Chimeric transcript discovery by paired-end transcriptome sequencing

Abstract

Recurrent gene fusions are a prevalent class of mutations arising from the juxtaposition of 2 distinct regions, which can generate novel functional transcripts that could serve as valuable therapeutic targets in cancer. Therefore, we aim to establish a sensitive, high-throughput methodology to comprehensively catalog functional gene fusions in cancer by evaluating a paired-end transcriptome sequencing strategy. Not only did a paired-end approach provide a greater dynamic range in comparison with single read based approaches, but it clearly distinguished the high-level ''driving'' gene fusions, such as BCR-ABL1 and TMPRSS2-ERG, from potential lower level ''passenger'' gene fusions. Also, the comprehensiveness of a paired-end approach enabled the discovery of 12 previously undescribed gene fusions in 4 commonly used cell lines that eluded previous approaches. Using the paired-end transcriptome sequencing approach, we observed readthrough mRNA chimeras, tissue-type restricted chimeras, converging transcripts, diverging transcripts, and overlapping mRNA transcripts. Last, we successfully used paired-end transcriptome sequencing to detect previously undescribed ETS gene fusions in prostate tumors. Together, this study establishes a highly specific and sensitive approach for accurately and comprehensively cataloguing chimeras within a sample using paired-end transcriptome sequencing.bioinformatics ͉ gene fusions ͉ prostate cancer ͉ breast cancer ͉ RNA-Seq

High-throughput detection of fusion genes in cancer using the Sequenom MassARRAY platform

“…4,[22][23][24] The majority of fusion genes identified by massively parallel sequencing, however, appear to exist at very low prevalence or represent private events (ie identified only in the index case). 7,9,10,14,15 This observation and the fact that chromosomal rearrangements are more frequently identified within amplified regions of the genome have led some to conclude that the majority of these rearrangements are likely to constitute passenger events. 7,8 The probability of a given expressed fusion gene to constitute an oncogenic driver is higher if it is recurrently found in cancers.…”

“…[2][3][4][5][6][7][8][9][10] For example, fusion genes involving ETS-family members, such as ERG (SLC45A3-ERG), ETV1 (TMPRSS2-ETV1), ETV4 (TMPRSS2-ETV4), and ETV5 (SLC45A3-ETV5), have been shown to be common in prostate carcinomas. 2,6,[11][12][13][14][15][16] Some special types of breast cancer have recently been shown to be characterized by the presence of recurrent fusion genes resulting from chromosomal translocations, including ETV6-NTRK3 in secretory carcinomas 5,[17][18][19][20][21] and MYB-NFIB in adenoid cystic carcinomas. 4,[22][23][24] The majority of fusion genes identified by massively parallel sequencing, however, appear to exist at very low prevalence or represent private events (ie identified only in the index case).…”

Harnessing Next Generation Sequencing in Climate Change: RNA-Seq Analysis of Heat Stress-Responsive Genes in Wheat (Triticum aestivum L.)

“…The Illumina transcriptome was mainly used for the sequencing of organisms whose genome was sequenced . It was confirmed in due course of time that the relatively short reads can be effectively assembled by the Illumina transcriptome, or whole genome de novo sequencing and assembly with the advantage of paired-end sequencing (Maher et al, 2009).…”

Transcriptome Sequencing

“…The same group also used a paired-end strategy using the Illumina system to identify chimeras (Maher et al, 2009b). Furthermore, Ruan et al (2009) recently demonstrated that PET technology can be used to detect fusion genes.…”

“…An important feature of the Illumina system is the paired-end strategy, in which the DNA in each cluster can be fully extended and flipped on the surface, and another round of sequencing can begin from the other end of the DNA molecule. In this case, 100 nucleotides from each end are obtained, which facilitates the discovery of, for example, fusion genes (Maher et al, 2009b). Like 454, Illumina features a barcoding system, where multiple samples can be tagged with a specific sequence and pooled together on a slide prior to sequencing.…”