Hybrid-seq deciphers the complex transcriptional profile of the human BRCA1 DNA repair associated gene.

Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of BRCA1 (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect BRCA1 alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of BRCA1 splice variants (BRCA1 sv.7 - sv.52), thus deciphering the transcriptional landscape of the human BRCA1 gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described BRCA1 mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, in silico analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.

The Transition from Cancer "omics" to "epi-omics" through Next- and Third-Generation Sequencing.

Deciphering cancer etiopathogenesis has proven to be an especially challenging task since the mechanisms that drive tumor development and progression are far from simple. An astonishing amount of research has revealed a wide spectrum of defects, including genomic abnormalities, epigenomic alterations, disturbance of gene transcription, as well as post-translational protein modifications, which cooperatively promote carcinogenesis. These findings suggest that the adoption of a multidimensional approach can provide a much more precise and comprehensive picture of the tumor landscape, hence serving as a powerful tool in cancer research and precision oncology. The introduction of next- and third-generation sequencing technologies paved the way for the decoding of genetic information and the elucidation of cancer-related cellular compounds and mechanisms. In the present review, we discuss the current and emerging applications of both generations of sequencing technologies, also referred to as massive parallel sequencing (MPS), in the fields of cancer genomics, transcriptomics and proteomics, as well as in the progressing realms of epi-omics. Finally, we provide a brief insight into the expanding scope of sequencing applications in personalized cancer medicine and pharmacogenomics.