Strand-specific deep sequencing of the transcriptome

Ana P. Vivancos; Marc Güell; Juliane C. Dohm; Luis Serrano; Heinz Himmelbauer

doi:10.1101/gr.094318.109

Strand-specific deep sequencing of the transcriptome

¹ Centre for Genomic Regulation (CRG), UPF, 08003 Barcelona, Spain;
² Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany;
³ Institució Catalana de Recerca i Estudis Avancats (ICREA), 08010 Barcelona, Spain

↵4 These authors contributed equally to this work.

Abstract

Several studies support that antisense-mediated regulation may affect a large proportion of genes. Using the Illumina next-generation 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.

Footnotes

↵5 Current address: Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, 08035 Barcelona, Spain.
↵6 Corresponding authors.

E-mail luis.serrano{at}crg.es.

E-mail heinz.himmelbauer{at}crg.es.
[Supplemental material is available online at http://www.genome.org. The tiling array CEL files from this study have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE14014, and the Illumina fastq files have been submitted to the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession no. SRA009091.]
Article published online before print. Article and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.094318.109.