BACKGROUND: Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform. RESULTS: We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol. CONCLUSION: We tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes.
BACKGROUND: Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform. RESULTS: We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol. CONCLUSION: We tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes.
Authors: J Guillermo Paez; Ming Lin; Rameen Beroukhim; Jeffrey C Lee; Xiaojun Zhao; Daniel J Richter; Stacey Gabriel; Paula Herman; Hidefumi Sasaki; David Altshuler; Cheng Li; Matthew Meyerson; William R Sellers Journal: Nucleic Acids Res Date: 2004-05-18 Impact factor: 16.971
Authors: Kun Zhang; Adam C Martiny; Nikos B Reppas; Kerrie W Barry; Joel Malek; Sallie W Chisholm; George M Church Journal: Nat Biotechnol Date: 2006-05-28 Impact factor: 54.908
Authors: Elena Bonora; Anna Maria Porcelli; Giuseppe Gasparre; Annalisa Biondi; Anna Ghelli; Valerio Carelli; Alessandra Baracca; Giovanni Tallini; Andrea Martinuzzi; Giorgio Lenaz; Michela Rugolo; Giovanni Romeo Journal: Cancer Res Date: 2006-06-15 Impact factor: 12.701
Authors: Irene Rodríguez; José M Lázaro; Luis Blanco; Satwik Kamtekar; Andrea J Berman; Jimin Wang; Thomas A Steitz; Margarita Salas; Miguel de Vega Journal: Proc Natl Acad Sci U S A Date: 2005-04-21 Impact factor: 11.205