Meissner B, Rogic S et al (2012) Whole transcriptome sequencing reveals recurrent NOTCH1 mutations in mantle cell lymphoma. Blood 119:1963–1971
Schmitz R, Young RM, Ceribelli M et al (2012) Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics. Nature 490:116–120
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63
Mortazavi A, Williams BA, McCue K et al (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5:621–628
Frischmeyer PA, Dietz HC (1999) Nonsense-mediated mRNA decay in health and disease. Hum Mol Genet 8:1893–1900
Garber M, Grabherr MG, Guttman M et al (2011) Computational methods for transcriptome annotation and quantification using RNA-seq. Nat Methods 8:469–477
Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760
Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
Larsson TP, Murray CG, Hill T et al (2005) Comparison of the current RefSeq, Ensembl and EST databases for counting genes and gene discovery. FEBS Lett 579:690–698
Robinson JT, Thorvaldsdottir H, Winckler W et al (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26
Kumar P, Henikoff S, Ng PC (2009) Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 4:1073–1081
Rui L, Schmitz R, Ceribelli M et al (2011) Malignant pirates of the immune system. Nat Immunol 12:933–940
Acknowledgments
This work was supported by the by the Dr. Mildred Scheel Stiftung für Krebsforschung (Deutsche Krebshilfe). We are grateful to Yuliya Kriga, Jyoti Shetty, Yongmei Zhao, John Powell, and George Wright who were instrumental in establishing the protocols described here.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Xiao, W., Tran, B., Staudt, L.M., Schmitz, R. (2013). High-Throughput RNA Sequencing in B-Cell Lymphomas. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 971. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-269-8_17
Download citation
DOI: https://doi.org/10.1007/978-1-62703-269-8_17
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-268-1
Online ISBN: 978-1-62703-269-8
eBook Packages: Springer Protocols