A transcriptional sketch of a primary human breast cancer by 454 deep sequencing.
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.
Guffanti
Alessandro
A
Institute of Biomedical Technologies, National Research Council, Milan, Italy. alessandro.guffanti@genomnia.com
Iacono
Michele
M
Pelucchi
Paride
P
Kim
Namshin
N
Soldà
Giulia
G
Croft
Larry J
LJ
Taft
Ryan J
RJ
Rizzi
Ermanno
E
Askarian-Amiri
Marjan
M
Bonnal
Raoul J
RJ
Callari
Maurizio
M
Mignone
Flavio
F
Pesole
Graziano
G
Bertalot
Giovanni
G
Bernardi
Luigi Rossi
LR
Albertini
Alberto
A
Lee
Christopher
C
Mattick
John S
JS
Zucchi
Ileana
I
De Bellis
Gianluca
G
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
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