Next-generation sequencers have revolutionized the way we do genomics. RNA-seq - a set of shotgun sequencing protocols developed for sequencing the transcriptome - is incredibly useful for gene finding, measuring expression (at the level of genes, transcripts, and alleles), alternative splicing studies, and noncoding RNA discovery. Here, we describe a simple strand-specific RNA-seq protocol for identifying and quantifying RNA species within a sample compatible with both SOLiD and Illumina Genome Analyzer second-generation DNA sequencers. Although several approaches exist for generating shotgun libraries of the transcriptome, the most used version to date involves cDNA fragmentation and linker ligation, which loses strand information. Maintaining strand information is critical to capture overlapping antisense transcription and discern the strand of novel transcribed regions (particularly important for unspliced noncoding RNAs). The strand-specific protocol presented here uses RNA fragmentation to generate short RNA fragments that are then converted to cDNA using an anchored random primer and a template switch primer. We discuss the application of the technology, and see great scope for its use in both model organisms and novel species. To date, RNA-seq has mostly been applied to human and mouse systems with applications in gene expression, transcript discovery, expressed single nucleotide polymorphisms, detection of gene fusions in cancer, and allelic usage studies. For other species with less genomic or transcriptomic information, RNA-seq provides a great tool for rapid annotation of a genome, providing massive-scale expressed sequence tag coverage in a few experiments. Even without a genome sequence, RNA-seq can in principal be used for building transcript contigs akin to UniGene assembled transcripts from the 1990s. With each of these applications in mind we discuss aspects of experimental design, such as read length, depth of sequencing, replicates, and analysis strategies needed to achieve each goal.
|Title of host publication||Tag-Based Next Generation Sequencing|
|Editors||M Harbers, G Kahl|
|Publisher||Wiley-VCH Verlag GmbH & Co. KGaA|
|Number of pages||18|
|Publication status||Published - 23 Jan 2012|