RNA‐Seq: A Method for Comprehensive Transcriptome Analysis

A recently developed technique called RNA Sequencing (RNA‐Seq) uses massively parallel sequencing to allow transcriptome analyses of genomes at a far higher resolution than is available with Sanger sequencing‐ and microarray‐based methods. In the RNA‐Seq method, complementary DNAs (cDNAs) generated from the RNA of interest are directly sequenced using next‐generation sequencing technologies. The reads obtained from this can then be aligned to a reference genome in order to construct a whole‐genome transcriptome map. RNA‐Seq has been used successfully to precisely quantify transcript levels, confirm or revise previously annotated 5′ and 3′ ends of genes, and map exon/intron boundaries. This unit describes protocols for performing RNA‐Seq using the Illumina sequencing platform. Curr. Protoc. Mol. Biol. 89:4.11.1‐4.11.13. © 2010 by John Wiley & Sons, Inc.

[1]  P. Green,et al.  Massively parallel sequencing of the polyadenylated transcriptome of C. elegans. , 2009, Genome research.

[2]  K. Nakai,et al.  Massive transcriptional start site analysis of human genes in hypoxia cells , 2009, Nucleic acids research.

[3]  M. Gerstein,et al.  RNA-Seq: a revolutionary tool for transcriptomics , 2009, Nature Reviews Genetics.

[4]  J. Bähler,et al.  Next-generation sequencing: applications beyond genomes , 2008, Biochemical Society transactions.

[5]  M. Stephens,et al.  RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. , 2008, Genome research.

[6]  M. Marra,et al.  Applications of next-generation sequencing technologies in functional genomics. , 2008, Genomics.

[7]  Marcel H. Schulz,et al.  A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome , 2008, Science.

[8]  Ryan D. Morin,et al.  Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. , 2008, BioTechniques.

[9]  J. Shendure.  The beginning of the end for microarrays? , 2008, Nature Methods.

[10]  B. Williams,et al.  Mapping and quantifying mammalian transcriptomes by RNA-Seq , 2008, Nature Methods.

[11]  I. Goodhead,et al.  Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution , 2008, Nature.

[12]  M. Gerstein,et al.  The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing , 2008, Science.

[13]  S. Ranade,et al.  Stem cell transcriptome profiling via massive-scale mRNA sequencing , 2008, Nature Methods.

[14]  R. Lister,et al.  Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis , 2008, Cell.

[15]  E. Mardis.  The impact of next-generation sequencing technology on genetics. , 2008, Trends in genetics : TIG.

[16]  Ruiqiang Li,et al.  SOAP: short oligonucleotide alignment program , 2008, Bioinform..

[17]  Wolfgang Huber,et al.  A high-resolution map of transcription in the yeast genome. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Thomas E. Royce,et al.  Global Identification of Human Transcribed Sequences with Genome Tiling Arrays , 2004, Science.

[19]  Joseph M. Dale,et al.  Empirical Analysis of Transcriptional Activity in the Arabidopsis Genome , 2003, Science.

[20]  W. J. Kent,et al.  BLAT--the BLAST-like alignment tool. , 2002, Genome research.