A simple method for directional transcriptome sequencing using Illumina technology.

Title: A simple method for directional transcriptome sequencing using Illumina technology.
Author(s): Croucher, NJ
Fookes, MC
Perkins, TT
Turner, DJ
Marguerat, SB
Keane, T
Quail, MA
He, M
Assefa, S
Bähler, J
Kingsley, RA
Parkhill, J
Bentley, SD
Dougan, G
Thomson, NR
Item Type: Journal Article
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.
Publication Date: 8-Oct-2009
Date of Acceptance: 15-Sep-2009
URI: http://hdl.handle.net/10044/1/21986
DOI: https://dx.doi.org/10.1093/nar/gkp811
ISSN: 1362-4962
Publisher: Oxford University Press
Start Page: e148
End Page: e148
Journal / Book Title: Nucleic Acids Research
Volume: 37
Issue: 22
Copyright Statement: © The Author(s) 2009. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Publication Status: Published
Appears in Collections:Clinical Sciences
Molecular Sciences
Faculty of Medicine
Epidemiology, Public Health and Primary Care



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