Fusion transcripts and transcribed retrotransposed loci discovered through comprehensive transcriptome analysis using Paired-End diTags (PETs)

Abstract

Identification of unconventional functional features such as fusion transcripts is a challenging task in the effort to annotate all functional DNA elements in the human genome. Paired-End diTag (PET) analysis possesses a unique capability to accurately and efficiently characterize the two ends of DNA fragments, which may have either normal or unusual compositions. This unique nature of PET analysis makes it an ideal tool for uncovering unconventional features residing in the human genome. Using the PET approach for comprehensive transcriptome analysis, we were able to identify fusion transcripts derived from genome rearrangements and actively expressed retrotransposed pseudogenes, which would be difficult to capture by other means. Here, we demonstrate this unique capability through the analysis of 865,000 individual transcripts in two types of cancer cells. In addition to the characterization of a large number of differentially expressed alternative 5′ and 3′ transcript variants and novel transcriptional units, we identified 70 fusion transcript candidates in this study. One was validated as the product of a fusion gene between BCAS4 and BCAS3 resulting from an amplification followed by a translocation event between the two loci, chr20q13 and chr17q23. Through an examination of PETs that mapped to multiple genomic locations, we identified 4055 retrotransposed loci in the human genome, of which at least three were found to be transcriptionally active. The PET mapping strategy presented here promises to be a useful tool in annotating the human genome, especially aberrations in human cancer genomes.

Transcriptional consequences of genomic structural aberrations in breast cancer

“…had previously pioneered pair-end-tag sequencing of full-length cDNA from the MCF-7 transcriptome (Ruan et al 2007). This RNA paired-end-tag (RNA-PET) approach captures the sequence of the ends of full-length transcripts with high sequencing depth and is complementary to the DNA-PET approach for identifying unconventional fusion transcripts.…”

“…To identify further FG and 39T-derived transcripts in MCF-7, we intersected DNA-PET data with GIS-PET and RNA-PET data. GIS-PET data sets for MCF-7 were described previously (Ng et al 2006;Ruan et al 2007). We compared 59 fused gene symbols of FG and 39T predicted in the DNA-PET data and those of the transcriptome data.…”

Sequence features that drive human promoter function and tissue specificity

“…To predict putative TSSs in silico, we first used published data that describe human transcribed sequences, such as full-length cDNAs and 59-end cDNA sequence tags (Carninci et al 2005;Ruan et al 2007;Wakaguri et al 2007), which specify TSSs when aligned to the genome. We aligned a database of more than 250,000 human cDNAs and predicted about 37,000 gene models, with about 22,000 gene models represented by two or more cDNAs.…”

Characteristics and Significance of Intergenic Polyadenylated RNA Transcription in Arabidopsis

“…Studies in yeast (Saccharomyces cerevisiae; David et al, 2006), animals (Bertone et al, 2004;Carninci et al, 2005), and plants (Yamada et al, 2003;Li et al, 2007;Matsui et al, 2008) have revealed the presence of a large number of unannotated, novel transcripts. These novel transcripts may represent alternatively spliced forms of known genes (Filichkin et al, 2010), products of antisense (Yamada et al, 2003) or bidirectional transcription (Xu et al, 2009), retained introns (Ner-Gaon et al, 2004;Filichkin et al, 2010), transcript fusions (Ruan et al, 2007), or intergenic transcriptional units (referred to hereafter as intergenic transcribed fragments [ITFs]). Among these novel transcripts, ITFs are unique in that they do not overlap with known genomic features and may represent novel genic sequences.…”