title: A simple method for directional transcriptome sequencing using Illumina technology.
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.
authors.affiliation: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, Cambridgeshire CB10 1SA, UK.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Croucher
authors.forename: Maria C
authors.initials: MC
authors.lastname: Fookes
authors.forename: Timothy T
authors.initials: TT
authors.lastname: Perkins
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Turner
authors.forename: Samuel B
authors.initials: SB
authors.lastname: Marguerat
authors.forename: Thomas
authors.initials: T
authors.lastname: Keane
authors.forename: Michael A
authors.initials: MA
authors.lastname: Quail
authors.forename: Miao
authors.initials: M
authors.lastname: He
authors.forename: Sammey
authors.initials: S
authors.lastname: Assefa
authors.forename: Jürg
authors.initials: J
authors.lastname: Bähler
authors.forename: Robert A
authors.initials: RA
authors.lastname: Kingsley
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Bentley
authors.forename: Gordon
authors.initials: G
authors.lastname: Dougan
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Thomson
mesh_terms: D018076:DNA, Complementary / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D012326:RNA Splicing; D012475:Salmonella / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013296:Streptococcus pneumoniae / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D018076:DNA, Complementary; D004277:DNA, Single-Stranded