High-Throughput RNA Sequencing in B-Cell Lymphomas
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.
Key wordsNext-generation sequencing Transcriptome Gene expression B cell B-cell lymphoma Immunoglobulin genes Mutation
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.
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