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    A simple method for directional transcriptome sequencing using Illumina technology

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    68
    Author
    Croucher, NJ; Fookes, MC; Perkins, TT; Turner, DJ; Marguerat, SB; Keane, T; Quail, MA; He, M; Assefa, S; Baehler, J; ...
    Date
    2009-12-01
    Source Title
    Nucleic Acids Research
    Publisher
    OXFORD UNIV PRESS
    University of Melbourne Author/s
    Dougan, Gordon
    Affiliation
    Microbiology and Immunology
    Metadata
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    Document Type
    Journal Article
    Citations
    Croucher, N. J., Fookes, M. C., Perkins, T. T., Turner, D. J., Marguerat, S. B., Keane, T., Quail, M. A., He, M., Assefa, S., Baehler, J., Kingsley, R. A., Parkhill, J., Bentley, S. D., Dougan, G. & Thomson, N. R. (2009). A simple method for directional transcriptome sequencing using Illumina technology. NUCLEIC ACIDS RESEARCH, 37 (22), https://doi.org/10.1093/nar/gkp811.
    Access Status
    Open Access
    URI
    http://hdl.handle.net/11343/260033
    DOI
    10.1093/nar/gkp811
    Abstract
    High-throughput sequencing of cDNA has been used to study eukaryotic transcription on a genome-wide scale to single base pair resolution. In order to compensate for the high ribonuclease activity in bacterial cells, we have devised an equivalent technique optimized for studying complete prokaryotic transcriptomes that minimizes the manipulation of the RNA sample. This new approach uses Illumina technology to sequence single-stranded (ss) cDNA, generating information on both the direction and level of transcription throughout the genome. The protocol, and associated data analysis programs, are freely available from http://www.sanger.ac.uk/Projects/Pathogens/Transcriptome/. We have successfully applied this method to the bacterial pathogens Salmonella bongori and Streptococcus pneumoniae and the yeast Schizosaccharomyces pombe. This method enables experimental validation of genetic features predicted in silico and allows the easy identification of novel transcripts throughout the genome. We also show that there is a high correlation between the level of gene expression calculated from ss-cDNA and double-stranded-cDNA sequencing, indicting that ss-cDNA sequencing is both robust and appropriate for use in quantitative studies of transcription. Hence, this simple method should prove a useful tool in aiding genome annotation and gene expression studies in both prokaryotes and eukaryotes.

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