6. Stranded RNA-Seq: Strand-Specific Shotgun Sequencing of RNA

  1. Dr. Matthias Harbers2,3 and
  2. Prof. Dr. Günter Kahl4,5,6
  1. Alistair R. R. Forrest

Published Online: 23 JAN 2012

DOI: 10.1002/9783527644582.ch6

Tag-Based Next Generation Sequencing

Tag-Based Next Generation Sequencing

How to Cite

Forrest, A. R. R. (2011) Stranded RNA-Seq: Strand-Specific Shotgun Sequencing of RNA, in Tag-Based Next Generation Sequencing (eds M. Harbers and G. Kahl), Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527644582.ch6

Editor Information

  1. 2

    4-2-6 Nishihara, Kashiwa-Shi, Chiba 277-0885, Japan

  2. 3

    DNAFORM Inc., Leading Venture Plaza 2, 75-1 Ono-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0046, Japan

  3. 4

    Mohrmühlgasse 3, 63500 Seligenstadt, Germany

  4. 5

    University of Frankfurt am Main Biocenter, Max-von-Lauestraße 9, 60439 Frankfurt am Main, Germany

  5. 6

    Frankfurt Biotechnology Innovation Center (FIZ), GenXPro Ltd, Altenhöferallee 3, 60438 Frankfurt am Main, Germany

Author Information

  1. RIKEN Yokohama Institute, Omics Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

Publication History

  1. Published Online: 23 JAN 2012
  2. Published Print: 14 DEC 2011

ISBN Information

Print ISBN: 9783527328192

Online ISBN: 9783527644582

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Keywords:

  • stranded RNA-Seq;
  • RNA strand-specific shotgun sequencing;
  • bioinformatic considerations;
  • applications

Summary

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.