title: A transcriptional sketch of a primary human breast cancer by 454 deep sequencing.
abstract: BACKGROUND
  The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)+ transcriptome. This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts.
  
  
  RESULTS
  We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA MALAT1, which is highly expressed in many human carcinomas.
  
  
  CONCLUSIONS
  Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown transcripts, splice isoforms, gene fusion events and ncRNAs, even at a relatively low sequence sampling.
authors.affiliation: Institute of Biomedical Technologies, National Research Council, Milan, Italy. alessandro.guffanti@genomnia.com
authors.forename: Alessandro
authors.initials: A
authors.lastname: Guffanti
authors.forename: Michele
authors.initials: M
authors.lastname: Iacono
authors.forename: Paride
authors.initials: P
authors.lastname: Pelucchi
authors.forename: Namshin
authors.initials: N
authors.lastname: Kim
authors.forename: Giulia
authors.initials: G
authors.lastname: Soldà
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Croft
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Taft
authors.forename: Ermanno
authors.initials: E
authors.lastname: Rizzi
authors.forename: Marjan
authors.initials: M
authors.lastname: Askarian-Amiri
authors.forename: Raoul J
authors.initials: RJ
authors.lastname: Bonnal
authors.forename: Maurizio
authors.initials: M
authors.lastname: Callari
authors.forename: Flavio
authors.initials: F
authors.lastname: Mignone
authors.forename: Graziano
authors.initials: G
authors.lastname: Pesole
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bertalot
authors.forename: Luigi Rossi
authors.initials: LR
authors.lastname: Bernardi
authors.forename: Alberto
authors.initials: A
authors.lastname: Albertini
authors.forename: Christopher
authors.initials: C
authors.lastname: Lee
authors.forename: John S
authors.initials: JS
authors.lastname: Mattick
authors.forename: Ileana
authors.initials: I
authors.lastname: Zucchi
authors.forename: Gianluca
authors.initials: G
authors.lastname: De Bellis
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics; D019295:Computational Biology; D003598:Cytoskeletal Proteins / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry; D030541:Databases, Genetic; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics; D022661:RNA, Untranslated / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic; D044767:Ubiquitin-Protein Ligases
chemical_list: D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D009687:Nuclear Proteins; D022661:RNA, Untranslated; D011495:Histone-Lysine N-Methyltransferase; C432968:NSD3 protein, human; C505833:UBR4 protein, human; D044767:Ubiquitin-Protein Ligases