Strand-specific deep sequencing of the transcriptome

Abstract

Several studies support that antisense-mediated regulation may affect a large proportion of genes. Using the Illumina nextgeneration sequencing platform, we developed DSSS (direct strand specific sequencing), a strand-specific protocol for transcriptome sequencing. We tested DSSS with RNA from two samples, prokaryotic (Mycoplasma pneumoniae) as well as eukaryotic (Mus musculus), and obtained data containing strand-specific information, using single-read and paired-end sequencing. We validated our results by comparison with a strand-specific tiling array data set for strain M129 of the simple prokaryote M. pneumoniae, and by quantitative PCR (qPCR). The results of DSSS were very well supported by the results from tiling arrays and qPCR. 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. DSSS data for mouse confirmed strand specificity of the protocol 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.

RNA Sequencing and Analysis

“…In this conventional approach, the strand orientation of the original RNA is lost as the sequencing reads derived from each cDNA strand are indistinguishable in an effort to maximize efficiency of reverse transcription. However, strand information can be particularly valuable for distinguishing overlapping transcripts on opposite strands, which is critical for de novo transcript discovery (Parkhomchuk et al 2009; Vivancos et al 2010; Mills et al 2013). Therefore, alternative library preparation protocols have since been developed that yield strand-specific reads.…”

New approaches to Prunus transcriptome analysis

“…A modified protocol of the Illumina/Solexa technology called DSSS (direct strand specific sequencing) has been recently described, allowing strand-specific transcriptome sequencing (Parkhomchuk et al 2009;Vivancos et al 2010). DSSS enables efficient antisense detection and precisely assigns transcription start points and untranslated regions.…”

Analysis and design of RNA sequencing experiments for identifying RNA editing and other single-nucleotide variants

“…Thus, RDDs in unannotated regions or in regions with bidirectional transcription (sense and antisense genes) cannot be accurately categorized into a specific type. To this end, strand-specific libraries (e.g., Parkhomchuk et al 2009;Levin et al 2010;Vivancos et al 2010;Zhong et al 2011) are advantageous, since the type of RDDs can be inferred directly from the reads independent of gene annotations. Indeed, our analysis of data from a strand-specific library derived from U87MG cells (data not shown) suggested that ∼7% of all identified RDDs are antisense to annotated genes and 40% reside in intergenic regions based on RefSeq annotation (as of June 29, 2012).…”