title: A platform independent RNA-Seq protocol for the detection of transcriptome complexity.
abstract: 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.
  
  
  CONCLUSIONS
  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.forename: Claudia
authors.initials: C
authors.lastname: Calabrese
authors.forename: Marina
authors.initials: M
authors.lastname: Mangiulli
authors.forename: Caterina
authors.initials: C
authors.lastname: Manzari
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Paluscio
authors.forename: Mariano Francesco
authors.initials: MF
authors.lastname: Caratozzolo
authors.forename: Flaviana
authors.initials: F
authors.lastname: Marzano
authors.forename: Ivana
authors.initials: I
authors.lastname: Kurelac
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: D'Erchia
authors.forename: Domenica
authors.initials: D
authors.lastname: D'Elia
authors.forename: Flavio
authors.initials: F
authors.lastname: Licciulli
authors.forename: Sabino
authors.initials: S
authors.lastname: Liuni
authors.forename: Ernesto
authors.initials: E
authors.lastname: Picardi
authors.forename: Marcella
authors.initials: M
authors.lastname: Attimonelli
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Gasparre
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Porcelli
authors.forename: Graziano
authors.initials: G
authors.lastname: Pesole
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Sbisà
authors.affiliation: Istituto di Tecnologie Biomediche (ITB), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy. apollonia.tullo@ba.itb.cnr.it.
authors.forename: Apollonia
authors.initials: A
authors.lastname: Tullo
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002874:Chromosome Mapping; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D064593:Heterografts; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D058977:Molecular Sequence Annotation; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome