Xiao Wenming, Tran Bao, Staudt Louis M, Schmitz Roland
Bioinformatics and Molecular Analysis Section, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2013;971:295-312. doi: 10.1007/978-1-62703-269-8_17.
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.
高通量mRNA测序（RNA-seq）使用大规模平行测序技术，对肿瘤的全基因组转录水平和突变状态进行无偏分析。在RNA-seq方法中，通过将数百万个扩增的DNA片段固定在固体表面并进行序列反应，利用互补DNA（cDNA）生成短序列读数。将得到的序列与参考基因组或转录本数据库进行比对，以全面描述所分析的转录组。本章介绍了使用Illumina测序平台进行RNA-seq的实验方案，给出了测序数据质量指标，并概述了用于序列比对、数字基因表达和突变发现的生物信息学流程。
