Fusion sequencing via terminator-assisted synthesis (FTAS-seq) identifies TMPRSS2 fusion partners in prostate cancer.

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.

Sensitive and accurate analysis of gene expression signatures enabled by oligonucleotide-labelled cDNA.

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.

Advanced preparation of fragment libraries enabled by oligonucleotide-modified 2',3'-dideoxynucleotides.

The ever-growing demand for inexpensive, rapid, and accurate exploration of genomes calls for refinement of existing sequencing techniques. The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. While modified nucleotides already were inherent tools in sequencing and imaging, further modification of nucleotides enabled the expansion into even more diverse applications. Herein we describe the design and synthesis of oligonucleotide-tethered 2',3'-dideoxynucleotide (ddONNTP) terminators bearing universal priming sites attached to the nucleobase, as well as their enzymatic incorporation and performance in read-through assays. In the context of NGS library preparation, the incorporation of ddONNTP fulfills two requirements at once: the fragmentation step is integrated into the workflow and the obtained fragments are readily labeled by platform-specific adapters. DNA polymerases can incorporate ddONNTP nucleotides, as shown by primer extension assays. More importantly, reading through the unnatural linkage during DNA synthesis was demonstrated, with 25-30% efficiency in single-cycle extension.

High-resolution microbiome analysis enabled by linking of 16S rRNA gene sequences with adjacent genomic contexts.

Sequence-based characterization of bacterial communities has long been a hostage of limitations of both 16S rRNA gene and whole metagenome sequencing. Neither approach is universally applicable, and the main efforts to resolve constraints have been devoted to improvement of computational prediction tools. Here, we present semi-targeted 16S rRNA sequencing (st16S-seq), a method designed for sequencing V1-V2 regions of the 16S rRNA gene along with the genomic locus upstream of the gene. By in silico analysis of 13 570 bacterial genome assemblies, we show that genome-linked 16S rRNA sequencing is superior to individual hypervariable regions or full-length gene sequences in terms of classification accuracy and identification of gene copy numbers. Using mock communities and soil samples we experimentally validate st16S-seq and benchmark it against the established microbial classification techniques. We show that st16S-seq delivers accurate estimation of 16S rRNA gene copy numbers, enables taxonomic resolution at the species level and closely approximates community structures obtainable by whole metagenome sequencing.

Enzymatic Synthesis of Chimeric DNA Oligonucleotides by in Vitro Transcription with dTTP, dCTP, dATP, and 2'-Fluoro Modified dGTP.

Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. In the present study, we selected T7 RNA polymerase mutants with reduced substrate specificity to employ an in vitro transcription reaction for the synthesis of chimeric DNA oligonucleotides, either individually or in pools. We performed in vitro evolution based on fluorescence-activated droplet sorting and identified mutations V783M, V783L, V689Q, and G555L as novel variants leading to relaxed substrate discrimination. Transcribed chimeric oligonucleotides were tested in PCR, and the quality of amplification products as well as fidelity of oligonucleotide synthesis were assessed by NGS. We concluded that enzymatically produced chimeric DNA transcripts contain significantly fewer deletions and insertions compared to chemically synthesized counterparts and can successfully serve as PCR primers, making the evolved enzymes superior for simple and cheap one-pot synthesis of multiple chimeric DNA oligonucleotides in parallel using a plethora of premixed templates.

Molecular Insights into miRNA-Driven Resistance to 5-Fluorouracil and Oxaliplatin Chemotherapy: miR-23b Modulates the Epithelial­Mesenchymal Transition of Colorectal Cancer Cells

Although treatment of colorectal cancer with 5-florouracil and oxaliplatin is widely used, it is frequently followed by a relapse. Therefore, there is an urgent need for profound understanding of chemotherapy resistance mechanisms as well as the profiling of predictive markers for individualized treatment. In this study, we identified the changes in 14 miRNAs in 5-fluouracil and 40 miRNAs in oxaliplatin-resistant cell lines by miRNA sequencing. The decrease in miR-224-5p expression in the 5-fluorouracil-resistant cells correlated with drug insensitivity due to its overexpression-induced drug-dependent apoptosis. On the other hand, the miR-23b/27b/24-1 cluster was overexpressed in oxaliplatin-resistant cells. The knockout of miR-23b led to the partial restoration of oxaliplatin susceptibility, showing the essential role of miR-23b in the development of drug resistance by this cluster. Proteomic analysis identified target genes of miR-23b and showed that endothelial-mesenchymal transition (EMT) was implicated in oxaliplatin insensibility. Data revealed that EMT markers, such as vimentin and SNAI2, were expressed moderately higher in the oxaliplatin-resistant cells and their expression increased further in the less drug-resistant cells, which had miR-23b knockout. This establishes that the balance of EMT contributes to the drug resistance, showing the importance of the miR-23b-mediated fine-tuning of EMT in oxaliplatin-resistant cancer cells.

Tissue-Specific miRNAs Regulate the Development of Thoracic Aortic Aneurysm: The Emerging Role of KLF4 Network.

MicroRNAs (miRNAs) are critical regulators of the functional pathways involved in the pathogenesis of cardiovascular diseases. Understanding of the disease-associated alterations in tissue and plasma will elucidate the roles of miRNA in modulation of gene expression throughout development of sporadic non-syndromic ascending thoracic aortic aneurysm (TAA). This will allow one to propose relevant biomarkers for diagnosis or new therapeutic targets for the treatment. The high-throughput sequencing revealed 20 and 17 TAA-specific miRNAs in tissue and plasma samples, respectively. qRT-PCR analysis in extended cohort revealed sex-related differences in miR-10a-5p, miR-126-3p, miR-155-5p and miR-148a-3p expression, which were the most significantly dysregulated in TAA tissues of male patients. Unexpectedly, the set of aneurysm-related miRNAs in TAA plasma did not resemble the tissue signature suggesting more complex organism response to the disease. Three of TAA-specific plasma miRNAs were found to be restored to normal level after aortic surgery, further signifying their relationship to the pathology. The panel of two plasma miRNAs, miR-122-3p, and miR-483-3p, could serve as a potential biomarker set (AUC = 0.84) for the ascending TAA. The miRNA-target enrichment analysis exposed TGF-ß signaling pathway as sturdily affected by abnormally expressed miRNAs in the TAA tissue. Nearly half of TAA-specific miRNAs potentially regulate a key component in TGF-ß signaling: TGF-ß receptors, SMADs and KLF4. Indeed, using immunohistochemistry analysis we detected increased KLF4 expression in 27% of TAA cells compared to 10% of non-TAA cells. In addition, qRT-PCR demonstrated a significant upregulation of ALK1 mRNA expression in TAA tissues. Overall, these observations indicate that the alterations in miRNA expression are sex-dependent and play an essential role in TAA via TGF-ß signaling.