Abstract
BackgroundThe 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.ResultsWe 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.ConclusionOur 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.
Highlights
The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status
Using the functional annotation strategies that are usually applied to microarray experiments functional annotation, we examined the genes identified by deep sequencing. 454 reads mapped to 6.067 RefSeq transcripts (Additional file 9) with counts per transcript ranging from 82 to 1, with median of 2
UCSC human genome 8 kb screenshot of a "gene desert region" on the X chromosome tagged as transcribed by the sequencing read 002770_3171_2414
Summary
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. 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. Transcriptome sequencing and annotation initiatives have challenged this view by discovering that most of the genome is actively transcribed to yield complex patterns of interlaced and overlapping transcripts, including tens of thousands long (>200 nt) non-protein-coding RNAs (ncRNA) [1,2,3]. Alternative splicing is often deregulated in cancer, probably as a consequence of quantitative alterations in the levels of expression of splicing regulators [21]; many examples of cancer-specific gene isoforms (CD44, BRCA1, survivin etc), whose expression seem to correlate with the disease, have been described in literature [22]
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