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Methods Mol Biol. 2019;1956:283-303. doi: 10.1007/978-1-4939-9151-8_13.

RNA Sequencing in B-Cell Lymphomas.

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Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Baylor College of Medicine, Houston, TX, USA.
Office of Intramural Research, Center for Information Technology, National Institutes of Health, Bethesda, MD, USA.
Institute of Pathology, University Hospital Giessen, Justus-Liebig-University Giessen, Giessen, Germany.


High-throughput mRNA sequencing (RNA-Seq) provides both qualitative and quantitative evaluation of the transcriptome. This method uses complementary DNA (cDNA) to generate several millions of short sequence reads that are aligned to a reference genome allowing the comprehensive characterization of the transcripts in a cell. RNA-Seq has a wide variety of applications which lead to a pervasive adoption of this method well beyond the genomics community and a deployment of this technique as a standard part of the toolkit applied in life sciences. This chapter describes a protocol to perform mRNA sequencing using the Illumina NextSeq or MiSeq platforms, presents sequencing data quality metrics, and outlines a bioinformatic pipeline for sequence alignment, digital gene expression, identification of gene fusions, detection of transcript isoforms, description and annotation of genetic variants, and de novo immunoglobulin gene assembly.


B cell; B-cell lymphoma; Gene expression; High-throughput sequencing; Immunoglobulin genes; Mutation; RNA-Seq; Transcriptome; VDJ

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