Huang Da Wei, Dawood Moez, Johnson Calvin A, Schmitz Roland
Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
Baylor College of Medicine, Houston, TX, USA.
Methods Mol Biol. 2019;1956:283-303. doi: 10.1007/978-1-4939-9151-8_13.
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.
高通量mRNA测序（RNA-Seq）可对转录组进行定性和定量评估。该方法利用互补DNA（cDNA）生成数百万条短序列读数，这些读数与参考基因组比对，从而全面表征细胞中的转录本。RNA-Seq有广泛的应用，这使得该方法在基因组学界之外也得到广泛采用，并作为生命科学应用工具包的标准组成部分进行部署。本章描述了使用Illumina NextSeq或MiSeq平台进行mRNA测序的方案，介绍了测序数据质量指标，并概述了用于序列比对、数字基因表达、基因融合鉴定、转录本异构体检测、遗传变异描述与注释以及从头免疫球蛋白基因组装的生物信息学流程。
