MPN_mice
Strand-specific deep sequencing of the transcriptome
Several studies support that antisense-mediated regulation may affect a large proportion of genes. However, methods available for transcriptome sequencing generally do not provide information on transcript orientation or are limited to the processing of short RNA fragments. Using the Solexa next-generation sequencing platform, we developed DSSS (Direct Strand Specific Sequencing), a strictly strand specific protocol for transcriptome sequencing. We tested DSSS with single stranded RNA fragments of 200 nt from two samples, prokaryotic (Mycoplasma pneumoniae) as well as eukaryotic (mouse), and obtained data containing strand specific information without 5’ to 3’ bias. We validated our results by comparison with a strand specific tiling array dataset for strain M129 of the simple prokaryote M. pneumoniae. The results of DSSS were very well supported by the results from tiling arrays. Moreover, DSSS provided higher dynamic range and single-base resolution, thus enabling efficient antisense detection and the precise mapping of transcription start sites and untranslated regions. Inspection of the data indicated the presence of at least x open reading frames (ORFs) that had previously not been annotated in the M129 genome, and antisense regulation was suggested for at least x loci. Pilot data for mouse confirmed strand-specificity of DSSS and the general applicability of the approach to studying eukaryotic transcription. We propose DSSS as a simple and efficient strategy for strand-specific transcriptome sequencing and as a tool for genome annotation exploiting the increased read lengths that next generation sequencing technology now is capable to deliver.