High-throughput RNA sequencing offers a comprehensive analysis of transcriptome complexity originated from regulatory events, such as differential gene expression, alternative polyadenylation and others, and allows the increase in diagnostic capacity and precision. For gene expression profiling applications that do not specifically require information on alternative splicing events, the mRNA 3′ termini counting approach is a cost-effective alternative to whole transcriptome sequencing. Here, we report MTAS-seq (mRNA sequencing via terminator-assisted synthesis) – a novel RNA-seq library preparation method directed towards mRNA 3′ termini. We demonstrate the specific enrichment for 3′-terminal regions by simple and quick single-tube protocol with built-in molecular barcoding to enable accurate estimation of transcript abundance. To achieve that, we synthesized oligonucleotide-modified dideoxynucleotides which enable the generation of cDNA libraries at the reverse transcription step. We validated the performance of MTAS-seq on well-characterized reference bulk RNA and further tested it with eukaryotic cell lysates.

Genetic rearrangements that fuse an androgen‐regulated promoter area with a protein‐coding portion of an originally androgen‐unaffected gene are frequent in prostate cancer, with the fusion between transmembrane serine protease 2 (TMPRSS2) and ETS transcription factor ERG (ERG) (TMPRSS2‐ERG fusion) being the most prevalent. Conventional hybridization‐ or amplification‐based methods can test for the presence of expected gene fusions, but the exploratory analysis of currently unknown fusion partners is often cost‐prohibitive. Here, we developed an innovative next‐generation sequencing (NGS)‐based approach for gene fusion analysis termed fusion sequencing via terminator‐assisted synthesis (FTAS‐seq). FTAS‐seq can be used to enrich the gene of interest while simultaneously profiling the whole spectrum of its 3′‐terminal fusion partners. Using this novel semi‐targeted RNA‐sequencing technique, we were able to identify 11 previously uncharacterized TMPRSS2 fusion partners and capture a range of TMPRSS2‐ERG isoforms. We tested the performance of FTAS‐seq with well‐characterized prostate cancer cell lines and utilized the technique for the analysis of patient RNA samples. FTAS‐seq chemistry combined with appropriate primer panels holds great potential as a tool for biomarker discovery that can support the development of personalized cancer therapies.