A transcriptional sketch of a primary human breast cancer by 454 deep sequencing
| Autor: | Pesole Graziano, Mignone Flavio, Callari Maurizio, Bonnal Raoul J, Askarian-Amiri Marjan, Rizzi Ermanno, Taft Ryan J, Croft Larry J, Soldà Giulia, Kim Namshin, Pelucchi Paride, Iacono Michele, Guffanti Alessandro, Bertalot Giovanni, Bernardi Luigi, Albertini Alberto, Lee Christopher, Mattick John S, Zucchi Ileana, De Bellis Gianluca |
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| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: | |
| Zdroj: | BMC Genomics, Vol 10, Iss 1, p 163 (2009) |
| Druh dokumentu: | article |
| ISSN: | 1471-2164 26970694 |
| DOI: | 10.1186/1471-2164-10-163 |
| Popis: | 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. Conclusion 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. |
| Databáze: | Directory of Open Access Journals |
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